The roles of integrin-like proteins, tyrosine phosphorylation and F-actin in hyphal tip growth A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biochemistry in the University of Canterbury By Kanueng Chitcholtan 2005 Table of Contents i Table of Contents Abstract??????????????????????????????v Abbreviations????????????????????????????vi Acknowledgements?????????????????????????viii Chapter 1 : General introduction .......................................................................1 1.1 Mechanism of tip growth and cell polarity.............................................1 1.2 Integrins.....................................................................................................6 1.2.1 Structure of integrins.......................................................................6 1.2.2 Integrin activation: Current model of outside-in and inside-out signalling ..................................................................................................................10 1.2.3 Integrin-like proteins in oomycetes and fungi...............................16 1.2.4 Integrin-like proteins in plants ......................................................19 1.3 Relationship of integrins and tyrosine phosphorylation .....................22 1.3.1 Hemidesmosomes and focal adhesion sites in animal cells ..........22 1.3.2 The presence of tight adhesions in oomycetes fungi and plants ...25 1.3.3 Protein cytoskeleton: actin and actin - binding proteins ...............27 1.3.4 Actin and cell polarity...................................................................28 1.3.5 Actin and tip growth......................................................................30 1.3.6 Actin-binding proteins (ABPs)......................................................31 1.4 Aims of Thesis .........................................................................................34 Table of Contents ii Chapter 2 : Immunocolocalisation of integrin-like proteins and phosphotyrosine containing proteins in A. bisexualis and S. cerevisiae....35 2.1 Introduction.............................................................................................35 2.1.1 Integrin-like proteins in oomycetes, fungi and plants...................35 2.1.2 The existence of adhesion sites in oomycetes, fungi and plants ...37 2.1.3 Tyrosine phosphorylation in hyphae and plants............................38 2.1.4 Adhesion sites in animal cells .......................................................39 2.2 Materials and Methods...........................................................................42 2.2.1 Cell cultures and antibodies ..........................................................42 2.2.2 Co-localisation of ?4 integrin-like protein and phosphotyrosine containing proteins....................................................................................43 2.2.3 Co-localisation of ?4 integrin-like protein and F-actin in A. bisexualis 44 2.2.4 Immunoblotting of Integrin-like proteins......................................44 2.2.5 Immunoblotting of phosphotyrosine containing proteins .............46 2.2.6 Immunoblotting of phosphotyrosine-containing proteins in the microsomal pellet fraction from A. bisexualis ..........................................47 2.3 Results......................................................................................................48 2.3.1 Immunocytochemistry of integrin-like protein and phosphotyrosine containing proteins in A. bisexualis ..........................................................48 2.3.2 Immunocytochemistry of Integrin-like protein and phosphotyrosine containing proteins in S. cerevisiae ..........................................................49 2.3.3 Immunoblotting of Integrin-like protein in A. bisexualis..............54 2.3.4 Immunoblotting of integrin-like protein in S. cerevisiae ..............54 2.3.5 Immunoblotting of phosphotyrosine containing proteins in A. bisexualis and S. cerevisiae .......................................................................................56 2.4 Discussion ................................................................................................58 Table of Contents iii Chapter 3 : The presence of protein tyrosine kinase activities in A. bisexualis and S. cerevisiae.............................................................................65 3.1 Introduction.............................................................................................65 3.1.1 Presence of protein tyrosine kinases in oomycetes, fungi, and plants 65 3.1.2 Tyrosine kinases in animal cells....................................................68 3.1.3 Conservative domains of SH2 and SH3 in tyrosine kinase and signaling proteins......................................................................................................70 3.1.4 Tyrosine kinase Inhibitors.............................................................72 3.2 Materials and Methods...........................................................................75 3.2.1 Immunocytochemistry of phosphotyrosine containing protein of A. bisexualis hyphae in the presence of tyrphostin 63 ..................................75 3.2.2 Protein extraction, partial purification and protein tyrosine kinase assay ..................................................................................................................75 3.2.3 Partially purified proteins..............................................................76 3.2.4 The detection of tyrosine kinase activity by ELISA .....................76 3.2.5 Detection of tyrosine kinase activity by a coupled assay..............77 3.2.6 The effect of tyrphostin on tip growth ..........................................78 3.2.7 The effect of tyrphostin on mycelial growth.................................78 3.3 Results......................................................................................................79 3.3.1 The effect of tyrphostin 63 on phosphotyrosine containing proteins in A. bisexualis hyphae......................................................................................79 3.3.2 The presence of protein tyrosine kinase activities in A. bisexualis and S. cerevisiae ..................................................................................................81 3.3.3 Extension rate of individual hyphae of A. bisexualis....................96 3.3.4 The effect of tyrphostins on mycelial area....................................98 3.4 Discussion ..............................................................................................101 Table of Contents iv Chapter 4 : The role of actin and actin-binding proteins in tip growth of A. bisexualis.........................................................................................................107 4.1 Introduction...........................................................................................107 4.1.1 Actin cytoskeleton.......................................................................107 4.1.2 Actin structure.............................................................................108 4.1.3 Organisation of dynamic actin in living cells..............................113 4.1.4 Actin and components of focal adhesion proteins ......................119 4.1.5 Actin and cell polarity.................................................................119 4.1.6 Actin and tip growth....................................................................120 4.2 Materials and Methods.........................................................................122 4.2.1 The effect of tyrphostins and TPA on actin staining...................122 4.2.2 Immunocytochemistry of talin-like protein in A. bisexualis.......122 4.2.3 Immunoblotting of talin-like protein in A. bisexualis and S. cerevisiae ................................................................................................................123 4.2.4 Co-localisation of actin and talin-like protein in A. bisexualis and S. cerevisiae ................................................................................................124 4.3 Results....................................................................................................126 4.3.1 The effect of tyrphostins on F-actin in A. bisexualis...................126 4.3.2 Immunocytochemistry of talin-like protein in A. bisexualis hyphae127 4.3.3 Immunoblotting of talin-like protein in A. bisexualis hyphae.....131 4.3.4 Co-localisation of talin-like protein and F-actin in A. bisexualis hyphae and S. cerevisiae .....................................................................................134 4.4 Discussion ..............................................................................................139 Chapter 5 : Conclusions.................................................................................143 References???????????????????????...152 Appendices???????????????????????..165 Abstract v Abstract Tip growth, the mechanism by which hyphae, pollen tubes, root hairs, and algal rhizoids extend, is a complex and dynamic process that is characterised by localised extension at the extreme apex of the cell and morphological polarity. Its complexity suggests that high degree of regulation is needed to ensure that the characteristics of a particular cell type are maintained during growth. Regulation is likely to come about through bidirectional interplay between the cell wall and cytoplasm, although the mechanisms by which such cross-talk might occur are unknown. Results of this thesis present immunocytochemical data that indicate the presence of, and a close association between ?4 integrin subunit-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall-membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the possible action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. In addition, results show A. bisexualis contains proteins epitopically similar to the rod domain of animal talin. However, these proteins do not co-localise with F-actin, and mainly locate at the sub-apical region in hyphae. For comparative purposes, Saccharomyces cerevisiae was also used to investigate the presence of ?4 integrin subunit-like proteins and tyrosine phosphorylation. Immunoblotting showed that S. cereviaise contains a protein, which is found in the microsomal pellet fraction, that cross reacts with anti-?4 integrin subunit antibody. Furthermore, there are a number of proteins containing phosphotyrosine residues. Immunocytochemistry shows that this anti-?4 integrin staining is at the cortical site but anti-phosphotyrosine residues are distributed throughout cells. On the basis of an ELISA and a coupled enzyme assay, it is suggested that a soluble fraction of S. cerevisiae contains tyrosine kinase activity. This activity is strongly inhibited by tyrphostins. Abbreviations vi Abbreviations ?C Degree Celsius ?l Microlitre ?M Micromolar A 280 Absorbance at 280 nm ADP Adenosine diphosphate ATP Adenosine triphosphate BLAST Basic Local Alignment Search Tool BSA Bovine serum albumin DMSO Dimethyl sulphoxide DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol-bis (?-aminoethyl ether) N, N, N, N,-tetraacetic acid ELISA Enzyme-Linked Immunosorbent Assay g Grams HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid IgG Immunoglobulin G kDa Kilodalton L Litre mg Milligram mins Minutes mL Millilitres mM Millimolar mV Millivolt NADH Nicotinamide adenine dinucleotide nm Nanometres nM Nanomolar PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffer saline PIPES Piperazine-N,N?-bis(2-ethanesulfonic acid) PMSF Phenylmethylsulfonyl fluoride Abbreviations vii PYG Peptone-Yeast extract-Glucose rpm Revolutions per minute S.D. Standard deviation SDS Sodium dodecyl sulfate v/v Volume/volume w/v Weight/volume YPD Yeast extract- Peptone- Dextrose Acknowledgements viii Acknowledgements For all his tremendous understanding, supporting, and guidance on many aspects relating to this project, and my life in the present and in the future, I would like to say a big thank you to my supervisor, Ashley Garrill. I also thank Sandra Jackson for contributing her knowledge to this thesis and assisting with confocal microscopic techniques. I would also like to thank my partner David Anderson for emotional, physical and financial supporting throughout my study. I would like to thank sexy Craig Galilee for ordering chemicals and many things that I can not remember. Also, this thesis would not be completed without assistance of the spunky Manfred Ingerfeld for helping me with microscopic techniques, so thank you spunky. I also thank Matt Walter for his tremendous assistance and guidance of printing. Also, this thesis can not be completed without a supporting from my good friends, Sophie Walker, Andy Cox, Nick Cuming, Tracy Putoczki , Hemma Nair, Shane Welsh, Fergus, Sandi, and to all my friends who I can not remember your names. Chapter 1 : General introduction 1.1 Mechanism of tip growth and cell polarity Tip growth is a major form of cell growth, characteristic of fungal and oomycete hyphae and plant cells such as root hairs and pollen tubes. The tip growth mechanism is complex and dynamic and includes localised expansion of the apical cell wall and plasma membrane (PM), the migration of the cytoplasm to keep up with the advancing tip, the movement of both organelles and exocytotic vesicles through the apical cytoplasm and the production and maintenance of polarised distributions of membrane proteins such as cell-wall fibril synthases and ion channels (Heath, 2000), (Heath and Skalamera, 2001). These processes lead to a highly polarised cell structure as depicted in Error! Reference source not found.. which shows a hyphal tip of Achlya bisexualis. Wall vesicles accumulate at the extreme tip of hyphae. Mitochondria are distributed at just behind the hyphal apex and nuclei are present in more subapical regions. The hyphal growth of oomycetes, fungi and plant cells is driven by the internal hydrostatic pressure called turgor pressure, the pressure exerted on the plastic wall at the tip causing deformation and thus localised extension at this site. Ray et al., (1972) stated that cell expansion is driven by turgor in the sense that the wall yields to the force exerted by the internal hydrostatic pressure. Turgor pressure is commonly defined as being the pressure exerted on the cell by the turgid protoplasm which is brought about the difference in the osmotic potential of the protoplasm and the external environment (Money, 1997). Hyphal organisms such as oomycetes and fungi encounter and must adapt to variation in the external osmotic potential when they grow in diverse environments. This can affect their ability to take up water during cellular expansion and maintain their internal turgor. Chapter 1: General introduction 2 Turgor pressure is a common property of fungi and oomycetes but turgor does not appear to be essential for hyphal growth. Some oomycetes such as A. bisexualis and Saprolegnia ferax do not regulate turgor after hyperosmotic treatment (Money and Harold, 1992; Lew et al., 2004; Money et al., 2004). In S. ferax a variety of structural and developmental events are unaffected by turgor loss. Furthermore, the distribution of F-actin in hyphal apices is not influenced by turgor reduction, nor is hyphal sensitivity to a range of compounds that disrupt the cytoskeleton and cyst germination occurs with normal frequency (Harold et al., 1996). Furthermore, the discovery of a wall-less amoeboid variant of N. crassa suggests that even ascomycetes have the innate ability to grow in the absence of turgor (Emerson, 1963), although N. crassa can regulate turgor pressure after hyperosmotic treatment. Thus it appears that the true fungi but not oomycetes are able to regulate turgor pressure. This difference may reflect their habitats representing two extremes in terms of osmotic challenge. N. crassa is a terrestrial fungus whereas A. bisexualis and S. ferax are present in fresh water. Thus data would suggest a single model of tip growth driven by turgor pressure may not be sufficient to explain hyphal extension in oomycetes and/or wall-less N. crassa. If the turgor pressure plays a non-essential role in hyphal growth in oomycetes then there may be other processes that function along side turgor pressure. One alternative pathway that hyphae of oomycetes may utilise is the polymerisation of actin. Hyphae of oomycetes and fungi have enrichments of actin located at their extreme tips. Error! Reference source not found..B shows F-actin in the apex of A. bisexualis hyphae. This has been used to suggest that actin polymerisation may generate a protrusive force. Chapter 1: General introduction 3 Chapter 1: General introduction 4 Actin polymerisation is a major force generating system in the expansion of animal cells. The best example is the movement of lamellipodium. Figure 1.1. shows the process of ameboid movement that involves the coordination of several components including F-actin, actin-binding proteins, integrins and signalling molecules, such as Ca 2+ . When animal cells grow on a solid substrate, they are able to move by extension of the leading edge. Actin assembles predominantly at the leading edge because the concentration of uncapped barbed end is high in this region and can be rate limiting for polymerisation (Chen et al., 2000). The uncapped barbed ends can be generated by de novo nucleation, severing of existing filaments and removal of barbed ends capping proteins. The actin related protein 2/3 (Arp2/3) plays a crucial role in movement of lamellipodium. The location of the Arp2/3 complex to the leading edge helps to provide the spatial organisation necessary for locomotion. The Arp2/3 complex is activated for actin nucleation by binding to proteins in the Wiscott-Aldrich Syndrome protein family, which are in turn activated by Cdc42 (Chen et al., 2000; Pantaloni et al., 2001). The details of actin polymerisation are addressed further in chapter 4. Several lines of evidence suggest that the growing hyphal tip may have some similarity with lamellipodia/amoeboid movements in animal cells. Some oomycete zoospore appear to escape from their sporangia by an amoeboid process (Gay and Greenwood, 1966). When secondary zoospores of Saprolegnia leave the germ tubes of the primary cysts, they seem to do so by amoeboid movement against the germ tube wall (Holloway and Heath, 1977a). Probably the most compelling argument for amoeboid based tip growth studies in the slime mutants of the fungus Neurospora. This mutant fails to produce a cell wall and thereby is devoid of turgor but they can proliferate in osmotically buffered media as protoplasts. The protoplasts can move across solid substrate like amoebae by producing tubular pseudopodia (Trevithick and Galsworthy, 1977). In common with lamellipodia/amoeboid movement this movement depends on substrate adhesion. The process is thus likely to involve adhesion molecules such as integrins, located at the plasma membrane. Chapter 1: General introduction 5 (A) (B) Figure 1.1. Representation of involvement of F-actin and integrins in movement of lamellipodium (from Alberts et al., 2004, Molecular Biology of the Cell 4 th Edition). Chapter 1: General introduction 6 1.2 Integrins Integrins are heterodimers of ? and ? subunits that consist of an extracellular domain that is responsible for ligand binding and a cytoplasmic domain that plays a vital role in signalling processes and protein-protein interactions at focal contacts and hemidesmosomes. Each individual subunit has a large amino terminal consisting of more than 700 residues forming the extracellular domain, and a cytoplasmic domain that in most cases consists of 20-70 residues. One exception is the cytoplasmic domain of the ?4 subunit that contains 1000 amino acid residues (Error! Reference source not found.. A and B). Integrins play a vital role in many processes including cell adhesion, cell migration, the control of cell differentiation, proliferation and programmed cell death. They are able to convey signals from the outside of the cell to the inside and conversely from the inside to the outside. These phenomena referred to as bidirectional signalling. 1.2.1 Structure of integrins There are 8 known ? subunits and 18 known ? subunits that are combined to form 24 distinct integrins. A hallmark of the integrins is the ability of individual family members to recognise multiple ligands. Error! Reference source not found.. summarises the members of integrins and the ligands that they interact with. The list includes a large number of extracellular matrix proteins reflecting the primary function of integrins in cell adhesion to extracellular matrices (Plow et al., 2000). Integrin-ligand interactions are determined by several factors. One major determinant of ligand binding specificity is the subunit composition of an integrin. One half of the integrin ? subunits contain an extra von Willebrand factor A-type domain (?A or I domain) and are known as ?A domains see Error! Reference source not found..B (Arnaout et al., 2002). A single bound divalent cation is found at the apex of ?A, coordinated by oxygen containing side chains from three loops, one containing an Asp-X-Ser-X-Ser motif (where X is any amino acid), the second an invariant threonine and the third an invariant aspartic acid. These residues are referred as the metal-ion-dependent adhesion site (MIDAS) motif (Emsley et al., 2000). Chapter 1: General introduction 7 In ?A lacking integrins, ligand binding is mediated by a predicted ?-propeller domain from the ? subunit and a predicted ?A-like domain (?A) from the ? subunit (Humphries et al., 2003). The MIDAS motif of ?A binds Mn 2+ or Mg 2+ with high (micromolar) affinity and Ca 2+ with low (millimolar) affinity. Three additional metal-binding regions were predicted in the extracellular segment of integrins: one in ?A, another in the EF-hand-like sequence at the bottom of the propeller and the third in the propeller?s central core (Arnaout et al., 2002). The ? subunit contains eight domains. A ?A domain contacts the top of the subunit?s propeller, thus forming the ?? heterodimers. The ?A projects from an I-set immunoglobulin-like ?hybrid? domain. Both the ?A and hybrid domains contribute to the head region (Liddington, 2002). The ? leg section is formed of six domains: an amino terminal PSI (plexins, semaphorins and integrins) domain lies at the base of the hybrid domain and is probably linked to the first of four epidermal growth factor (EGF) domains by a disulfide bond. PSI, EGF1 and EGF2 domains are disordered. EGF3 and EGF4 each contains a core of six cysteine residues linked in a Cys1-Cys3, Cys2-Cys4, Cys5-Cys6 pattern (Arnaout et al., 2002). The structure determined by Xiong et al., (2001) was obtained in a Ca 2+ buffer and lacked bound ligand. This condition usually produces inactive integrins. Subsequent structures obtained after diffusing cycloRGDF and Mn 2+ into the crystal showed cycloRGDF bound at the ?? interface with the arginine residue binding the propeller domain of the ? subunit and the aspartate joining the coordination sphere of a Mn 2+ ion bound at the MIDAS site (Xiong et al., 2002). Chapter 1: General introduction 8 Chapter 1: General introduction 9 Chapter 1: General introduction 10 1.2.2 Integrin activation: Current model of outside-in and inside-out signalling Xiong et al. (2001, 2002) suggested that the bent form is the active form of the integrin. However, others have argued that it is more likely to be an inactive state, based on the details of the conformation of the ?-I/A domain and the fact that it was crystallised in the absence of ligand (Shimaoka et al., 2000; Liddington and Ginsberg, 2002). By mapping epitopes for activation specific monoclonal antibodies to specific residues in I-EGF repeats 2 and 3, it has been shown that these residues would be buried in the bent form of the integrin (Beglova et al., 2002). Beglova et al, (2002) proposed that the bent form represents the inactive state and that activation occurs by a ?switch-blade? opening of the integrin into an extended shape and a separation of the legs. Such a conformational change could expose the epitopes for activation-specific antibodies, many of which are known to bind to the I-EGF repeats or to the PSI domain. (Takagi et al., 2002) showed that integrins clamped in the inactive state predominantly adopt a bent shape as seen by electron microscopy (EM), whereas integrins activated by Mn 2+ or by cycloRGDF were predominantly in an extended form. They showed that the claimed bent form did not bind ligand, whereas the activated extended form did. Finally they presented evidence that integrins on cell surfaces can be trapped in a bent and inactive state by an engineered disulfide bond that when released, allows their activation. These results confirm well with the idea that the bent form seen in the crystal represents the inactive state of the integrin and that activation comprises straightening and separation of the legs. These concepts are schematised in Error! Reference source not found.. , which shows two ways in which the bent form might be related to the membrane. These differ in the orientation of the membrane-proximal ?ankles? of the legs relative to the membrane; this is unknown at present. In the switch-blade or ?flick-knife? model, the ?calves? of each leg are perpendicular to the membrane and the head domain is very close to the cell surface. In a variant ?angle-poise? model, the legs are bent over closer to the membrane and extend like an angle-poise lamp during activation. The latter model would place the head domain in a better position to interact with macromolecular ligand (Hynes, 2002). Chapter 1: General introduction 11 Chapter 1: General introduction 12 For some time, it has been suggested that the interaction of ?? tails of integrins lead to altered states of integrin activation. (Lu et al., 2001b) replaced the cytoplasmic domains of ?L and ?2 with acidic and basic peptides that form an ?-helical coiled-coil heterodimers, thus clasping the integrin tail region together (Error! Reference source not found..a). Expression of these mutated integrin subunits on the surface of cells resulted in cell adhesion molecule 1 (ICAM-1), unless activated externally. If, however, both subunits were expressed with basic coiled-coil domains that do not interact, the cells constitutively bound ligand. A similar approach was taken to study the effect of C- terminal opening on soluble ?5?1 activation in the absence of cellular factors (Takagi et al., 2001). This study used integrin that was truncated N-terminally to the transmembrane domain and clasped with the acid-base coiled-coil heterodimeric system (Error! Reference source not found..b). Enzymatic cleavage of the constraint led to increased fibronectin binding to the soluble integrin, compared with the clasped integrin. In addition, it was shown that activation of integrin by unclasping correlated with an approximate 14 nm separation of the integrin leg regions (Takagi et al., 2001; Travis et al., 2003). This suggests that conformational changes in integrins can account for major affinity changes in integrin irrespective of integrin clustering or avidity changes. These two studies therefore demonstrate that releasing the C-terminal constraint on integrins can mimic ?inside-out? signalling, and suggest that the mechanism of inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains. A number of intracellular proteins have been reported to bind to integrin cytoplasmic domains, with many influencing integrin function. Such molecules have been implicated in both outside-in and inside-out signaling processes. Several molecules including calcium- and integrin- binding protein (CIB), ?3-endonexin, cytohesin-1 and talin, have been shown to bind to integrin ?-subunit cytoplasmic tails resulting in activation of the integrin. (O'Toole et al., 1991; O'Toole et al., 1994) have shown that the short cytoplasmic domain of ?IIb acts as a negative regulator of activation. A constitutively active integrin could be produced if the entire domain or the highly conserved sequence was deleted. O'Toole et al., (1994) also suggested that the interaction of the cytoplasmic domain of integrins with proteins at focal adhesion sites was a central control point in the Chapter 1: General introduction 13 activation process. (Horwitz et al., 1986) found that the talin co-localizes with integrins at focal adhesion sites and possibly forms a link between integrins and the actin cytoskeleton. (Vinogradova et al., 2002) have proposed a model for the activating properties of talin, which was previously demonstrated to bind to the ?3 tail through its N-terminal head domain, leading to integrin activation. (Vinogradova et al., 2002) verified that a fragment of talin could activate ?IIb?3 and also demonstrated that it disrupted the ?? complex observed using NMR. Furthermore, the substantial transferred nuclear overhauser effects (NOEs), which detect protein-protein interactions through space, for the ?IIb-?3 mixture was lost in the presence of talin. The authors concluded that talin effectively competed with the ?IIb tail for binding to the ?3 tail, thereby disrupting the interaction between the two tails. This mechanism could be controlled at the level of talin because its integrin binding site is masked in the whole molecule. Binding of talin to inositol phospholipids or proteolytic cleavage of talin resulted in unmasking of this integrin binding site (Calderwood et al., 1999; Martel et al., 2001; Yan et al., 2001). The authors therefore suggested a model in which one of a series of agonist signals would result in a conformational change in talin, leading to exposure of its integrin binding site. Subsequent binding of talin to the ?3 tail would in turn lead to separation of the integrin tails and integrin activation (Error! Reference source not found..) A recent study has determined the crystal structure of interacting fragments of talin and ?3 cytoplasmic domain. The phosphotyrosine binding (PTB)-like domain of talin was found to interact directly with a central motif of the ?3 tail, encompassing the NPLY motif (Hynes, 2002). Chapter 1: General introduction 14 Chapter 1: General introduction 15 Chapter 1: General introduction 16 1.2.3 Integrin-like proteins in oomycete and fungi The presence of proteins with epitopic similarity to metazoan integrins in oomycetes, fungal and plant cells has been suggested by their cross-reactivity to antibodies raised against animal integrins, so the term of ?integrin-like protein? has been used in oomycetes, fungi and plant literature (Table 1.1.). (Kaminskyj and Heath, 1995) found that the filamentous oomycete Saprolegnia ferax has integrin-like proteins that are located at the tip. Western blots cross reacted with ? 1 - integrin antibody and a protein band at 120 kDa was obtained under reducing conditions. However, the failure of RGD-containing peptides to disrupt growth and adhesion in S. ferax suggests that either this protein is insensitive to RGD-containing proteins or that it does not play a role in growth or adhesion (Bachewich and Heath, 1997). Preliminary data with A. bisexualis however suggests that RGD-containing peptides do inhibit tip extension and affect tip morphology. Fungi such as Uromyces and Mucor rouxii have been shown to be sensitive to RGD- containing peptides (Correa et al., 1996; Pereyra et al., 2003). (Degousee et al., 2000) investigated integrin-like proteins in the filamentous ascomycete N. crassa, immunofluorescence showed that integrin-like proteins are located at the plasma membrane/cell wall and are enriched at the extreme tip; with more punctuate staining in the subapical region. These authors also found that in western blots a protein band at ~63 kDa cross reacted with the anti-? 1 integrin antibody, presumably corresponding to a fragment of the reduced form of the protein or an integrin homologue. The bean rust Uromyces appendiculatus has also been shown to contain integrin-like proteins, which were detected by two different ?1 antibodies, one from chicken and the other from human, a similar 95 kDa band was obtained. With respect to studies on non-filamentous fungi, (Marcantonio and Hynes, 1988) found that anti-integrin ?1 directed against the COOH-terminal domain of chicken cross reacted with a 95 kDa protein present in a membrane extract from C. albicans, but not from S. cerevisiae. (Gale et al., 1996) isolated a gene encoding a putative integrin-like protein in Chapter 1: General introduction 17 C. albicans by screening a genomic library with conserved sequences from the transmembrane and cytoplasmic domains of the human ?M. These authors found the predicted polypeptide (?Int1p) of 188 kDa which contains several motifs common to ?M and ?X: a putative I domain, two EF-hand divalent cation-binding site, a transmembrane domain and a cytoplasmic tail with a single tyrosine residue. More recent evidence showed that C. albicans has ?v?3 and ?v?5 integrin-like proteins, which recognise vitronectin in vitro. Moreover, the engagement of such integrin-like proteins consequently activates protein tyrosine phosphorylation (Santoni et al., 2002). Chapter 1: General introduction 18 Table 1.1. Integrin-like proteins found in fungi and oomycetes. Species Antibodies used Detected molecular weight (kDa) References Saccharomyces cerevisiae ?1 none Marcantonio et al,1988 Candida albicans ? 1 95 Marcantonio et al,1988 Neurospora crassa ? 1 63, 120 Degous?e et al,2000 Uromyces appendiculatus ? 1 95 Corr?a et al,1996 Saprolegnia ferax ? 1 120, 178 Kaminskyj et al,1995 Chapter 1: General introduction 19 1.2.4 Integrin-like proteins in plants In plants, it is apparent that the cell wall plays a key role in both cell-cell communication and the maintenance cell shape. A large number of proteins-associated with the cell wall have been investigated these include WAK-like kinases, cellulose synthases, glucanases, arabinogalactan proteins (AGPs), pectin and glycine-rich protein (GRP) all of which are candidates as wall membrane linkers that could function in cell wall/cytoplasmic communication (Kohorn, 2000). Recently, several lines of evidence have emerged suggesting that plants also contain integrin-like proteins which could also fulfil such a role. Furthermore, integrin-like proteins found in plants have a range of molecular weight similar to those found in animal cells (Table 1.2). (Sun et al., 2000) used confocal microscopy to image antibodies against the ?v, ?3, and ? 1 integrin subunits. These gave prominent staining at the tips of pollen tube in lily and tobacco. These authors previously showed that antibodies raised against the human VnR and against the ? 3 , and ?v integrin subunits cross reacted with proteins in the PM fractions of Hemerocallis citrina (Sun et al., 1998). Sun et al (2000) also found that anti- ?3, anti-?v and anti-VnR antibodies all recognised a ~150 kDa band in western blots in both H. citrina Baroni and in L. davidii Duch under non-reducing conditions. However, under reducing condition the anti-?3 antibody also cross reacted with a minor band at 95- 97 kDa in H. citrina Baroni in addition to a major 150 kDa band. Other groups have identified putative integrin-like proteins of 100, 58, and 84-116 kDa in plasma membrane fractions from Arabidopsis. There appears to be a high concentration of integrin-like protein in the root of Arabidopsis. In onion cells, (Gens et al., 1996) have described an integrin-like protein using monoclonal antibody against the cytoplasmic domain of ? 1 integrin in animal cells. Gens et al., (1996) found two protein bands of 105-110 and 115-125 kDa that cross reacted with the ?1 integrin antibody. These authors concluded that the protein bands from onion cells very likely represent protein antigenically analogous to ? 1 integrin from animal cells. (Lynch et al., 1998) showed the presence of a similar ? 1 integrin-like protein localised inside amyloplast of Chapter 1: General introduction 20 tobacco NT-1 suspension culture, callus cells and whole-root caps. A distinct band at 110 kDa in western blots suggested size homology with the known ? 1 integrin from chicken embryo crude extract. With respect to RGD sensitivity, (Labour? et al., 1999) found that when maize calluses were grown in the presence of the RGD peptide, important morphological changes were observed indicating the presence of a putative RGD-binding receptor. Immunoblotting showed a protein band at 120 kDa under reduced conditions using the anti-?1 antibody and protein bands at 30 and 60 kDa using anti-?IIb?3 antibody. Only the 60 kDa was observed when using anti-?3 antibodies. Chapter 1: General introduction 21 Table 1.2. Integrin-like proteins of plant species. Plant Species Antibody used Detected molecular weights (kDa) References Arabidopsis ? 1 58, 84- 116,100 Katembe et al 1997, Swatzell et al,1999 Onion Cells ? 1 105-125 Gens et al,1996 Nicotiana tabacum ? 1 110 Lynch et al,1998 Lilium davidii ? 3 150 Sun et al,2000 ?v 150 Hemerocallis citrina ? 3 95-97, 150 Sun et al,2000 ?v 150 Pisum sativum ? 1 60,74,86 Kiba et al,1998 Maize calluses ? 1 120 Labour?,1999 ? 3 60 ?IIb ? 3 30,60 Chapter 1: General introduction 22 1.3 Relationship of integrins and tyrosine phosphorylation Protein phosphorylation regulates a variety of cellular processes and is known to be involved in both eukaryotes and prokaryotes. In eukaryotes, signal transduction involving phosphorylation is generally initiated from the cell surface to the nucleus for development and function of cells (Nakashima et al., 2005). The signal is transferred through the cell in a cascade of threonine/serine and tyrosine phosphorylation events. In many cases these events lead to altered gene expression. 1.3.1 Hemidesmosomes and focal adhesion sites in animal cells In animal cells, adhesive interactions between cells and the surrounding extracellular matrix (ECM) proteins via integrins plays a vital role in cell migration, cell proliferation and differentiation, cell survival, blood clotting and inflammatory responses. Some of the integrin-mediated signal transduction pathways such as tyrosine phosphorylation have been shown to be initiated at cell junctions such as hemidesmosomes and focal adhesion sites (Error! Reference source not found.). 1.3.1.1 Presence of ?4 integrin in hemidesmosomes The ?4 integrin subunit associates exclusively with the ?6 subunit and is a receptor for the laminins (Plow et al., 2000). The ?4 cytoplasmic domain is distinct both in size (approximately 1000 amino acid residues) and structure from any other integrin subunit. From a functional prospective, it is known that ?6?4 is essential for the organisation and maintenance of epithelial architecture. In many epithelia, ?6?4 mediates the formation of stable adhesives structures termed hemidesmosomes on the basal cell surface that link the intermediate filament cytoskeleton with laminins in the basement membrane (Green and Jones, 1996) (Error! Reference source not found.. A). A significant breakthrough was the finding that ?6?4 can also associate with F-actin and is localised at the leading edges of Chapter 1: General introduction 23 invasive carcinoma cells (Mercurio et al., 2004). Moreover, ?6?4 mediates the migration of such cells through its ability to associate with the actin cytoskeleton and promotes the formation and stabilisation of filopodia and lamellipodia (Mercurio and Rabinovitz, 2001). A link between ?6?4-stimulation of invasion and signal transduction is though to activate phosphoinositide 3-OH kinases (PI3-K), a key signalling molecule and that activity of PI3-K is essential for invasion (Mercurio et al., 2004) One important function of PI3-K with respect to invasion appears to be its ability to regulate actin dynamics via the Rho- family of GTPases (Mercurio and Rabinovitz, 2001). 1.3.1.2 Presence of integrins in focal adhesion sites Unlike the ?4 integrin subunit, the ?1 integrin subunit, which has formed the basis for a number of studies in oomycetes, fungi and plants, can associate with several ? subunits. It typically associates with the ligand fibronectin and forms a major component of focal adhesion sites, which mediate cell to ECM adhesion (Hynes, 1992). The aggregation of actin stress fibers and associated intracellular proteins at these sites are thought to contribute to the spreading and migration of cells. The physical interaction of the cytoplasmic tail of integrins and proteins at focal adhesion sites indirectly links to the actin cytoskeleton via actin-binding proteins. Talin is a major structural element of focal adhesions and it is also the first actin binding protein that was shown to directly interact with the cytoplasmic domain of an integrin (Horwitz et al., 1986). However, both hemidesmosomes and focal adhesion sites play more than a structural role in anchoring the cell to the ECM, as ligand binding of integrin leads to the activation of a range of biochemical signalling events, including elevation of intracellular pH and Ca 2+ , activation of protein kinases, changes in lipid metabolism and ultimately, changes in gene expression. Chapter 1: General introduction 24 Chapter 1: General introduction 25 Talin consists of an NH 2 -terminal about 47 kDa globular head domain and about 196 kDa, COOH-terminal (Ree et al., 1990). The talin head domain has a band 4.1, ezrin, radixin, meosin homology (FERM) domain and this domain also can activate integrins Chapter 1: General introduction 26 (Calderwood et al., 1999; Calderwood et al., 2002). Talin also binds to actin thus indirectly providing a linkage between integrins and the actin cytoskeleton. The binding of integrins to other families of actin-binding proteins such as ?-actinin has also been well characterized. Like talin, ?-actinin co-localises with integrins in focal adhesions by interacting with ? cytoplasmic domain tails (Cattelino et al., 1999). However, the binding affinity of ?-actinin to the ? domain of integrin is less than that of talin (Goldmann, 2000). Additionally, integrins bind indirectly to several proteins at focal adhesion sites, these include vinculin, filamin, paxillin, actin and tensin (Calderwood et al., 2000; Critchley, 2000). Integrin-?-tails do not only provide a link to the actin cytoskeleton but they also regulate outside-in and inside-out signalling. However, integrins themselves have no enzymatic activity in their cytoplasmic tails. This suggests that for integrins to mediate a signal, they require direct binding of signalling proteins. A focal adhesion kinase (FAK) has been reported to bind to the integrin ? domain (Vuori, 1998; Parsons, 2003) and indeed FAK appears to play a central role in integrin-mediated signal transduction in animal cells. This kinase is tyrosine phosphorylated and its tyrosine kinase activity is enhanced upon integrin engagement (Parsons., 2003). FAK also binds to several signalling molecules that include paxillin, Grb2-SOS protein complex, phosphoinositide 3-kinases (PI 3-kinase) phospholipase C (PLC), Grb 7, CAS, ASAP/GRAF and Src (Schaller, 2001). 1.3.2 The presence of tight adhesions in oomycetes, fungi and plants Recent evidence suggests that oomycetes and fungi have a membrane skeleton linking to cell wall polymers forming tight adhesions, which can be detected following plasmolysis. The adhesions are found abundantly in regions of active wall expansion at the growing hyphal tips. In oomycetes such as S. ferax and A. bisexualis and fungi such as N. crassa, the apical cytoplasm often remains firmly attached to the apical walls following plasmolysis as the rest of the protoplasm retracts sub apically (Heath, 1987; Kaminskyj and Heath, 1995; Bachewich and Heath, 1997). Chapter 1: General introduction 27 It has been suggested that such attachments may be analogous to focal contacts in animal cells (Heath and Steinberg, 1999). Such adhesion sites in oomycetes and fungi have been found to contain actin and integrin-like proteins, it can thus be hypothesised that signal transduction processes common to focal adhesions, such as tyrosine phosphorylation, might also occur at hyphal tips. Evidence is accumulating to support the hypothesis that a tip-high Ca 2+ gradient is generated and maintained in fungi internally by IP3. Alteration of IP3 results in the reduction of tip morphogenesis and growth (Silverman-Gavrila and Lew, 2002). For the oomycete S. ferax hyphal tip growth was correlated with the difference between tip-localised Ca 2+ and basal Ca 2+ although in this organism the tip high Ca 2+ -gradient may be generated by localised mechanosensitive ion channels (Garrill et al., 1993). Other signalling molecules such as GTPases (e.g. Cdc42, Rac, and Rho) and G-protein subunits are key regulators in the signalling pathway that links extracellular growth signals to the assembly and organisation of the actin cytoskeleton, and these are found in several filamentous fungi (Raudaskoski et al., 2001). These signalling molecules are down stream molecules, which are regulated by tyrosine phosphorylation in animal cells. Tyrosine kinases such as Wee1 or Swe1p have been found in S. cerevisiae (Parker et al., 1993) The cell cycle delay that is induced by the morphogenesis checkpoint requires Swe1p. Checkpoint control of mitosis in S. cerevisiae is regulated by tyrosine phosphorylation of Cdc2 by Swe1p and dephosphorylation by a Cdc25-related phosphatase (McMillan et al., 1999). In this organism it has also been (Dailey et al., 1990) shown that p40 is a protein kinase with features that resemble known protein- serine kinase, which has tyrosine kinase activity toward an exogenous tyrosine containing peptide such as poly(Glu:Tyr 4:1 ). In addition, (Santoni et al., 2002) recently showed that Candida albicans expresses a focal adhesion kinase (Fak)-like protein that is antigenically related to the animal Fak p125 . With regard to hyphal tip growth, there is as yet no direct evidence showing the involvement of tyrosine kinases and/or tyrosine phosphorylation in this process. However, we can not rule out the possible role of such kinases and it is interesting to note recent evidence shows that tyrosine kinase inhibitors alter the development of fucoid algae including the genus Fucus and Pelvetia (Corellou et al., 2000). Furthermore, Chapter 1: General introduction 28 several signalling molecules, especially Ca 2+ play a role in the tip growth process (Fowler and Quatrano, 1997). In plants, several kinases that regulate many different aspects of metabolism growth and division have been isolated and characterised (Stone and Walker, 1995). Although protein phosphorylation seems to occur less abundantly on tyrosine than on serine or threonine residues and although conventional tyrosine kinases do not appear in sequence data from higher plants, tyrosine phosphorylation and tyrosine kinase activities have been reported in several plant species such as pea (Torruella et al., 1985; H?kansson and Allen, 1995), alfalfa (Duerr et al., 1993), tobacco (Zhang et al., 1996), maize (Trojanek et al., 1996), and coconut (Islas-Flores et al., 1998). 1.3.3 Protein cytoskeleton: actin and actin - binding proteins Filamentous organisms grow in a highly polarised fashion and give rise to tubular hyphae. Cytoskeletal elements in particular actin microfilaments are organised to support such growth. The current understanding of cytoskeletal organisation during polarised cell growth has greatly benefited from studies on the budding and the fission yeasts (Sawin, 1999). However, the growth pattern of filamentous growth differs from those of budding or fission yeast in that polarised growth has to be maintained (Xiang and Plamann, 2003). Actin is one of the most abundant proteins in all eukaryotes. Under physiological conditions, actin, a 43-kDa globular protein, polymerises itself into polar, helical filaments in which subunits are connected by a 167? rotation and 2.7 nm axial rise (Pantaloni et al., 2001). Irreversible hydrolysis of the bound ATP associated with polymerisation is at the origin of treadmilling and destabilises the filament. Treadmilling plays a crucial role in the function of actin in motility. Chapter 1: General introduction 29 1.3.4 Actin and cell polarity The budding yeast S. cerevisiae has proven a useful model system for investigating the molecular mechanisms involved in cell polarisation. The mechanisms underlying polarised morphogenesis highlights several important features that are likely to apply to filamentous organisms as well. In S. cerevisiae, cortical landmark proteins that generate a positional signal such as Bud3p, Bud4p and Axl2p or a bipolar budding pattern such as Bud8p, Bud9p and Rax2p have been well characterised and are summarised in Error! Reference source not found.. (Harris and Momany, 2004). The published genomes of filamentous species shows that these markers are poorly conserved in those species ( e.g. Aspergillus nidulans) (Harris and Momany, 2004). The lack of strong positional marker homologues might indicate that filamentous fungi do not use landmark proteins. In S. cerevisiae, once a positional landmark is established, it is transduced via the Cdc42 Rho GTPase module to the morphogenetic machinery. The active Cdc42-GTP transduces signals to multiple downstream effectors that in turn signal to the actin cytoskeleton. Among the best- characterised effectors are the p21-activated kinases (PAKs), Ste20p, and Cla4p, which among other functions may control actin cytoskeletal organisation by regulating the polarisome (Goehring et al., 2003). Cdc42 module proteins are highly conserved in A. nidulans and other filamentous fungi (Harris and Momany, 2004) and deletion of CDC42 is lethal in other fungi (Bassilana et al., 2003). Chapter 1: General introduction 30 Chapter 1: General introduction 31 1.3.5 Actin and tip growth Actin is thought to play a major role in tip growth of oomycetes, fungi, pollen tubes, and root hairs. In the oomycetes, growing tips contain a fine network of fibrils most concentrated at the extreme tip which become less abundant and more widely separated sub-apically (Heath, 1987). These apical fibrils (which have been called an F-actin cap) are oriented parallel to the long axis of the hyphae and lie exclusively adjacent to the plasma membrane. In non-growing tips, the actin cap is replaced by PM-associated plaques similar to those found in sub-apical regions of all hyphae. Conversely, the actin cap assembles prior to new tip formation when branches are produced (Bachewich and Heath, 1998). This pattern of actin is seen when hyphae are fixed with formaldehyde then stained with fluorochrome-labeled phalloidin. An alternative procedure has been used involving the introduction of rhodamine-labeled phalloidin (RP) into living hyphae in both S. ferax and Phytophthora sporangia (Jackson and Heath, 1990a, 1993a; Jackson and Hardham, 1998). However, this method is not thought to reveal all actin. Recently, hyphae of the oomycete A. bisexualis fixed with combinations of formaldehyde and methylglyoxal, showed an actin-depleted zone within the actin caps (Yu et al., 2004). This may suggest that in the growing tips of oomycetes there may be a discontinuity in the actin cap, which may reflect sites of vesicles accumulation. In the eufungi, there are no reports of an apical fibrillar cap in hyphal tips. Instead, the actin is always seen to be concentrated into peripheral plaques. In some species these occur over the entire apex, being most concentrated at the extreme apex (Heath, 1990; Heath and Skalamera, 2001), but in some species such plaques are absent from the extreme apex but become most abundant about 5 ?m back from the apex (Srinivasan et al., 1996; Torralba et al., 1998). In N. crassa, the presence of a structure that is rich in vesicles and F-actin, the Spitzenk?rper staining is only seen in growing hyphae where the plaques are absent from the extreme tip (Degousee et al., 2000), however, plaques reoccur when Spitzenk?rper is absent. Chapter 1: General introduction 32 In fact the Spitzenk?rper appears to be a part of the actin cytoskeleton and also is highly motile (Lopez-Franco and Bracker, 1996). It might also suggest that there are other undetected populations of actin in the apices of the eufungi. The presence of actin as either filaments or plaques at tips suggests that it plays critical functions in the tip growth process including the control of surface expansion and protrusion, the regulation of exocytosis, the direction of vesicle and organelle motility, an involvement in cytoplasmic migration and the regulation of plasma membrane protein distribution (Heath, 2000). Disruptions of actin by phalloidin, cytochalasins and latrunculins alters the morphogenesis of tips and terminates tip extension (Heath, 2000). 1.3.6 Actin-binding proteins (ABPs) The elongation of actin towards a growing tip is analogous to the distribution shown in motile structures of animal cells such as lamellipodia, filopodia, phagosomes and pinosomes. These cells require the assembly of actin filaments at the leading edge of the cell. Actin polymerisation is regulated and maintained by actin-binding proteins. Many actin-binding proteins have been identified and studied, and have been isolated from organisms across the evolutionary spectrum. ABPs regulate filament assembly and disassembly (e.g. profilin, thymosin ?4, ADF/cofilin, gelsolin, capping protein), de novo nucleation of actin filaments ( e.g. Arp2/3, WASP), cross-link or bundle actin filament (e.g. fimbrin, filamin, villin, ?-actinin) or are involved in the formation of focal adhesions ( e.g. vinculin, talin, paxillin, integrin). These ABPs are found in animal cells although not all of these ABPs are found in oomycetes, fungi and plants. The treadmilling of actin filaments is maintained by the actin depolymerising factor (ADF)/cofilin. ADF/cofilin is an 18 kDa, ubiquitous, conserved actin-binding protein, which is responsible for the rapid turnover of actin filaments in the cell. ADF/cofilin interacts preferentially with G-actin and F-actin in the ADP bound form (Carlier et al., 2003). ADF accelerates pointed end depolymerisation, which is the rate limiting step in the treadmilling ATPase cycle (Pantaloni et al., 2001). As a result, a higher steady-state concentration of monomeric ATP-actin is established that supports faster barbed end growth, balancing faster pointed-end depolymerisation (Figure 1.2.). Chapter 1: General introduction 33 Figure 1.2. Schematic diagram for the general activity of ADF/cofilin. ADF/cofilin servers actin filaments and enhances the disassociation of actin monomers from the pointed end and provides monomers for assembly at the barbed end of an actin filament. ADP ADP ATP ADP ATP ADP ADP ADP ADP Pi ATP ATP Barbed end Depolymerisation Polymerisation ADF Pointed end Chapter 1: General introduction 34 Plant ADF/cofilin family members share approximately 30 % amino acid identity with the vertebrate family members (Danyluk et al., 1996). ADF/cofilin has been found in pollen tubes and root hair of maize (Hussey et al., 2002) , tobacco (Chen et al., 2002), Arabidopsis (Hussey et al., 2002) and yeast (Du and Frieden, 1998). The oomycetes Phytophthora infestans and Phytophthora sojae have nucleotide sequences of ADF analogous to those found in Lilium longiflorum and A. thaliana. (http://www.pfgd.org). Profilin is an actin-binding protein, with functions that cooperate with those of ADF. Profilin specifically interacts with ATP-G actin. The profilin-actin complex productively associates with barbed ends (Carlier et al., 2003). It enhances the process of the treadmilling of actin filaments and improves actin-based motility. The actin related protein (Arp2/3) is a complex of actin-binding proteins, which is responsible for branching of actin filaments. The Arp2/3 complex is the downstream target of a variety of signalling pathway leading to actin polymerisation. Early studies provided evidence that the Arp2/3 complex was required for the motility and integrity of S. cerevisiae actin patches (Winter et al., 1997). Recently, genetic studies suggest that plant Arp2/3 complexes have similar functions to those of mammals (Millard et al., 2004). The key components of actin patch formation also include the Wiscott-Aldrich syndrome protein (WASP) family and Cdc42, which is a small GTPase. These actin regulators including the WASP homologue Las17/Bee1p and myosin Myo3p, have been found in yeast (Harris and Momany, 2004). Las17p is necessary for actin assembly in permeabilised cell assays, which is though to reflect a role in patch assembly (Weaver et al., 2003). Chapter 1: General introduction 35 1.4 Aims of Thesis The aims of the research described in the thesis were to firstly identify the presence of ?4 integrin subunit-like proteins in the oomycete Achlya bisexualis. Also, the relationship between these integrin-like proteins, F-actin and tyrosine phosphorylation and their role in tip growth were examined using immunocytochemistry, immunoblotting, ELISA, and coupled enzyme assays. Preliminary work was also carried out looking for similar proteins in yeasts with a view to creating a platform for future studies of their roles in budding and pseudohyphal growth. Chapter 2 : Immunocolocalisation of integrin-like proteins and phosphotyrosine containing proteins in A. bisexualis and S. cerevisiae 2.1 Introduction Tip growth, the mechanism by which hyphae, pollen tubes, root hairs and algal rhizoids extend, is a complex and dynamic process that is characterised by localised extension at the extreme apex of the cell and morphological polarity (Geitmann et al., 2001). Its complexity suggestions that a high degree of regulation is needed to ensure that the characteristics of a particular cell type are maintained during growth. Regulation is likely to come about through bidirectional interplay between the cell wall and cytoplasm, although the mechanisms by which such cross-talk might occur are unknown. There have been suggests that this may involve proteins with functional similarity to the integrins of the metazoan (Heath and Steinberg, 1999). 2.1.1 Integrin-like proteins in oomycetes, fungi and plants With respect to the above, the ?6?4 integrin is of particular interest in that it plays a role in cell adhesion and given the appropriate signal can function in cell migration. The ?6?4 is primarily concentrated at the basement membrane zone and is a component of hemidesmosomes (Watt, 2002). The ?6?4 binds with cytoskeleton proteins such as intermediate filaments and F-actin. One of the mechanisms by which ?6?4 promotes migration is through the activation of phophatidylinositol 3-kinase (PI3-K) and signalling via tyrosine phosphorylation and the mitogen activated protein kinase (MAPK) pathway (Watt, 2002). As detailed in chapter 1 and at greater length below, the presence of proteins with epitopic similarity to metazoan integrins in oomycete, fungal and plant cells Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 37 has been suggested by their cross-reactivity to antibodies raised against animal integrins although none of these studies have addressed the ?6 or ?4 integrin subunits. There are also a number of investigations that show an inhibitory effect of RGD- containing peptides on cell growth and development in many plant and fungal species (Santoni et al., 1994; Santoni et al., 1995; Correa et al., 1996; Gil et al., 1996; Canut et al., 1998; Faik et al., 1998; Kiba et al., 1998; Labour? et al., 1999; Pereyra et al., 2003). Evidence for the presence of integrin-like proteins in filamentous organisms is mostly derived using antibodies from animal integrins. Using immunoblotting or immunocytochemistry, it has been suggested that oomycetes, filamentous fungi, yeasts, and plants have integrin-like proteins that may be located at the plasma membrane- cell wall adhesion sites. In the oomycete S. ferax, immunoblotting showed that a polypeptide of about 178 kDa that had cross reactivity with the anti-?1 antibody. This integrin-like protein is located in the plasma membrane and at cell wall adhesion sites (Kaminskyj and Heath, 1995). However, RGD-containing peptides did not affect tip extension and tip morphology in this organism (Bachewich and Heath, 1997). Using a human ?M cDNA as a probe, (Gale et al., 1996) detected a C. albicans DNA sequence expressing an integrin-like domain. The full length of gene, the termed ?INT1? was detected and encoded a protein with a molecular weight of about 188 kDa (Gale et al., 1996). However, the amino acid sequence identity was low in comparison to animal integrin amino acid sequences. A putative fibronectin receptor molecular weight of about 120 kDa expressed by C. albicans was recognised by anti-?1 antibody (Santoni et al., 1995) and a protein with a molecular weight about 84 kDa has been recognised by anti ?5 antibody (Santoni et al., 1998). In S. cerevisiae a gene termed USO1 was identified by immunoscreening of an expression library of an integrin-I domain that is conserved in animal integrins. However, the biological function of USO1 protein appears to be in intracellular transport and it is mainly present in cytosolic rather than plasma membrane fractions (Sapperstein et al., 1996). A peptide of 95 kDa from the filamentous fungus Uromyces appendiculatus was recognised by an anti-?1 antibody (Correa et al., 1996) and under reducing condition a peptide of about 63 kDa identified from PM fractions of N. crassa treated with the anti- Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 38 ?1 antibody. However, immunoblotting of an unboiled PM extract showed a band at about 120 kDa (Degousee et al., 2000). A cDNA clone, AtELP1 (Arabidopsis thaliana EGF receptor-like protein) from A. thaliana has similarity to a highly conserved region of animal ?-integrins (Laval et al., 1999). Immunoblotting analysis showed that a protein of 60 kDa from A. thaliana cross reacts with an anti-?3 antibody. (Faik et al., 1998; Kiba et al., 1998) found that plasma membrane proteins from Pisum sativum at molecular weight at 60-, 74- and 86- kDa were recognised by anti-?1 antibody. 2.1.2 The existence of adhesion sites in oomycetes, fungi and plants In oomycetes and fungi, there are a number of pieces of evidence that indicate the presence of adhesion sites and that these are more abundant or are stronger in the hyphal tips. Firstly, plasmolysis leaves remnants of the plasma membrane and actin attached to the cell wall. These remnants are greater in number at the tips than sub-apical Hechtian strands. Furthermore, plasmolysis draws the cell wall inward in a region just behind the apex, where the plasma membrane remains attached to the wall via very broad adhesions (Kaminskyj and Heath, 1995; Bachewich and Heath, 1997). In addition, any contractions of cytoplasm are predominantly unidirectional towards the tips indicating potential sites of attachment. In fungi, it is possible that these attachments contain integrins or integrin-like proteins as fungal cell walls contain proteins with putative integrin-binding sites, comparable to those of animal ECM molecules (Laurent et al., 1999). This is also supported by the observed effects of RGD peptides on growth or differentiation in several fungi (Correa et al., 1996; Hostetter, 2000; Mellersh and Heath, 2001; Pereyra et al., 2003). In addition F- actin is often concentrated in the apical region of hyphae and associated with the apical plasma membrane (Kaminskyj and Heath, 1995). The data above are consistent with structures in walled cells that are comparable to focal adhesions of animal cells. In plants, physical connections between the cell wall and the plasma membrane have been observed in a number of ways including electron microscopy of cells showing the Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 39 appression of PM against the cell wall (Roberts, 1990). In plants, it is assumed that turgor pressure is largely responsible for appression, as disruption of turgor by osmotic shock induces plasmolysis and results in separation of the membrane from the cell wall (Roberts, 1990). The exact nature of the contact site is unknown, although there have been suggestions of homology between these and focal adhesion sites in animal cells. This suggestion is supported by the observed effects of treatment with RGD-containing peptides which can influence several physiological events in plants (Schindler et al., 1989; Wayne et al., 1992). Significantly these include a loss of plasma membrane-cell wall adhesion in plasmolysed Arabidopsis cells and a loss of the thin Hechtian strands that form during onion cell plasmolysis (Canut et al., 1998). A similar loss of localised plasma membrane-cell wall adhesions in plasmolysed zygotes of the alga Pelvetia after RGD-containing peptide treatments indicate that this type of interaction may not just be restricted to plants (Henry et al., 1996). More recently, two proline-rich cell wall proteins from bean, that were up-regulated during osmotic stress, were shown to interact with the plasma membrane in a manner that was disrupted specifically by RGD-containing peptides, even though these cell wall proteins apparently lack an RGD motif (Garc?a-G?mez et al., 2000). This may suggest that although the molecules involved in plasma membrane-cell wall/ECM interactions in plants and animal may differ in their exact structure, plant may possess functionally analogous cell wall/ECM proteins that contain an RGD-like configuration. 2.1.3 Tyrosine phosphorylation in hyphae and plants The formation of polarised cells such as hyphae requires the specification of growth sites, followed by the recruitment of morphogenetic machinery for localised the cell wall deposition. Once established, polarity must be strictly maintained during hyphal extension. Polarity maintenance implies the existence of novel regulatory molecules that include the Ras- related GTPases (Cdc42p), G-proteins, calcium, actin and actin-binding proteins and also the involvement of protein phosphorylation. One such type of phosphorylation, tyrosine phosphorylation is still to be shown in hyphae and indeed is still a controversial area in oomycete, fungal and plant biology (see chapter 3). There are Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 40 indications that numerous phosphorylations may be present in growing fungal tips. These include the elevated levels of Ca 2+ and IP3, phospholipase C (PLC), the presence of other protein kinases, actin and actin-binding proteins (Foissner et al., 2002; Silverman-Gavrila and Lew, 2002; Harris and Momany, 2004). Recent evidence also suggests that tyrosine phosphorylation may be important with respect to embryogenesis in zygotes of fucoid algae (Corellou et al., 2000). It is worth noting that tyrosine phosphorylation has been less well reported than phosphorylation of serine or threonine residues and conventional tyrosine kinases do not appear in the sequence data from higher plants (Luan et al., 2001; Luan, 2002). There is however evidence of tyrosine phosphorylation in a number of plant species such as pea (Torruella et al., 1985; H?kansson and Allen, 1995), alfalfa (Duerr et al., 1993), tobacco (Zhang et al., 1996), maize (Trojanek et al., 1996), and coconut (Islas-Flores et al., 1998). 2.1.4 Adhesion sites in animal cells In animal cells, focal adhesion sites play a vital role in integrin mediated signalling pathways. Many of the signalling proteins regulated by integrins occur at focal adhesion sites and also are involved in signal transduction pathways. Engagement of integrins with their cognate ligands at these sites induces a number of signalling events within the cell, including changes of pH and intracellular Ca 2+ concentration, stimulation of tyrosine phosphorylation of a number of cellular proteins, and induction of gene expression. Protein phosphorylation is one of the earliest events detected in response to integrin stimulation. Tyrosine phosphorylation has been shown to be a common and perhaps ubiquitous response to integrin engagement in many cell types. Several proteins that associate with integrin-nucleated protein complexes contain a domain termed Src homology 2 (SH2) and 3 (SH3), that specifically mediates protein-protein coupling. The SH2 domain binds to proteins through interactions with specific peptide motifs containing phosphotyrosine, whereas SH3 domains bind to short proline-rich region on their protein targets. Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 41 The focal adhesion kinase (FAK) appears to play a central role in integrin-mediated signal transduction. This kinase is tyrosine-phosphorylated, and its tyrosine kinase activity is enhanced upon integrin engagement. Integrin-induced phosphorylation of FAK requires the cytoplasmic domain of the ? integrin subunits. The targeting of FAK to focal adhesions appears to involve multiple binding interactions. The N-terminal of FAK binds to cytoplasmic domains of integrin, whereas the C-terminal provides the binding site for the cytoskeletal protein paxillin and in turn, several proteins bind to paxillin through SH2 and SH3 domains (Vuori, 1998). The identification of the cytoskeletal proteins paxillin and tensin as substrate for tyrosine kinases suggests a possible mechanism for the assembly and regulation of integrin-mediated signalling complexes and pathways (DeMali et al., 2003). Many signalling proteins that contain the SH2 and SH3 domains carry additional catalytic domains. These include Grb2, mSOS1, a guanine nucleotide exchange factor (GNEF), Crk, and C3G. The involvement of Grb2 and mSOS1 with FAK suggests that integrins may regulate the Ras-MAP kinase pathways. The activation of this pathway subsequently regulates cytoskeleton change and gene expression. In addition, MAP-kinase phosphorylates and activates cytoplasmic phospholipase A 2 (cPLA 2 ) to yield arachidonic acid and lysophospholipid. Integrin-FAK complexes also regulate the association of phosphatidylinositol (PI)-3 kinase, which phosphorylates either PI(4)phosphate (PIP) to generate PI(3,4)P 2 or PI(4,5)bisphosphate (PIP 2 ) to generate PI(3,4,5)P 3 . The production of PIP 2 by the engagement of integrins could be important for actin polymerisation via the regulation of actin-binding proteins such as profilin. In addition, phospholipase C (PLC) uses PIP 2 as substrate to generate the important secondary messenger diacylglycerol (DAG) and inositol triphosphate (IP 3 ) which in turn enhances the activity of protein kinase C (PKC). The activation of PKC regulates cell spreading and tyrosine phosphorylation of FAK (Clark and Brugge, 1995). In addition, a link between ?6?4-stimulation of invasion and signal transduction at hemidesmosome is thought to activate phosphoinositide 3-OH kinase (PI3-K), a key signalling molecule and that activity of PI3-K is essential for invasion (Mercurio et al., 2004) One important function of PI3-K with respect to invasion appears to be its ability Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 42 to regulate actin dynamics via the Rho-family of GTPases (Mercurio and Rabinovitz, 2001). Due to the limited evidence for the presence of integrin-like proteins and tyrosine phosphorylation in oomycete organisms, experiments were carried out to investigate the presence of these in the oomycete A. bisexualis. For comparative purpose, experiments were also carried out on cells of the yeast S. cerevisiae. The antibody that was used in these experiments was raised against amino acid residues at 28 to 128 of the N- terminal of the human integrin ?4 subunit. Unlike integrin ?1 subunits, the integrin ?4 subunit forms with only one alpha subunit, ?6, and specifically binds with laminins at a conserved RGD sequence. The cytoplasmic domain of the ?4 subunit is composed of about 1000 amino acid residues which compares with about 50 amino acids in most of the other ? subunits (Hynes, 2002). A comparison of the amino acid residues at 28 to 128 between ?4 and ?1 subunits using BLATP programme, which is available at www. ncbi.nlm.nih.gov/ shows 30% identity. This chapter shows that the oomycete A. bisexualis and yeast S. cerevisiae contain proteins that are epitopically related to the ?4 subunit of animals. Preliminary data is presented regarding the size of these proteins. Results in this chapter also show the presence of adhesion sites which may be analogous to focal adhesions of animal cells. This is supported by the co-localisation of the integrin-like protein and F-actin in cell wall/plasma membrane adhesion of plasmolysed hyphae of A. bisexualis. Furthermore, phosphotyrosine containing proteins are present in both apical and sub-apical regions and these proteins co-localise with integrin-like proteins. Finally, immunoblotting identified several proteins extracted from hyphae of A. bisexualis that contained phosphotyrosine residues. Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 43 2.2 Materials and Methods 2.2.1 Cell cultures and antibodies A. bisexualis: Stock cultures of a female strain of A. bisexualis Coker, isolated in New Zealand from Xenopus laevis dung were grown on PYG (3 g/L glucose, 1.25 g/L Bacto Peptone, 1.25 g/L Yeast extract) agar and maintained at 20 ?C in the dark. Prior to experiments, hyphae were sub-cultured on PYG agar over laid with cellophane (Hallmark brand) sheets and allowed to grow at 20 ?C for 5 days. Cellophane sheets were boiled 3 times and then were autoclaved prior to culturing. This was necessary to remove any manufacturing residues. S. cerevisiae: Yeast cells (strain SY1129(F131)) were kept in 50 % glycerol and stored at -80 ?C. Prior to use, cells were subcultured on YPD (20 g/L glucose, 20 g/L Bacto Peptone, 10 g/L Yeast extract) agar at 30 ?C overnight. Antibodies Mouse monoclonal IgG 1 antibody raised against amino acids 28-128 of the integrin ?4 subunit of human origin was purchased from Santa Cruz Biotechnology, Inc and was used as a primary antibody. Alexa? 488 goat anti-mouse IgG (H+L) conjugate was purchased from Molecular Probes and used as secondary antibody. Rabbit IgG polyclonal antibody raised against phosphotyrosine was purchased from Santa Cruz Biotechnology, Inc, and chicken anti-rabbit poly IgG antibody conjugated with Texas Red was purchased from Sigma. A mouse monoclonal IgG1 antibody raised against phosphotyrosine purchased from Sigma was used for immunoblotting. Actin was stained using Texas red-phalloidin (Molecular probes). Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 44 2.2.2 Co-localisation of ?4 integrin-like protein and phosphotyrosine containing proteins A. bisexualis: Prior to experiments, hyphae were grown for 5 days and then cut 1 cm behind the growing margin and allowed to grow in PYG media for 60 mins to enable recovery. For plasmolysis experiments, mycelial sheets were incubated with 0.5-0.7 M sorbitol for 20-30 mins. Plasmolysed hyphae were visualised under the light microscope. Mycelial sheets were fixed in 4% formaldehyde, 0.5 % methylglyoxal in saline buffer containing 60 mM PIPES pH 6.8, 2 mM EGTA, 2 mM MgCl 2 , 137 mM NaCl, and 0.268 mM KCl for 45 mins at room temperature. Fixed mycelial sheets were rinsed for 4 times for 5 mins with a washing solution ( 0.05% v/v Tween-20 in saline) then the cell wall was digested for 15 mins in 10 mg/ml Driselase (Sigma) and membranes were permeabilised in 0.1% (v/v) Triton X-100 (Biorad) for 20 mins. The mycelial sheets were again rinsed in the washing solution 2 times for 5 mins and blocked for 30-45 mins in blocking solution containing 2.5 % (w/v) skimmed milk powder (BioRad) and 5% (w/v) BSA (Sigma) in saline and washed 4 times for 5 mins after blocking. The mycelial sheets were incubated with a 1:100 dilution of the primary mouse monoclonal IgG 1 antibody raised against the integrin ?4 subunit, and rabbit IgG polyclonal antibody raised against phosphotyrosine, at 4 ?C overnight. The mycelial sheets were rinsed 5 times for 5 mins and incubated with a 1:1000 dilution of secondary antibody Alexa TM 488 goat anti-mouse IgG (H+L) conjugate. After washing twice, they were then incubated with a 1:500 dilution of chicken anti-rabbit poly IgG antibody conjugated with Texas Red. Both secondary antibodies were incubated for 90 mins at room temperature and washed with 20 ml of washing buffer and this was repeated 3 times for 15 mins. Before microscopic examination, anti-fading solution (containing 0.1 % (w/v) of p-phenylenediamine dihydrochloride) was added to prevent photo bleaching. S. cerevisiae: Yeast cells strain (SY1129(F131)) were streaked onto a YPD agar plate and incubated overnight at 30 ?C, from this an isolated colony was selected and cultured in 10 ml YPD media overnight at 30 ?C. Cells were centrifuged at 5,000 g for 30 mins Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 45 and the pellet was washed twice with ice-cold 40 mM PBS pH 7.0. Yeast cells were fixed in fixing solution as described previously for A. bisexualis. After washing with the saline buffer, fixed cells were incubated in KS solution (containing 100 mM KPO 4 buffer pH 7.0, 1.2 M sorbitol) at 4?C overnight. The cell wall was digested with 10 mg/ml Driselase in KS solution containing 0.5 % (v/v) ?-mercapthoethanol for 30 mins at room temperature. Cells were centrifuged and resuspended in 0.5 % v/v Triton-X in 100 mM KPO 4 buffer pH 7.0 and incubated at room temperature for 20 mins. Cells were washed and fixed to slides by incubating at 45?C for 30 mins. The slide was then washed 3 times and blocked with blocking solution ( containing 1% w/v BSA, 0.05 % v/v Tween-20 and 40 mM PBS pH 7.0) for 45 mins at room temperature then the slides were washed 5 times for 5 mins and incubated with primary antibodies and secondary antibodies as described previously for A. bisexualis. Anti-fading solution was added prior to visualisation under the microscope. 2.2.3 Co-localisation of ?4 integrin-like protein and F-actin in A. bisexualis The procedure was similar to the above except that only the ?4-integrin antibody was used in the overnight incubation. Furthermore, after incubation with the secondary antibody conjugated with Alexa 488 and washing, hyphae were incubated in the dark, with Texas red-phalloidin made in buffer containing 60 mM PIPES pH 6.8, 2 mM EGTA, 2 mM MgCl 2 , 137 mM NaCl, and 0.268 mM KCl for 30 mins at room temperature and washed as described above. 2.2.4 Immunoblotting of Integrin-like proteins A. bisexualis: Forty plates of A. bisexualis grown on PYG overlaid with cellophane were cut 1 cm from the margin of mycelia and frozen with liquid nitrogen, then ground. The powder was then re-suspended in buffer containing 50 mM Tris-HCl pH 6.2, 10 mM NaF, 7 mM EDTA, 2 mM DTT, 20 % glycerol, 7 mM ?-mercaptoethanol, 2 mM PMSF, 250 mM sucrose and centrifuged at 10,000 g for 45 mins and further centrifuged with Backman L8-M ultracentrifuge SW28 rotor at 100,000 g for 2 hrs. The microsomal Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 46 pellet was re-suspended with the same buffer. Soluble crude fractions before and after centrifugation at 100,000 g and microsomal fraction were then precipitated on ice by addition of trichloroacetic acid (TCA) to a final concentration of 10% for 30 mins. After centrifugation the precipitates were mixed with 5x Laemmli buffer and heated for 5 mins in a boiling water bath. Proteins were separated on a 5% stacking gel and 12% separating polyacrylamide gel. After SDS/PAGE, proteins were transferred to a nitrocellulose membrane in a buffer containing 15.6 mM Tris-Base and 120 mM glycine and the transfer was run at 30 V at 4 ?C overnight. Transferred proteins were stained with 0.1% w/v Ponceau red to check for homogeneity of the transfer. The nitrocellulose blots were then blocked in 10 mM PBS containing 5 % BSA and 0.05 % Tween-20 for 2 hours at room temperature then washed with Tris- Buffer saline (TBS) 2 times for 20 mins. The nitrocellulose membrane was incubated overnight at 4 ?C with a 1:250 dilution of mouse monoclonal IgG 1 antibody raised against the integrin ?4 subunit as primary antibody. The membrane was visualised by Western Breeze TM , (Western Immunodetection System, Invitrogen). S. cerevisiae: Yeast cells (strain SY1129(F131)) were streaked onto a YPD agar plate and incubated overnight at 30 ?C, from these, an isolated colony was selected and cultured in 10 ml YPD media overnight at 30 ?C. Then, 10 ml of the overnight culture was poured into 1 L YPD media and further incubated at 30 ?C overnight on an orbital shaker. Cells were centrifuged at 5,000 g for 30 mins and the pellet was washed twice with ice-cold 40 mM PBS pH 7.0. The pellet was then re-suspended with buffer as described for A. bisexualis and vortexed with pre-chilled ice cold glass beads for 20 mins (1 min vortexing and 1 min on ice) and then further sonicated for 30 mins on ice. Lysed cells were then centrifuged at 10,000 g for 30 mins and then further centrifuged at 100,000 g to obtain the microsomal pellet. The microsomal pellet was then re-suspended in buffer as described for A. bisexualis. Electrophoresis and immunoblotting was carried out as described for A. bisexualis. Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 47 2.2.5 Immunoblotting of phosphotyrosine containing proteins A. bisexualis: The growing edge of A. bisexualis mycelia were cut, quick frozen with liquid nitrogen, ground and extracted with extraction buffer containing 30 mM Tris-HCl, pH 7.1, 20 mM NaF, 5 mM EGTA, 7 mM EDTA, 2 mM DTT, 50 mM KCl, 2 mM Na 3 VO 4 , 2 mM PMSF, 20% glycerol. The extract was centrifuged at 10,000 g for 30 mins, the supernatant was then applied to a phosphocellulose column (1cm X 15 cm) that had been equilibrated with extraction buffer. The column was washed with washing buffer containing 30 mM Tris-HCl, pH 7.1, 7 mM EDTA, 6 mM 2-mercapthoethanol, 10 % glycerol and 1 mM Na 3 VO 4 until the A 280 of the elutent was below 0.1, and the column was then eluted with a gradient of 0 to 1 M NaCl in eluting buffer containing 40 mM Hepes pH 7.0, 7 mM EDTA, 2 mM Na 3 VO 4 , 20 % glycerol, 2 mM DTT. Fractions of 2 ml were collected. S. cerevisiae: Yeast cells (strain SY1129(F131)) were streaked onto a YPD agar plate and incubated overnight at 30 ?C, from this an isolated colony was selected and cultured in 10 ml YPD media overnight at 30 ?C. Then, 10 ml of the overnight culture was poured into 1 L YPD media and further incubated at 30 ?C overnight on an orbital shaker. Cells were centrifuged at 5,000 g for 30 mins and the pellet was washed twice with ice- cold 40 mM PBS pH 7.0. The pellet was then re-suspended in the extraction buffer as above and vortexed with pre-chilled iced cold glass beads for 20 mins and further sonicated for 30 mins. Lysed cells were then centrifuged at 10,000 g for 30 mins. The soluble crude fraction was partially purified with a phosphocellulose column as described above. Each fraction was tested for protein content using a Bradford assay (Deutscher, 1990). Fractions containing proteins were then pooled. One ml of pooled fractions was precipitated on ice by addition of trichloroacetic acid (TCA) to a final concentration 10% for 30 mins. After centrifugation the precipitates were mixed with 5x Laemmli buffer, heated 5 mins in a boiling water bath. Proteins were separated on a 5% stacking gel and 12% separating polyacrylamide gel. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane in a buffer containing 15.6 mM Tris-Base and 120 mM glycine and the transfer run at 30 V at 4 ?C overnight. Transferred proteins were stained with Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 48 0.1% w/v Ponceau red to check for homogeneity of the transfer. The nitrocellulose blots were then blocked in 10 mM PBS containing 5 % BSA and 0.05 % Tween-20 for 2 hours at room temperature then washed with Tris- Buffer saline (TBS). The renaturation of transferred proteins was carried out as described by (Trojanek et al., 1996). The nitrocellulose membranes were incubated with 50 ?M of ATP for 60 mins at room temperature to look for auto-phosphorylation and washed with TBS. The membrane was incubated with a 1:2000 dilution of mouse monoclonal IgG1 anti-PY antibody (Sigma) as primary antibody and visualised by Western Breeze TM , (Western Immunodetection System, Invitrogen). 2.2.6 Immunoblotting of phosphotyrosine-containing proteins in the microsomal pellet fraction from A. bisexualis A. bisexualis: culturing, microsomal pellet extraction, SDS-PAGE, and protein blotting were as described in section 2.2.4. After blocking the nitrocellulose membrane, the membrane was incubated overnight at 4 ?C with a 1/2000 dilution of mouse monoclonal IgG1 anti-phosphotyrosine antibody purchased from Sigma. Phosphotyrosine containing proteins were then visualised by Western Breeze TM , (Western Immunodetection System, Invitrogen). Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 49 2.3 Results 2.3.1 Immunocytochemistry of integrin-like protein and phosphotyrosine containing proteins in A. bisexualis Hyphae of A. bisexualis stained with anti- ?4 integrin antibody showed two distinctive staining patterns. In approximately 45 % of hyphae (15 out of 33 hyphae) staining was highly visible at the extreme apex (Error! Reference source not found.. A). The remaining 55% (18 out of 33) had no obvious tip high concentration and staining was more common in the sub apical regions (Error! Reference source not found.. B). Control hyphae were exposed to secondary antibody only and showed no fluorescence. If integrin-like proteins form connections between the cytoplasm and cell wall, then after hyphae are plasmolysed with sorbitol, the cytoplasmic remnants that remain attached to the wall should contain integrin-like proteins. Typically when A. bisexualis hyphae are plasmolysed, the protoplasm does not pull cleanly away from the cell wall and there are distinct wall-membrane attachment sites. When plasmolysed hyphae were stained again two discrete patterns were observed with wall membrane fragments and associated staining present predominantly at the tips (46% of hyphae, n= 100) (Error! Reference source not found.. A) or in more sub apical regions (54% of hyphae, n=100) (Error! Reference source not found.. B). These wall-membrane fragments were also found to contain F-actin, which co-localised with the ?4 integrin-like protein in both intact and plasmolysed cells (Error! Reference source not found..). F-actin may thus form the cytoplasmic continuum of the wall-membrane attachments. In both intact and plasmolysed hyphae simultaneous staining with antisera directed against integrin and phospholated tyrosine residues and merging of the respective images indicated that integrin-like proteins and phosphotyrosine co-localise (Error! Reference source not found.. and Error! Reference source not found..). This was perhaps not surprising given the abundance of staining in some cells (Error! Reference source not found. A), but it was also the case when staining occurred in more discrete spots (Error! Reference source not found. B). Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 50 Some integrin-like protein patches did not show co-localisation with phosphorylated tyrosine and conversely some phosphotyrosine staining was not associated with integrin like staining (Error! Reference source not found.. B). 2.3.2 Immunocytochemistry of Integrin-like protein and phosphotyrosine containing proteins in S. cerevisiae Using the anti-?4 antibody to detect integrin-like proteins in S. cerevisiae cells, immunofluorescence of integrin-like proteins was localised to the cortical regions of cells (Error! Reference source not found.. ?4-integrin). This may suggest that a polypeptide which is responsible for the staining may associate with the cell wall/plasma membrane. Unlike staining of integrin-like protein, staining of phosphotyrosine containing proteins are located throughout cells (Error! Reference source not found.. p-Tyr). This may suggest that at least some of the phosphotyrosine containing proteins are soluble proteins. However, some of the phosphotyrosine containing proteins may also be located at the cell wall/plasma membrane (Error! Reference source not found.. arrows Merged). Controls were carried out by incubating cells with secondary antibody only and these showed no staining. Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 51 Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 52 Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 53 Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 54 Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 55 Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 56 2.3.3 Immunoblotting of Integrin-like protein in A. bisexualis Proteins were extracted from A. bisexualis hyphae, and were subjected to ultracentrifugation to obtain microsomal pellets. Extractions were carried out in both the presence and absence of DTT and ?-mercapthoethanol. Only two polypeptide bands that showed cross reactivity with anti-?4 antibody were present and these were present with or without the reducing compounds. A soluble fraction obtained after the 10,000g centrifugation contained polypeptides with molecular weights of 65 kDa and 85 kDa (Error! Reference source not found.. Lane A). However, after the soluble fraction was further centrifuged at the 100,000 g to obtain microsomal pellets, only the 85 kDa was found (Error! Reference source not found.. Lane B). This indicates that the 85 kDa polypeptide may be plasma membrane associated. The 65 kDa remained in the soluble fraction after centrifugation at the 100,000g (Error! Reference source not found.. Lane C). 2.3.4 Immunoblotting of integrin-like protein in S. cerevisiae Immunoblotting showed that only one polypeptide of about 45 kDa that cross reacted with anti-?4 antibody was present in the microsomal fraction S. cerevisiae (Error! Reference source not found..). The crude fraction obtained centrifugation after at 10,000g and after 100,000 g was also subjected to immunoblotting. However, there were no polypeptides that were recognised by the antibody in these (Data not shown). Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 57 Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 58 2.3.5 Immunoblotting of phosphotyrosine containing proteins in A. bisexualis and S. cerevisiae Soluble fractions of proteins obtained from A. bisexualis hyphae and S. cerevisiae cells were separated with SDS-PAGE and then tested for phosphorylation of tyrosine residues by immunoblotting, using the monoclonal anti-phosphotyrosine antibody. In the absence of ATP, four major bands at 26, 46, 60, and 85 kDa from A. bisexualis were present (Error! Reference source not found.. A). To verify autophosphorylation of these proteins, experiments were carried out in which nitrocellulose membranes were incubated with ATP prior to staining with the anti-phosphotyrosine antibody. The immunoblotting analysis shows that the intensity of these four protein bands was greater in the presence of ATP. Similar results were also obtained from immunoblotting analysis of yeast proteins (Error! Reference source not found.. B). With yeast extracts, in the absence of ATP, there were three major bands at about 35, 60, and 90 kDa that contained phosphotyrosine residues and again these proteins show greater staining intensity in the presence of ATP (Error! Reference source not found.. B). To evaluate tyrosine phosphorylation of integrin-like proteins in A. bisexualis, the soluble crude fraction was subjected to ultracentrifugation at 100,000g for 2 hours to obtain a microsomal pellet fraction. Then proteins from the microsomal pellet were immunobloted with anti-phosphotyrosine antibody. The 85 kDa protein from the crude fraction (Error! Reference source not found.. (C), lane A) contains phosphotyrosine residues. Moreover, the 85 kDa band still appears in the microsomal pellet fraction but the intensity of the band is weak (Error! Reference source not found.. (C), lane B). This could be a breaking down of plasma membrane containing proteins during separation by SDS-PAGE. Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 59 Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 60 2.4 Discussion The presented results indicate that hyphae of the oomycete A. bisexualis and yeast S. cerevisiae contain proteins that are epitopically similar to the integrin ?4 subunit of human. Specifically this similarity is between amino acid residues 28-128. In A. bisexualis, these integrin-like proteins co-localise with phosphotyrosine containing proteins but this does not appear to be the case for S. cerevisiae. Integrin-like proteins stain in hyphae of A. bisexualis in both apical region and sub- apical regions. After plasmolysis, the staining was present at wall-membrane attachment sites suggesting a role in wall-membrane attachment. This staining is very similar to that in the oomycete S. ferax that was treated with antibody raised against the integrin ?1 cytoplasmic domain subunit (Kaminskyj and Heath, 1995). In S. ferax, this integrin-like protein also appeared to mediate attachment between the cytoskeleton and the cell wall. This conclusion was supported by the peripheral location of the integrin patches, the strong resistance to plasmolysis in integrin-rich sporangial tips relative to hyphal tips, the formation of integrin-like proteins and actin-rich cytoplasmic remnants that attach to the cell walls following plasmolysis and finally the colocalisation of actin and integrin-like proteins in hyphal and sporangial tips. Integrin-like proteins may therefore function in tip morphogenesis, since this is one suggested role for F-actin. This is supported by preliminary data suggesting that the peptide RGDS, which affects integrin-extracellular matrix interactions in animal cells, interferes with growth and morphology in A. bisexualis hyphae. The appearance of integrin-like protein staining and its co-localisation with F-actin in A. bisexualis also fits the above model. In around one half of hyphae (Error! Reference source not found.. A), the antibody staining is highly concentrated at the extreme tip and located at the cell wall/plasma membrane sites. Similar staining is found in the fungus Neurospora crassa, in which the anti-?1-integrin antibody showed fine PM-associated spots which were tip-high and tended to fuse into almost continuous caps at the extreme tips, with more separated sub apical staining (Degousee et al., 2000). In plasmolysed hyphae, staining was present in patches located at the cell wall and remnants of cytoplasm (Error! Reference source not found.. A and B). Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 61 Immunoblotting has identified candidate proteins responsible for the above staining. The ?4-integrin antibody cross reacted with protein bands at about 65 and 85 kDa regardless of the presence/absence of reducing agents. These polypeptide masses differ from similar proteins identified in S. ferax using the anti ?1-integrin antibody. However, the same molecular weight proteins would be surprising. This is because different antibodies were used, the ?4- integrin is bigger than ?1-integrin in animal cells, and there is diversity in the sizes of ?1-integrin-like proteins in fungi. The 85 kDa protein is however close in weight to the 95 kDa proteins detected with anti ?1-integrin antibody in Candida albicans (Marcantonio and Hynes, 1988). Western blot analysis with antibody specific to the ?v or ?5 subunit detected a heterodimer of 130/84 kDa (Santoni et al., 1998). The protein bands found in A. bisexualis are also close to the weights of 80 kDa found in Chara and 84 kDa Arabidopsis (Katembe et al., 1997). (Marcantonio and Hynes, 1988) investigated the degree of cross-reactivity to integrin antibodies in yeast cells. Immunoblotting experiments showed no specific bands in membrane extracts of S. cerevisiae using an antibody raised against the cytoplasmic domain of chicken integrin ?1 subunit. Furthermore, there is an absence of integrin sequence in the yeast genome database (http://www.yeastgenome.org). The result from immunoblotting and immunofluorescence in the presence study indicates that S. cerevisiae contains a polypeptide that cross-reacts with anti-?4 integrin antibody. A protein of about 45 kDa present in a microsomal pellet fraction was recognised by the antibody. Immunofluorescent images indicate that this protein is located mainly in cortical regions. Using the amino acid sequence at position 28 to 128 of the human ?4- integrin as the bait to detect any proteins from the yeast protein sequence database, eight protein profiles have been found (Table 2.1.) Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 62 Table 2.1. Proteins in the S. cerevisiae database that show sequence similarity to the human ?4 integrin. Sequence names Sequence identities (%) Protein molecular weight (kDa) Protein YMR178W 29 31.1 Hypothetical protein YBR150C 32 126.9 Probable Zinc-finger protein YNL267W 31 119.9 Phosphatidylinositol 4-kinase YLR098C 22 74.4 Zinc-finger protein YDR272W 32 31.3 Glyoxalase (Glo2p) YGL116W 25 67.3 Cdc20 YLR030W 36 29.9 Unknown protein YDR047W 39 41.3 Uroporphyrinogen decarboxylase (Hem12) Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 63 Of these only one protein is a transmembrane protein that has a mass close to 45 kDa. This protein is called Hem12p. Hem12p is composed of 362 amino acid residues and has a molecular mass of 41.349 kDa. It contains a transmembrane domain between positions 67 to 93. These amino acid sequences in the transmembrane domain have 39 % identity to the ?4-integrin between residues 75-110. The hem12 gene encodes uroporphyrinogen decarboxylase which catalyses the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis (Garey et al., 1992). As such, Hem12 does not appear to play a role in morphogenesis. One integrin-like gene, uso1, has been isolated from S. cerevisiae by screening an expression library with two antibodies against leukocyte integrins ?X and ?M (Hostetter et al., 1995). However, USO1 is a cytosolic rather than plasma a membrane associated protein and has been shown to play a role in intracellular transport of vesicles (Sapperstein et al., 1996). Another integrin-like gene, int1, from Candida albicans encodes a protein of about 188 kDa. Again, the INT1 protein has limited similarity to vertebrate integrins (Gale et al., 1996). However, it localises to the cell surface and has been shown to be involved in adhesion of yeast to human epithelial cells and in filamentous growth (Hostetter, 1999). Tyrosine phosphorylation plays an important role in cell signal transduction, regulation of growth and development in animals (Vuori, 1998; Watt, 2002). The relationship between tyrosine phosphorylation and cell polarity in filamentous organisms are still unknown. This thesis presents the first evidence that suggests tyrosine phosphorylation occurs in oomycete hyphae. This tyrosine phosphorylation appears to occur at or close to integrin-like proteins and can occurs at the apex of the hyphae. This may suggest that integrin-like protein itself might have phosphotyrosine residues. This data is consistent with the observation that the cytoplasmic tail of integrins have conserved tyrosine residues that can be phosphorylated and these play a vital role in signalling pathways in animal cells (Calderwood et al., 1999; Calderwood et al., 2002). Tip growth of hyphae, pollen tubes and root hairs is an example of polarised cell growth. Polarity establishment presumably requires the specification of growth sites, Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 64 followed by the recruitment of the morphogenesis machinery necessary for localised cell wall deposition and once polarity is established, it must be maintained during tip extension. The mechanisms underlying polarised morphogenesis have been extensively characterised in the yeasts S. cerevisiae and S. pombe (Palmieri and Haarer, 1998; Verde, 2001). There are several types of signalling molecules involved in polarisation, these include G-protein, Cdc42p, Cdc24p, Rho GTPase, Rac, Ras, actin, and actin-binding proteins (Harris and Momany, 2004). In animal cells, all these signalling molecules are down regulated by integrin- tyrosine phosphorylation regulation (Clark and Brugge, 1995). Furthermore, actin dynamics are regulated either by reversible tyrosine phosphorylation or by phosphorylation of actin-binding proteins (Kameyama et al., 2000), Other secondary signalling molecules such Ca 2+ also play a vital role in tip polarity. Elevation of Ca 2+ levels have been found in several hyphae (Lew, 1999; Silverman- Gavrila and Lew, 2002). Accordingly, Ca 2+ -dependent protein kinases are required for germination and pollen tube growth and are co-localised with F-actin in pollen tubes (Putnam-Evans et al., 1989; Estruch et al., 1994). Since, the growing tip of A. bisexualis contains by F-actin, there should be actin-binding protein coordination with maintenance of F-actin in the growing tip. Even though, tyrosine phosphorylation is still unknown in filamentous organisms such as in oomycetes and fungi, recent reports showed that in the dimorphic fungus C. albicans, integrin-like proteins and tyrosine phosphorylation co-localised at cortical regions of the yeast cells and a reduction of tyrosine phosphorylation was observed when yeast cells were incubated with tyrosine kinase inhibitors (Santoni et al., 2002). Furthermore, embryogenesis of the fucoid algae Fucus and Pelvetia requires tyrosine phosphorylation and the germination of embryos were prevented by tyrosine kinase inhibitors (Corellou et al., 2000). The data from this study also suggests that there are several cytosolic proteins that have proteins containing phosphotyrosine residues. Immunoblot analysis shows partially purified soluble fractions of protein from hyphae of A. bisexualis have four such proteins of 26, 46, 60, and 85 kDa. Also these proteins are able to be autophosphorylated in the Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 65 presence of ATP. The 26 kDa protein is interesting as it appears to be the predominant band and mostly comes out when the phosphocellulose column is used for purification. However, it does not have any tyrosine kinase activity, and this will be discussed further in the Chapter 3. Phosphotyrosine-containing proteins have been reported in several plant species. The 26 kDa protein in A. bisexualis has a protein mass close to that of phosphotyrosine- containing proteins found in pea mitochondria in respiratory redox signalling (H?kansson and Allen, 1995). The proteins at 46 and 60 kDa found in A. bisexualis are close in mass to those found in transformed roots of Catharanthus roseus (Rodriguez-Zapata and Hern?ndez-Sotomayor, 1998) and in maize seedlings that contain a group of proteins ranging from 45 to 60 kDa (Trojanek et al., 1996). Recently, proteins at 38, 39, 48, and 75 kDa that are tyrosine phosphorylated have been found during phytohormone- stimulated cell proliferation in Arabidopsis hypocotyls (Hang et al., 2003). The data also shows that the 85 kDa protein is phosphotyrosine-containing protein with or without ATP. It is very interesting that this protein band is approximately the same size as the integrin-like protein band. To evaluate the 85 kDa phosphotyrosine-containing protein, the microsomal pellet obtained after ultracentrifugation was used to detect phosphotyrosine residues. Immunoblotting shows that the microsomal pellet has a band but it is weaker than that from the crude and there is high back ground signal. This may be explained by the fact that there may be breakage of plasma membrane during separation by SDS-PAGE. However the data is consistent with the integrin-like protein and the phosphotyrosine containing protein being the same protein. Results also showed that S. cerevisiae contains phosphotyrosine proteins and these are distributed throughout the cell. However, no co-localisation between integrin-like proteins and phosphotyrosine containing proteins was observed in S. cerevisiae. There is an absence of integrin sequence in a yeast sequence database but phosphotyrosine containing proteins have been found in yeast. In fungi, the mitogen-activated protein (MAP) kinases are known to play major roles in regulating growth and differentiation processes (Xu, 2000). Although no conventional tyrosine kinases exist in S. cerevisiae, tyrosine phosphorylation has been shown to be essential in controlling the activity of MAP kinases in yeast. MAP kinase activation requires phosphorylation on Chapter 2: Immunocolocalisation of Integrin-like proteins and Phosphotyrosine containing Proteins in A. bisexualis and S. cerevisiae 66 tyrosine and threonine residues catalysed the dual-specificity MAP kinase kinase (MAPKK)(Cobb and Goldsmith, 1995). MAPKKs such as Wee1 or Swe1p have tyrosine kinase activity (Parker et al., 1993). The cell cycle delay induced by the morphogenesis checkpoint requires Swe1p. Checkpoint control of mitosis in S. cerevisiae is regulated by the tyrosine phosphorylation of Cdc2 by Swe1p and dephosphorylation by Cdc25-related phosphatase (McMillan et al., 1999). Chapter 3 : The presence of protein tyrosine kinase activities in A. bisexualis and S. cerevisiae 3.1 Introduction Signal transduction regulates cellular processes in response to external/internal stimuli and is vital for the growth and development of all organisms. It is thought that the reversible phosphorylation of proteins is a crucial component of almost all signalling pathways in living cells. Protein phosphorylation is regulated through the concerted action of kinases and phosphatases. Most protein phosphorylation in cells occurs on residues of serine and threonine with a minor proportion at residues of tyrosine. Despite this tyrosine phosphorylation is very important in animal systems. The introduction of chapter will address the possible existence of tyrosine kinases in oomycetes, fungi, and plants. This will be followed by a consideration of tyrosine kinases found in animal cells. 3.1.1 Presence of protein tyrosine kinases in oomycetes, fungi, and plants The previous chapter suggested the presence of proteins that are epitopically similar to the ?4 integrin subunit and an association between this and phosphorylated tyrosine residues. This suggests that A. bisexualis may contain a tyrosine kinase. While there is no sequence data available for A. bisexualis, the oomycete Phytophthora sojae shows nucleotide sequences that resemble tyrosine kinases found in Dictyostelium discoideum, Caenorhabditis elegans, Homo sapiens, Rattus norvegicus, and Mus musculus (http://cogeme.ex.ac.uk). Although the protein products of these sequences in Phytophthora have not yet been characterised, it raises the possibility that oomycetes may have tyrosine kinases (Table 3.1.). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 68 Table 3.1. DNA sequences of the oomycete P. sojae resemble sequences of tyrosine kinases found in various eukaryotic organisms. Amino acid residues 481-1052 of the human focal adhesion kinase (FAK) were used to detect nucleotide sequences from oomycete and fungal databases, which are available at http://cogeme.ex.ac.uk. Sequences of Phytophthora sojae Sequence identities (%) Protein Ps31050460 40 Putative tyrosine kinase-like protein from D. discoideum Ps9834403 38 Tyrosine kinase family member (kin-24) from C. elegans Ps30499946 35 Src-related kinase lacking C-terminal regulatory N- terminal myristylation sites from Homo sapiens Ps30499946 34 Src-related kinase lacking C-terminal regulatory N- terminal myristylation sites from R. norvegicus Ps30499946 34 Tyrosine ?specific protein kinase from M. musculus Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 69 Unlike the Phytophthora genome, analysis of the budding yeast genomic sequence indicates that this organism may lack true tyrosine kinases. However, some serine/threonine kinases in S. cerevisiae such as Swe1 (Wee1), Ste7, Rad53, and Mck1 also have tyrosine kinase activity. For instance, Swe1 (Wee1 in the fission yeast) phosphorylates a conserved tyrosine on Cdc28p (Cdc2 in the fission yeast), thereby preventing cells entry into mitosis until cells reach a critical size (Harvey and Kellogg, 2003). In the yeast mating pathway, the Ste7 which is the MAPKK, phosphorylates two MAPKs (Kss1 and Fus3) on conserved serine and tyrosine (Kusari et al., 2004) and this results in the fusion of two haploid cells opposite mating types to produce a diploid cell (Pan et al., 2000; Maleri et al., 2004). Mck1 kinase functions in a variety of processes including the cell cycle delay by Ca 2+ , segregation of mitotic chromosome, and diploid cells for expression a gene encoding an early meiotic activator (Mizunuma et al., 2001). Furthermore the Ser/Thr kinases represent virtually all of the kinases described in filamentous fungi. These play a role in MAPK pathways that are very important in filamentous fungi, controlling pathways control diverse growth and differentiation processes and survival and pathogenesis in fungal pathogens (Xu, 2000). In plants, mitogen-activated protein kinase (MAPK) is a serine/threonine kinase that plays a major role in various physiological, developmental and hormonal responses. The activation of MAPK correlates with stimulatory treatments such as pathogen infection, wounding, low temperature, drought, hyper- and hypo-osmolarity, high salinity, touch and exposure to reactive oxygen species (Munnik and Meijer, 2001; Romeis, 2001; Ichimura and Shinozaki, 2002; Rakwal and Arawal, 2003). Arabidopsis shares components of conserved MAPK with human and yeast. MAPK cascades are minimally composed of three kinase modules, MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK), and MAP kinase (MAPK), which are linked in various ways to upstream receptors and downstream targets. MAPKKKs are serine/threonine kinases and activate MAPKKs through the phosphorylation of serine and threonine residues. Importantly with respect to the present study, MAPKKs are dual-specificity kinases that phosphorylate MAPKs on threonine and tyrosine residues. Activated MAPKs can phosphorylate a variety of substrates, including transcription factors, protein kinases and cytoskeleton proteins (Jonak et al., 2002). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 70 In Arabidopsis, there are 417 genes that have been referred to as receptor kinases (Cock et al., 2002). The structures of plant receptor kinases (PRKs), which consist of an extracellular domain, a single membrane-spanning domain and a cytosolic kinase domain, resemble those of the receptor tyrosine kinase and receptor serine/threonine kinase families in metazoans. A common feature of the receptor kinase families in plants and those of animals is that the kinase domains are homologous although, in contrast, the extracellular domains can be highly diverse. Recent studies indicate that PRKs can autophosphorylate in response to ligand binding both in vitro and in vivo and there is evidence that phosphorylation can occur within receptor oligomers (Cock et al., 2002). Even though, the presence of true tyrosine kinases in plants remains controversial, there is evidence suggesting the presence of tyrosine kinase activity in plant species such as pea (Torruella et al., 1985; H?kansson and Allen, 1995), alfalfa (Duerr et al., 1993), tobacco (Zhang et al., 1996), maize (Trojanek et al., 1996), and coconut (Islas-Flores et al., 1998). Recent evidence also shows that tyrosine kinase inhibitors alter the development of fucoid algae such as Fucus and Pelvetia (Corellou et al., 2000). However, the question still remains to be answered as to whether this activity is due to an actual tyrosine kinase or a dual-specificity kinase such as MAPKK. 3.1.2 Tyrosine kinases in animal cells There are two types of tyrosine kinases in animal cells; the transmembrane tyrosine kinase receptors, and the non-receptor tyrosine kinases. Receptor tyrosine kinases include the epidermal growth factor receptor (EGFR), insulin receptors, platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1) and colony-stimulating factor 1 (CSF-1). All of the receptor tyrosine kinases are induced by binding of specific ligand and it appears that ligand-induced activation of the kinase domains and its signalling potential are mediated by receptor oligomerisation. Receptor oligomerisation permits the transmission of a conformation change from the extracellular domain to the cytoplasmic domain without requiring alterations in the positioning of amino acid residues within the transmembrane domain. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 71 Receptor tyrosine kinases catalyse the phosphorylation of exogenous substrate as well as tyrosine residues within their own polypeptide chains. Most of receptor tyrosine kinases share common domains, which include extracellular, transmembrane, juxtamembrane, tyrosine kinase and carboxy-terminal tail domains. The extracellular domains of the insulin receptor and EGFR share homologous cysteine- rich sequence repeat domains, but the extracellular ligand binding domains of the PDGF receptors have an immunoglobulin-like structure. The transmembrane domain functions in anchoring the receptor in the plane of the plasma membrane; thereby it connects the extracellular environment with internal compartments of the cell. The juxtamembrane domain is involved in modulation of receptor functions by heterologous stimuli or known as receptor transmodulation (Chuen Wong and Guillaud, 2004). The tyrosine kinase domain is the most conserved portion of all receptor tyrosine kinase molecules. The conserved region contains a consensus sequence, GlyXGlyXXGlyX(15-20)Lys that functions as an ATP- binding site. Replacement of the lysine residue of the ATP- binding site in the EGFR, insulin and PDGF receptors completely abolishes their kinase activities. The carboxy-terminal tail is among the most divergent between all known receptor tyrosine kinases. Several autophosphorylation sites have been identified in the EGFR and in the insulin receptor. Tyrosine residues are conserved in the carboxy-terminal tails within each receptor tyrosine kinase subclass. The autophosphorylation of conserved tyrosine residues at the carboxy-terminal tails plays a role in maintaining the activated state of the insulin kinase activity. EGFR autophosphorylation sites are clustered at the carboxy-terminal tail and appear to modulate the interaction of activated EGFR with substrates and other proteins. Furthermore, the autophosphorylation sites of EGFR compete with exogenous substrate for the substrate binding site of the kinase domain. Hence, autophosphorylation appears to release an internal constraint by establishing a conformation of the receptor that is competent to interact with and phosphorylated cellular substrates (Grandis and Sok, 2004). As mentioned above other types of tyrosine kinases are non-receptor tyrosine kinases. Several non-receptor tyrosine kinases have been found in animal cells and one which has received particular interest is sarcoma protein (Src), a non-receptor tyrosine kinase. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 72 Src is expressed ubiquitously; however, brain, osteoclasts, and platelets express higher levels of this protein than most other cells. There are 11 members of the Src-family kinases in humans. These include Blk, Brk, Fgr, Frk, Fyn, Hck, Lck, Lyn, Src, Srm, and Yes. Src, Fyn, and Yes are expressed in all cell types. Srm is found in keratinocytes, and Blk, Fgr, Hck, Lck, and Lyn are primarily found in hematopoietic cells. Frk occurs chiefly in bladder, brain, breast, colon, and lymphoid cells. Brk occurs in colon, prostate and the small intestine (Roskoski, 2004). 3.1.3 Conservative domains of SH2 and SH3 in tyrosine kinase and signaling proteins Both receptor and non-receptor tyrosine kinases select their targets by the identification of a conserved protein module of approximately 100 amino acids in the Src homology 2 (SH2) domain and of about 60 amino acids in the Src homology 3 (SH3). These SH2 and SH3 domains are found in a remarkably diverse group of cytoplasmic signalling proteins (Pawson and Gish, 1992). SH2 domains bind to distinct amino acid sequences C- terminal to the phosphotyrosine where as SH3 binds to sequences of proline rich regions (Lee, 2005). Src tyrosine kinase, SH2 and SH3 domains have four important functions. First, they constrain the activity of the enzyme via intracellular contacts. Second, proteins that contain SH2 and SH3 ligands can bind to the SH3 and SH3 domains of Src and attract them to specific cellular location. Third, as a result of displacing the intracellular SH2 and SH3 domains, proteins can activate Src activity. Finally, proteins containing SH2 and SH3 ligands can enhance their ability to function as substrates for Src tyrosine kinase (Roskoski, 2004). As a result of the tyrosine phosphorylation events, Src tyrosine kinase and the docking proteins residing in focal adhesions form a network of protein-protein interactions that connect to multiple downstream biochemical signalling pathways (Figure 3.1.). There are a number of biochemical pathways that can be activated downstream of Src. These include signalling components that are well known from the field of growth factor receptor signalling; the PI3-kinase, PLC? and MAP kinase pathways (Schaller, 2001). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 73 Figure 3.1. Cellular dependent pathways that occur as a result of focal adhesion kinase (FAK) and Src interaction (Vuori, 1998). Paxillin FAK Src PI3-K Grb2 SOS CAS CSK RAS AKT Cell survival Erk Cell proliferation Gene expression Crk C3G RAP1 Cytoskeletal Change Rac JNK Gene expression Integrin Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 74 3.1.4 Tyrosine kinase Inhibitors Based on animal models, the crucial role of tyrosine phosphorylation by protein tyrosine kinases therefore presents an attractive target for developing an inhibitor. The compounds quercetin, genistein, lavendustin A, erbstatin, and herbimycin A, are tyrosine kinase inhibitors that exhibit broad specificity in micromolar range (Levitzki and Gazit, 1995). Derivatives of quercetin inhibit only tyrosine kinases. Genistein and lavendustin A are competitive inhibitors of ATP in the kinase reaction and compete non- competitively with the protein substrates. These compounds are broad-spectrum tyrosine kinase inhibitors, probably because the ATP binding domain is highly conserved among tyrosine kinases (Levitzki and Gazit, 1995). Synthetic inhibitors such as tyrphostins have been made that are effective both in vivo and in vitro. The structures of some of these compounds are shown in Figure 3.2. Tyrphostins are tyrosine kinases inhibitors, have been used extensively for the last decade. They specifically bind to the binding site of tyrosine but not the site of ATP binding (Levitzki, 1990). Tyrphostins can inhibit EGFR, the insulin receptor, and PDGF receptor. Tyrphostins also block tyrosine phosphorylation of host cells, in which lipopolysaccharides of gram-negative bacteria cause the development of sepsis ( septic shock) (Levitzki and Gazit, 1995). Furthermore, tyrphostins inhibit the EGF induced Ca 2+ mobilisation concomitant with the inhibition of phosphatidylinositol bisphosphate breakdown to inositol phosphates (Levitzki, 1990). In this study, tyrphostins and genistein were employed to investigate presence of in vivo and in vitro tyrosine kinase activity in whole cells and cell fractions of A. bisexualis. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 75 OH CN CN OOH OH O OH Tyrphostin 63 Genistein OH OH CN NH 2 CN NC OH OH CN NH 2 CN NC OH Tyrphostin 25 Tyrphostin 51 PD098059 O O NH 2 OCH 3 Figure 3.2. Structure of tyrosine kinase inhibitors Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 76 This chapter firstly shows that tyrosine phosphorylation in hyphae of A. bisexualis is reduced by tyrphostin 63. Using various biochemical techniques, the presence of tyrosine kinase activities in crude and partially purified fractions obtained from A. bisexualis hyphae and S. cerevisiae were identified. These activities were strongly inhibited by tyrphostin 25 and tyrphostin 51. Mycelial growth as well as extension of individual hyphae was reduced by tyrphostin 63 and tyrphostin 25. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 77 3.2 Materials and Methods Tyrosine kinase assay kits, phosphocellulose powder, and poly(Glu:Tyr) 4:1 were purchased from Sigma. The Centriprep desalting column was purchased from Amicon Bioseparation, Millipore. 3.2.1 Immunocytochemistry of phosphotyrosine containing protein of A. bisexualis hyphae in the presence of tyrphostin 63 Methods were as described in Chapter 2. Hyphae were incubated with 1 mM tyrphostin 63 prior to fixation. 3.2.2 Protein extraction, partial purification and protein tyrosine kinase assay A. bisexualis Cytosolic crude fraction: The growing edge of A. bisexualis mycelia were cut, quick frozen with liquid nitrogen, ground and extracted with buffer A ( 30 mM Tris-HCl, pH 7.1, 20 mM NaF, 5 mM EGTA, 7 mM EDTA, 2 mM DTT, 50 mM KCl, 2 mM NaVO 3 , 2 mM PMSF, 20% glycerol). The extract was centrifuged at 10,000 g for 30 mins, the supernatant and pellets obtained were stored on ice prior to being assayed. Triton-X crude fraction: The pellet was further re-suspended in buffer A, containing 2 % Triton X-100 and gently shaken on ice for 60 mins. The re-suspended pellet was then centrifuged at 900 g for 20 mins and the supernatant was collected, this was referred to as the Triton X-100 soluble fraction. S. cerevisiae Yeast cells (strain SY1129 (F131)) were streaked on YPD agar plates and incubated overnight at 30 ?C. An isolated colony was selected and then cultured in 10 ml YPD media overnight at 30 ?C. After this, 10 ml of the overnight culture was poured into 1 L Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 78 YPD media and further incubated at 30 ?C overnight on an orbital shaker. Cells were then centrifuged at 5,000 g for 30 mins and the pellet obtained was washed twice with ice-cold 40 mM PBS pH 7.0. The pellet was then re-suspended with the extraction buffer as above and vortexed with pre-chilled ice cold glass beads for 20 mins and further sonicated for 30 mins. Lysed cells were then centrifuged at 10,000 g for 30 mins. 3.2.3 Partially purified proteins The supernatants obtained as described above were then applied to a phosphocellulose (Sigma) column (1cm X 15 cm) that had been equilibrated with buffer A. The column was washed with buffer B (washing buffer containing 30 mM Tris-HCl, pH 7.1, 7 mM EDTA, 6 mM 2-mercapthoethanol, 10 % glycerol 1 mM Na 3 VO 4 ) until the A 280 of the elutent was below 0.1, and was then eluted with a gradient of zero to 1 M NaCl in buffer C (eluting buffer contained 40 mM Hepes pH 7.0, 7 mM EDTA, 2 mM Na 3 VO 4 , 20 % glycerol, 2 mM DTT). Fractions of 2 ml were collected and the kinase activity was determined by ELISA (see below). Fractions containing tyrosine kinase were then desalted using a Centriprep column, which was continuously centrifuged at 900 g for 1 hour and the column was refilled with buffer without salt and the process repeated 5 times. The desalted fraction was then applied to a poly(Glu:Tyr)4:1, which was pre- equilibrated with buffer C. The column was then washed with 150 ml of buffer C. The bound protein was then eluted with buffer C containing 0.5 mM NaCl. One ml of the fraction was collected and tested for tyrosine kinase activity by ELISA. 3.2.4 The detection of tyrosine kinase activity by ELISA For the tyrosine kinase ELISA, 96-microtiter wells were coated with 150 ?l of 1.25 ?M poly(Glu:Tyr) 4:1 (Sigma) and incubated at 45 ?C overnight. Wells were then blocked with blocking solution (5% skim milk, 10 mM PBS pH 7.2) for 45 mins at 37 ?C and washed with a washing buffer (10 mM PBS pH 7.2, 0.05% Tween- 20) 5 times. The phosphorylation reaction (the final volume of which is 250 ?l) contains 300 ?M ATP, tyrosine kinase buffer (50 mM Hepes pH 7.0, 20 mM MgCl 2 , 1 mM MnCl 2 , 1 mM Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 79 Na 3 VO 4 ) and 50 ?l of the soluble crude fraction, the Triton X-100 soluble fraction or 10 ?l (2 units) of growth epidermal factor receptor (EGFR) which was used as a positive control. Wells were incubated at 37 ?C for 45 mins. Wells were then washed with a washing buffer 5 times. Phosphotyrosine was probed by the addition of a 1:2000 dilution of anti-phosphotyrosine antibody conjugated with horse radish peroxidase (HRP) for 45 mins at 37 ?C. After this wells were washed 5 times with a washing buffer. HRP colour development solution (Sigma Fast OPD and Sigma fast Tablets) was added and incubated in the dark for 7 mins, then 2.5 N of H 2 SO 4 was added. Colorimetric change was determined at a wavelength of 492 nm with the microtiter plate reader (Multiskan ? MCC/340). For assays in the presence of inhibitors, either soluble crude or Triton X100 soluble fractions were pre-incubated with different concentrations of tyrphostins for 10 mins on ice, then assayed on the poly(Glu:Tyr) 4:1 - coated wells as described previously. The control contained DMSO at a concentration similar to those in the tyrphostin solutions. 3.2.5 Detection of tyrosine kinase activity by a coupled assay For these reactions a phosphorylation mixture was prepared using non-coated microtiter plates. Each reaction mixture contained 1 mM ATP, 1.25 ?M poly (Glu:Tyr) 4:1 , 100 ?l of the appropriate cell fractions and tyrosine kinase buffer to a final volume of 300 ?l. For inhibition by tyrphostins, the appropriate concentration of tyrphostin was added. The reaction was incubated at 37 ?C for 45 mins. The coupled assay was carried out to a final volume of 1 ml in a plastic cuvette. The coupled assay mixture contained 0.3 mM NADH, 1 mM phosphoenolpyruvate (PEP), 20 units of pyruvate kinase (PK), 7-10 units of lactic dehydrogenase (LDH) and buffer (containing 100 mM Hepes, pH 7.5, 20 mM MgCl 2 , 1 mM 2-mercaptoethanol (Biorad), 20 ?g/ml BSA and 5 mM MnCl 2 ). The coupled assay was initiated by the addition of 300 ?l of the phosphorylation mixture into 700 ?l of the coupled assay mixture and then the reaction was monitored by following the reduction of NADH at 340 nm for 2-3 mins. From this the initial reaction rate was calculated. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 80 3.2.6 The effect of tyrphostin on tip growth The growing margin of a mycelial sheet of A. bisexualis growing on cellophane overlaying PYG agar was cut about 1 cm behind tips and mounted in a flow-through chamber with 2 % low melting point agar in PYG media. The mounted hyphae were left at room temperature for 2 hours in PYG media. The control hyphae were exposed to 1% dimethyl sulphoxide (DMSO) in PYG media. The stock solution of tyrphostins were made in DMSO and freshly diluted in PYG media to give the required concentration of tyrphostin. To monitor hyphal growth rate, hyphae were grown in PYG media for 30 mins and then incubated with the required concentration of tyrphostin for 1 hour. After 1 hour, the inhibitor solution was replaced with PYG media. Hyphae were then allowed to grow in PYG media for another 1 hour. The rate of extension of the hyphal tip was monitored at 5 mins intervals using a 10x objective lens microscope (Olympus BH2). Statistical analyses were carried out using Statistix 8. 3.2.7 The effect of tyrphostin on mycelial growth An agar plug of growing mycelia of A. bisexualis was inoculated onto PYG agar plates on which the media agar was overlaid by cellophane. Control plates contained 1% DMSO. The experimental plates contained different concentrations of tyrphostin 63 and 25 in the agar. Mycelia were imaged after 36 hours incubation at 20 ?C. Digital images were generated using a Microtek Scanmaker V6USL Scanner (Microtek Lab Incorporated). Stored digitised images were processed using a combination of Metamorph (Universal Imaging Corporation, West Chester, PA, USA) and Adobe PhotoShop (Adobe Systems Incorporated) software prior to calculation of mycelial area which was carried out using ImageJ software that is available in the public domain via the NIH. The website for software download is ftp://rsbweb.nih.gov/pub/image-J/. Mycelial area was calculated by counting the number of black pixels in each image and multiplying this by the area that each pixel represented. The area of the inoculation plug was subtracted from the obtained value. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 81 3.3 Results 3.3.1 The effect of tyrphostin 63 on phosphotyrosine containing proteins in A. bisexualis hyphae To investigate the possible role of a tyrosine kinase in the phosphorylation of tyrosine that was observed in Chapter 2, hyphae were firstly incubated with 1 mM tyrphostin 63 for 20 mins in the dark at room temperature prior to fixation. Hyphae were then double stained with two primary antibodies, an anti-?4 integrin antibody and an anti- phosphotyrosine antibody. In hyphae that were treated with tyrphostin 63, the phosphotyrosine staining was diminished but the ?4-integrin staining remained (Error! Reference source not found..). Hyphal tips were rounded, a shape that is common in slowly growing or non-growing hyphae (Error! Reference source not found.. A, B). The effect of tyrphostin on growth is described in more detail below. Hyphae that were grown in the absence of tyrphostin had the long tapered tip that is characteristic of A. bisexualis. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 82 Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 83 3.3.2 The presence of protein tyrosine kinase activities in A. bisexualis and S. cerevisiae 3.3.2.1 Protein tyrosine kinase activity in A. bisexualis by ELISA. Protein extracts taken from the growing margin of five day old cultures were tested for tyrosine kinase activity using an ELISA method, the details of which are shown in Appendix 1. The study did not attempt to characterise tyrosine kinases but looked for the presence of tyrosine kinase activity in cell fractions. As a consequence, a time course of reaction as well as kinetic properties of enzymes was not fully addressed due to time constraints. This is clearly an area of some interest for future studies. The incubation time for the phosphorylation reaction was 45 mins (as described previously in (Cleaveland et al., 1990). Cytosolic crude and Triton-X soluble fractions showed significant levels of tyrosine kinase activity (Figure 3.3. and Figure 3.7.) respectively. In the cytosolic crude fraction, tyrosine kinase activity decreased significantly in the presence of 2 mM tyrphostin 63 (P<0.0001, n= 7) and 1 mM tyrphostin 25 (P<0.0001, n= 7) (Figure 3.3.). However, 2 mM tyrphostin 51 did not have (p>0.05, n= 7) any inhibitory effect on kinase activity. The inhibitory effect was also tested on EGFR which was used as a control. In the absence of inhibitors, 2 units of EGFR gave an absorbance at 492 nm of 1.12 ? 0.38. In contrast, in the presence of 0.25 mM tyrphostin 25 and 51, it gave values of 0.1 ? 0.04, and 0.25 ? 0.09, respectively. Tyrosine kinase activity was also tested in the presence of 1% (v/v) DMSO which was the highest concentration required for solubility of the tyrphostins. DMSO did not inhibit tyrosine kinase activity at this concentration. To further investigate the kinase activity genistein was used which is another tyrosine kinase inhibitor, inhibiting kinases by competing at ATP binding sites. At 0.5 mM of genistein, tyrosine kinase activity was significantly (p<0.0001, n= 7) reduced in the cytosolic crude fraction by 62%. Moreover, 1 mM of genistein significantly (p<0.0001, n= 7) inhibited about 75% of kinase activity (Figure 3.4.). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 84 0 20 40 60 80 100 120 Control 2 mM Tyrphostin 51 2 mM Tyrphostin 63 1 mM Tyrphostin 25 % of K i n a s e A c t i vi t y Figure 3.3. The tyrosine kinase activity of cytosolic crude fractions from A. bisexualis as determined by ELISA. Control contains 1% (v/v) DMSO. Data are presented as percentage relative to the control. The Absorbance at 492 nm value for the control was 0.257 ? 0.035. Averaged absorbance of kinase activities at 492 mm tyrphostin 51, tyrphostin 63 and tyrphostin 25 are, 0.218 ? 0.0298, 0.17 ? 0.035, and 0.131 ? 0.018, respectively. Data are shown as mean absorbance values ? SD. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 85 0 20 40 60 80 100 120 Control 0.5 mM 1 mM Genistein % o f K i n a s e A c ti v i ty Figure 3.4. The inhibitory effect of genistein on tyrosine kinase activity in a cytosolic crude fraction of A. bisexualis as determined using an ELISA. Control contains 1% (v/v) DMSO. Tyrosine kinase activity was determined at the absorbance of 492 mm. Averaged absorbance of control, 0.5 mM, and 1 mM of genistein are 0.152 ? 0.021, 0.0573 ? 0.019, and 0.0384 ? 0.025, respectively. Data are shown as mean absorbance values ? SD. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 86 A Triton X-100 soluble fraction was also tested for tyrosine kinase activity. The method for using Triton X-100 to extract proteins present in the cell debris was modified from that previously described in (Torruella et al., 1985) and (He et al., 1996). The cell debris of A. bisexualis mycelia that remained after the removal of the cytosolic crude fractions was extracted with 2% Triton X-100 buffer and centrifuged at 900 g for 20 mins at 4 ?C. It was found that the centrifugation used affected kinase activity. A number of centrifugal forces were tested and it was found that kinase activity was absent when centrifugation was higher than 900 g. The Triton X-100 soluble fraction showed a very high level of tyrosine kinase activity (Figure 3.7.), giving 1.4 ? 0.26 (n=7) absorbance at 492 nm. In the presence of 0.25 mM of tyrphostin 63, kinase activity was significantly (p<0.001, n= 7) reduced by 25%. Furthermore, at 0.25 mM two additional tyrphostins, tyrphostin 25 and tyrphostin 51 also strongly inhibited kinase activity. Tyrphostin 25 and tyrphostin 51 significantly reduced (p<0.0001, n= 7) tyrosine kinase activity by 85%. Partial purification of both the cytosolic crude and Triton X-100 soluble fractions was attempted using a phosphocellulose column. An SDS-PAGE showed that the partially purified fraction contained proteins with molecular masses ranging from 26 to 85 kDa (Error! Reference source not found.. A, lane 2). However, none of these are likely to be kinases as the partially purified fractions of the soluble crude did not have any tyrosine kinase activity that was detectable by ELISA. Prior to applying the soluble crude fraction into a phosphocellulose column, the activity of the soluble crude was tested by ELISA and gave 0.2-0.3 absorbance at 492 nm. This absorbance was low in comparison with the absorbance obtained from the Triton X-100 soluble fraction, which as described above is about 0.9-1.4 at 492 nm. The partially purified fraction of Triton X-100 soluble still showed tyrosine kinase activity as determined by ELISA. An SDS-PAGE revealed several protein bands in the partially purified Triton X-100 soluble fractions (Error! Reference source not found.. B, lane 2). A number of bands were observed with especially prominent ones at 28, 34, 41 and 88 kDa which may possibly contribute to the tyrosine kinase activity. The tyrosine kinase activity from the partially purified Triton X-100 fraction was reduced in the dose dependent manner by tyrphostin 25 and tyrphostin 51 (Figure 3.6.). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 87 Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 88 0 20 40 60 80 100 120 Control Tyrphostin 63 Tyrphostin 25 Tyrphostin 51 % o f K i n a s e A c ti v i ty Figure 3.5. Inhibitory effect of tyrphostins on tyrosine kinase activity obtained from the Triton X-100 soluble fraction of A. bisexualis as determined using ELISA. All concentrations of tyrphostins contain 0.24% (v/v) of DMSO. The original absorbance at 492 nm of control, tyrphostin 63, tyrphostin 25, and tyrphostin 51 are 1.4 ? 0.26, 1.03 ? 0.056, 0.222 ? 0.031, and 0.20 ? 0.038, respectively. Data are shown as mean absorbance values ? SD Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 89 0 20 40 60 80 100 120 140 0 8 16 31 63 125 250 Concentrations (?M) % of K i n a s e A c t i vi t y tyrphostin 25 tyrphostin 51 Figure 3.6. Dose dependent inhibition by tyrphostin 25 and tyrphostin 51 on kinase activity of the partially purified fractions of the Triton X-100 soluble fraction from A. bisexualis. The kinase activity was determined by ELISA. The absorbance at each concentration is the average of three experiments. Data are presented as percentages relative to the control. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 90 3.3.2.2 Protein tyrosine kinase activity in A. bisexualis by the coupled assay Kinase activities were also determined using a coupled assay method. The method used was modified from that of (Qiu and Miller, 2002). If tyrosine kinases are present in A. bisexualis extracts, in the presence of poly(Glu:Tyr) 4:1 and ATP, tyrosine kinase should phosphorylate poly(Glu:Tyr) 4:1 and release ADP. This reaction is coupled to the dephosphorylation of phosphoenolpyruvate (PEP) by pyruvate kinase (PK). The pyruvate produced is subsequently utilised by lactate dehydrogenase (LDH) with oxidation of NADH. The oxidation of NADH is then measured by spectrophotometer at 340 nm. The production of ADP from the kinase reaction is thus reflected by the amount of NAD + produced (see appendix 1). The soluble crude fraction of A. bisexualis, in the presence of poly(Glu:Tyr) 4:1 and ATP, gave tyrosine kinase activity of 10.2 ? 2.4 x 10 -2 units/ml (n=7) (Figure 3.9.). In the presence of ATP alone, the soluble crude fraction was able to utilise ATP through autophosphorylation. Autophosphorylation led to ?activity? of 8.6 ? 1.3 x 10 -2 units/ml (n=7). There is no significant (p>0.05, n= 7) difference between kinase activities with or without poly(Glu:Tyr) 4:1 . Thus it is likely that most of phosphorylating activity in the soluble crude fraction is due to autophosphorylation. Since the soluble crude is therefore likely to have little kinase activity, inhibitory experiments were not carried out. In contrast the Triton X-100 soluble fraction had tyrosine kinase activity. A value of 4 ? 0.89 x 10 -2 (n=8) units/ml was obtained in the presence of poly(Glu:Tyr) 4:1 and ATP compared to 2 ? 0.74 x 10 -2 (n=8) units/ml with ATP alone. These values are significant (p<0.001, n= 8) difference. In order to further evaluate the tyrosine kinase activity present in the Triton X-100 soluble fraction, 0.25 mM of tyrphostin was incubated with the Triton X-100 fraction on ice for 10 mins prior to the phosphorylation reaction. In the presence of tyrphostins 25 and 51, tyrosine kinase activity was significantly (p<0.0001 and p<0.0001, respectively, n= 8) reduced (Figure 3.8.). This suggests that the tyrosine kinase activity present in the Triton X-100 soluble fraction is sensitive to tyrphostins. Concentrations of tyrphostin and DMSO that the fractions were exposed to were not found to inhibit the activity of PK or LDH in the coupled assay. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 91 0 20 40 60 80 100 120 With poly(Glu:Tyr)4:1 Without poly(Glu:Tyr)4:1 % of K i n a se s A c t i vi t y Figure 3.7. Tyrosine kinase activity of the soluble crude fraction of A. bisexualis with and without the exogenous substrate poly(Glu:Tyr) 4:1 . The production of ADP due to phosphorylation reaction was determined using the coupled assay. The original kinase activities from the phosphorylation reaction is 10.2 ? 2.4x10 -2 (n=8) units/ml with poly(Glu:Tyr) 4:1 and 8.6 ? 1.3x10 -2 (n=8) units/ml without poly(Glu:Tyr) 4:1 . Data are shown as mean absorbance values ? SD. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 92 0 20 40 60 80 100 120 140 Control Autophosphorylation Tyrphostin 25 Tyrphostin 51 % of K i n a s e A c t i vi t y Figure 3.8. Inhibitory effect of tyrphostins on tyrosine kinase activity in the Triton X- 100 soluble fractions of A. bisexualis. 0.25 mM of tyrphostin 25 and tyrphostin 51 was used. Original kinase activities are 3.88 ? 0.89x10 -2 units/ml for the control, 1.79 ? 0.66x10 -2 units/ml for tyrphostin 25, 0.758 ? 0.21x10 -2 units/ml for tyrphostin 51 and 2.05 ? 0.74x10 -2 units/ml for autophosphorylation. Data are shown as mean absorbance values ? SD. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 93 3.3.2.3 Protein tyrosine kinase activities in S. cerevisiae as determined by ELISA and the coupled assay The soluble crude fraction of yeast was partially purified using a combination of a phosphocellulose column, a Sephadex G-100 column and a poly(Glut:Tyr) 4:1 affinity column. Fractions containing tyrosine kinase activity from the phosphocellulose column were desalted using a Centriprep column. Then, the desalting fractions were applied to a poly(Glu:Tyr) 4:1 affinity column and eluted with 0.5 M NaCl. Tyrosine kinase activity was determined by ELISA (Figure 3.9.). Tyrosine kinase activity of fractions obtained from each step are also summarised in Error! Reference source not found.. The purification fold was increased from 130-fold by the phosphocellulose of phosphocellulose column to 230-fold by the affinity column. The proteins present in the above fractions were examined by SDS-PAGE (Error! Reference source not found..) There are five major bands that are possibly responsible for kinase activity. Proteins at 93, 50, 45, and 35 kDa bound to the poly(Glu:Tyr) 4:1 - Sepharose column since these proteins showed weak or no bands in the unbound fraction washed out from the poly(Glu:Tyr) 4:1 -Sepharose column (Error! Reference source not found.. lane 4). Especially, the 93 kDa was absent in unbound fractions. The 35 kDa protein showed a stronger band in the bound fraction but there was a trace of this in the unbound fraction. Furthermore, 0.5 mM of tyrphostin 25 and genistein significantly (p<0.001, n=8) reduced kinase activity in partially purified fractions after passing through a poly(Glu:Tyr) 4:1 - Sepharose affinity column by 54 % and 58 % respectively. However, at 1 mM tyrphostin 63 significantly reduced kinase activity only 38 % (p<0.05, n=8). In order to confirm, phosphorylation by tyrosine kinases present in partially purified fractions in the yeast, MAPKK inhibitor; PD098059 was incubated with cell fractions. At 0.5 mM PD098959 significantly (p<0.001) reduced kinase activity by 52 % (Figure 3.10.). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 94 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Crude phosphocellulose column Affinity column K i n a s e a c ti v i t y a t 492 n m Figure 3.9. The tyrosine kinase activity obtained after various purification steps of S. cerevisiae as determined by ELISA. Averaged absorbance at 492 nm of tyrosine kinase activities crude, phosphocellulose column and affinity column are 0.119 ? 0.034 (n= 6), 0.654 ? 0.09 (n=9), and 0.613 ? 0.148 (n=9), respectively. Data are shown as mean absorbance values ? SD. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 95 Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 96 Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 97 0 20 40 60 80 100 120 140 control 0.5 mM PD98059 0.5 mM tyrphostin 25 0.5 mM genistein 1 mM tyrphostin 63 % o f K i n a s e A c ti v i ty Figure 3.10. The effect of inhibitors on tyrosine kinase activity of S. cerevisiae. Tyrosine kinase activity was determined by ELISA. Original absorbances after treatment with PD98059, tyrphostin 25, genistein, and tyrphostin 63 are 0.21 ? 0.04, 0.2 ? 0.02, 0.19 ? 0.04 and 0.28 ? 0.03 respectively. The control was 0.44 ? 0.07. Both control and inhibitors contain 1.6 % (v/v) of DMSO. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 98 3.3.3 Extension rate of individual hyphae of A. bisexualis To evaluate the possible role of tyrosine phosphorylation in the regulation of tip growth tyrphostin 63 and tyrphostin 25 were added in growth media. The rate of tip extension of hyphae was measured in a perfusion chamber containing PYG low melting point agar. Hyphae were allowed a 60 mins recovery period. To begin the experiment, mycelia were allowed to grow for 30 mins; the tip extension was monitored at 5 mins intervals. After 30 mins, tyrphostins dissolved in PYG media were added (Figure 3.14. black arrow) and the rate of tip growth measured for 60 mins. After that tyrphostin was withdraw (Figure 3.14. red arrow) and the perfusion chamber was gently washed twice with 700 ul of PYG media and hyphae were allowed to grow in PYG media containing 1 % DMSO for another 60 mins. The growth rates showed in Figure 3.14 were obtained from the mean values of 10 individual hyphae. Tyrphostin 63 significantly (P<0.0001, n= 7) reduced the rate of tip growth of (Figure 3.14.), in a dose dependent manner. After 5 mins administrating 0.5 mM tyrphostin 63 to hyphae, tip growth declined from 5.2 to 5.0 ?m/min and then continually declined after 60 mins to 3.3 ?m/min. After removal of the inhibitor, tip growth did not recovery after 60 mins and reduced further to about 2.9 ?m/min. At 1mM tyrphostin 63, tip growth reduced to a greater extent than 0.5 mM. Five minutes after administrating the inhibitor, tip growth was 4.6 ?m/min and it continued to reduce to 2.5 ?m/min after 60 mins. Tip growth reduced further to 1.8 ?m/min 60 mins after removal of the inhibitor. At both 0.5 and 1 mM tyrphostin 63 affected the tip morphology. Tip morphology changed into a blunt shape after about 10-20 mins. Swelling of sub-apical regions was visible after 20-30 mins. Tyrphostin 25 also affected tip growth. Hyphae were treated with 1.5 mM tyrphostin 25, tip growth gradually declined from 4.4 ?m/min to 3.6 ?m/min after 60 mins treatment. However, after the inhibitor was removed, tip growth was stable and started to recovery back to 3.8 ?m/min. At 0.75 mM tyrphostin 25 had little effect on tip growth. Blunt tips and swelling were observed in hyphae treated with 1.5 mM tyrphostin 25. Control hyphae were grown in Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 99 the presence of 1% (v/v) of DMSO. At this concentration, hyphae grow on averaged rate 5.2 ?m/min. There was no change of tip morphogenesis in the control hyphae. 0 20 40 60 80 100 120 0 2040608010120140 Time (minutes) % of grow t h rat e control 1mM Tyrphostin 63 0.5mM Tyrphostin 63 1.5mM Tyrphostin 25 0.75mM Tyrphostin 25 Figure 3.11. The effect of tyrphostin 63 and tyrphostin 25 on tip growth of hyphae of A. bisexualis. Inhibitors were added at the black arrow and removed at the red arrow. Data are shown as the average value of 7 hyphae. Growth rate of the control was 5.2 ?m/min. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 100 3.3.4 The effect of tyrphostins on mycelial area Tyrphostin 63 and tyrphostin 25 were tested to investigate their effect on the growth of mycelium (Error! Reference source not found..). After 36 hrs post inoculation, the mycelia were imaged. The area of mycelia was calculated. At 0.5 and 1mM tyrphostin 63, the area of mycelia was significantly (P< 0.0001, n= 7) reduced by 73 % and 95% respectively. Mycelial area was also significantly (P<0.0001, n= 7) decreased by 67% with 1.5 mM tyrphostin 25. However, 0.75 mM of tyrphostin 25 (P>0.05, n= 7) showed no effect on mycelial area (Figure 3.12.). The mycelial area of A. bisexualis was reduced in the presence of tyrphostin 63 and the reduction was dose-dependent. It is very interesting to note that tyrphostin 25, another potential tyrosine kinase inhibitor showed less of an inhibitory effect. This should not be expected since tyrphostin 25 has lower IC 50 than tyrphostin 63 toward EGFR (Levitzki, 1990). One possible explanation for this is that the ability of the inhibitors to cross the cell wall and then the plasma membrane are different. Base on the structures, tyrphostin 63 has a less polar side chain (1-OH) in comparison with tyrphostin 25 (2-OH). Therefore, tyrphostin 63 is likely to cross the cell wall and plasma membrane easier than tyrphostin 25. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 101 Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 102 Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 103 0 20 40 60 80 100 120 0 0.5 1 0.75 1.5 Tyrphostin 63 (mM) Tyrphostin 25 (mM) % o f A.bi se x u a l i s my c e l i u m Figure 3.12. Mycelial area of A. bisexualis grown in the presence of tyrphostin 63 and tyrphostin 25. The control contained 1% (v/v) of DMSO. In the presence of tyrphostin 63, at 0.5 mM and 1 mM significantly reduced (p<0.0001, n= 7) the mycelial area of A. bisexualis. Also tyrphostin 25, at 1.5 mM significantly reduced (p<0.0001, n=7) the area of mycelia but 0.75 mM did not have any inhibitory affect (p>0.05, n=7). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 104 3.4 Discussion The previous chapter suggested the presence of proteins that contain phosphorylated tyrosine residues in A. bisexualis. This would suggest the presence of kinases that facilitate such phosphorylation. In the present chapter the possibility that tyrphostin inhibits the tyrosine kinase activity which was responsible for tyrosine phosphorylation was investigated. Hyphae that were treated with tyrphostin 63 for 20 mins before fixation showed a reduction of phosphotyrosine staining while staining of integrin-like proteins was not affected. This data suggest that tyrosine phosphorylation is sensitive to the presence of tyrphostins. The tyrphostins are a group of synthetic compounds that were originally designed to bind the substrate subsite in the kinase domain of the epidermal growth factor receptor, against which tyrphostins 25, 51 and 63 have IC 50 values of 3 ?M, 0.8 ?M and 6.5 mM respectively (Levitzki, 1990). They have since been used to inhibit a number of other tyrosine kinases, with the sensitivity to a particular tyrphostin dependant upon the identity of the kinase (Levitzki and Gazit, 1995). The results in this thesis show that the kinase activity is more sensitive to tyrphostins 25 and 51 than to tyrphostin 63, which is in keeping with a number of animal tyrosine kinases. In other walled cells tyrphostins have been found to block the cell cycle of S. cerevisiae (Fujimura, 1997) and to inhibit a STY dual specificity kinase in peanut (Rudrabhatla and Rajasekharan, 2004). In Fucus, tyrphostin 25 was found to have no effect on either germination of embryos or the elongation of rhizoids, although a number of other tyrosine kinase inhibitors affected germination and two of these had a moderate inhibitory effect on rhizoid elongation (Corellou et al., 2000). It is possible that the lack of effect of tyrphostin reflects the concentration that was used (10 ?M) as inhibition of tip growth of A. bisexualis occurs at low millimolar concentrations. This study also shows that tyrosine phosphorylating activity was present in cytosolic crude and Triton X-100 soluble fractions. The level of kinase activity in the cytosolic crude fraction was low in comparison with activity present in the Triton X-100 fraction. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 105 This contrasts with results from Fucus embryos where attempts to characterise tyrosine kinase activity in cytosolic crude fraction were unsuccessful (Corellou et al., 2000). In the cytosolic crude fraction of A. bisexualis, inhibition of kinase by tyrphostins required high dose of inhibitors relative to dose of inhibitor used in the Triton X-100 fraction. This may suggest that the kinase activity present in the cytosolic crude fraction comes from a dual-specific kinase. Other possible reasons for less kinase activity in the cytosolic crude fraction are the presence of tyrosine phosphatases or a high background of autophosphorylation. Tyrosine phosphatases have been reported in fungi and plants (Dickman and Yarden, 1999; Luan et al., 2001). Even though, there are no report of tyrosine phosphatases in oomycetes, this can not be ruled out since they are highly conserved in eukaryotic cells (Luan et al., 2001). Genistein was also found to reduce tyrosine kinase activity in the cytosolic crude fraction. Unlike tyrphostins, genistein is designed to bind at the ATP binding site of tyrosine kinases and it can specifically bind to both receptor and non-receptor tyrosine kinases (Levitzki, 1990). However, one question that needs to be considered is what is the relative specificity of genistein to kinases? Genistein can inhibit serine/threonine kinases such as a protein kinase C in animal cells at concentrations greater than 300 ?M (Akiyama et al., 1987). In this study, 0.5 and 1 mM was used, both of which concentration significantly reduced kinase activity. It is thus possible that the kinase activity in the cytosolic crude is due to dual specific kinases. The other possibility is the process of autophosphorylation may be responsible for activity in the cytosolic crude fraction. The coupled assay shows that the cytosolic crude fraction has a high back ground level of autophosphorylation. In the presence of both poly(Glu:Tyr) 4:1 and ATP, kinase activity is very high, as well as activity obtained in the presence of ATP alone. This data suggest that the phosphate groups of ATP are not transferred to poly(Glu:Tyr) 4:1 but instead they are possibly transferred to proteins, which are present in the cytosolic crude fraction. The autophosphorylation as shown by the coupled assay has also been shown on tyrosine kinases obtained from animal cells (Barker et al., 1995; Qiu and Miller, 2002). However, it is possible that several kinases, including tyrosine kinase in animal cells and MAP kinases in animals, plants and fungi Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 106 are able to autophosphorylate on serine, threonine and tyrosine residues (Dickman and Yarden, 1999). The data from this study also shows that a phosphocellulose column partially purified cytosolic crude fraction of A. bisexualis does not contain tyrosine kinase activity. There are several protein bands present in a partially purified fraction one of which has a mass of 26 kDa. This peptide also contains phosphotyrosine residues as previously described in chapter 2. A 26 kDa peptide was also found in Papaver rhoeas but this is most likely to be phosphorylated at serine/threonine residues (Rudd et al., 1996). Despite of the absence of tyrosine kinase activity in the cytosolic fraction, the cell wall that remained after removal of the cytosolic fraction was extracted using buffer containing Triton X-100. The purpose of employing Triton X-100 is to extract proteins that are associated between the cell wall and plasma membrane. The method of this extraction was modified from that described previously by (Torruella et al., 1985; He et al., 1996). The Triton X-100 soluble fraction contained very high tyrosine kinase activity relative to activity found in the cytosolic fraction as determined by ELISA. Furthermore, this activity was sensitive to tyrphostin 25 and tyrphostin 51 but less sensitive to tyrphostin 63. A partially purified Triton X-100 fraction also showed tyrosine phosphorylating activity and this activity was reduced in a dose dependent manner by tyrphostin 25 and tyrphostin 51. An SDS-PAGE showed prominent protein bands at 28, 34, 41, and 88 kDa, it is possible that these peptide bands may be responsible for the kinase activity in the partially purified Triton X-100 soluble fraction. To date there is no strong evidence to support the presence of tyrosine kinases similar to those of animals in oomycete, fungi and plants. However, the oomycete Phytophthora has nucleotide sequences that resemble tyrosine kinases present in Dictyostelium discoideum, Caenorhabditis elegans, Homo sapiens, Rattus norvegicus, and Mus musculus. The identities of these Phytophthora sequences are Ps31050460, Ps9834403, and Ps30499946, which can be found at http://cogeme.ex.ac.uk. A membrane-bound insulin binding protein of 67 kDa has been purified from the fungus N. crassa but the protein possessed no tyrosine kinase activity with poly(Glu:Tyr) 4:1 Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 107 substrate (Kole et al., 1991). In plants a high level of apparent tyrosine kinase activity has been reported pea plantlets (Torruella et al., 1985). However, author did not address the question of what proteins might be responsible for the kinase activity as there was no SDS-PAGE presented and the kinase activity assay did not include tests for autophosphorylation. Even though the structure of plant receptor kinases (PRKs), which consists of an extracellular domain, a single membrane-spanning domain and a cytosolic kinase domain resembles receptor tyrosine kinases in animal cells, PRKs belong to the serine/threonine kinase family (Cock et al., 2002). Furthermore, no receptor kinases have been found in fungi such as yeast and N. crassa (Shiu and Bleecker, 2001). One interesting group of plant receptors are the wall associated kinases (WAKs), which function as mediators between the cell wall and plasma membrane. As such these are integral membrane proteins. WAK1 is most abundant in plant tissue and has a protein mass of about 68 kDa and can only be extracted by boiling plant material in buffer containing 4% (w/v) SDS (He et al., 1996). The kinase activity shown in the Triton X-100 soluble fraction of A. bisexualis may not be a WAK like-kinase as SDS-PAGE did not show any proteins at 68 kDa. However, this possibility can not be ruled out since the cell wall of oomycetes is more similar to the plant cell wall than the fungal cell wall. For comparison, the kinase activity from the budding yeast S. cerevisiae was determined. Although the S. cerevisiae genome does not contain an animal type tyrosine kinase gene sequence, it appears that some serine/threonine kinases are able to phosphorylate tyrosine residues of the poly(Glu:Tyr) 4:1 synthetic peptide substrate. The data from this study suggest that partially purified fractions of yeast have high levels of tyrosine kinase activity. An SDS-PAGE shows proteins of 35, 45, 50, and 93 kDa that may be responsible for this activity. The 93 kDa protein is close in size to Swe1, which has a mass of about 92.5 kDa. Swe1 is a serine/tyrosine dual-specificity kinase and plays a vital role in the mitotic process in S. cerevisiae. Swe1 is a negative regulator of the Cdc28 protein kinase by phosphorylation of Tyr-19 in the ATP binding site and consequently blocks the cells entry into mitosis (Sia et al., 1998; Shulewitz et al., 1999). Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 108 Several serine/threonine kinases have been found in yeast, which are able to phosphorylate poly(Glu:Tyr) 4:1 . Chip technology has been used to identify 27 kinases able to phosphorylate poly(Glu:Tyr) 4:1 (Magherini et al., 2004). Tyrosine kinase activity in S. cerevisiae had previously been detected by using poly(Glu:Tyr) 4:1 as a substrate for in vitro phosphorylation assays (Schieven et al., 1986). The purified protein tyrosine kinase activity is associated with a 40 kDa yeast protein with features that resemble protein-serine kinases (Dailey et al., 1990). In addition the calmodulin-dependent protein kinase and cAMP-dependent protein kinase are able to phosphorylate poly(Glu:Tyr) 4:1 on tyrosine (Stern et al., 1991). To further address kinase activity present in the partially purified fraction of yeast, the inhibitory effect of various kinase inhibitors was investigated. Both tyrphostin 25 and genistein strongly inhibited the kinase activity. In contrast, tyrphostin 63 significantly reduced kinase activity. In order to investigate whether that the activity present in yeast comes from tyrosine -like kinases or serine/threonine kinases, an inhibitor of the MAP kinase kinase PD098059 was included in the phosphorylation assay. The data shows that PD098059 significantly inhibits kinase activity when compared to tyrphostin 25 and genistein. This may suggest that the kinase activity present in yeast comes from a tyrosine-like kinase possibly comes from MAPKK. PD098059 is designed to specifically bind to MAP kinase kinase (MAPKK), and acts as a non-competitive inhibitor (Alessi et al., 1995). It does not affect the activities of some serine/threonine kinases, tyrosine kinases and phosphatidylinositol 3-kinase in animal cells (Alessi et al., 1995). In addition to inhibition of tyrosine phosphorylation, tyrphostin 63 also showed inhibition of tip growth. At both 0.5 and 1 mM, it reduced the rate over 50%. At 1.5 mM tyrphostin 25 reduced tip growth rates by 30%. Hyphae treated with tyrphostin 63 took a longer time for hyphae to recover after removal of the inhibitor compared with hyphae treated with tyrphostin 25. This suggests that the binding of this inhibitor is reversible. The exact target of tyrphostins in hyphae is still unknown although it may well be proteins that have identified using immunocytochemistry and biochemical assays. Treatment of hyphae with tyrphostins also resulted in a change in hyphal morphology. Both tyrphostins induced sub-apical swelling and subsequently tips formed a blunt shape. Chapter 3: The Presence of Protein Tyrosine kinase activities in A. bisexualis and S. cerevisiae 109 This swelling of sub-apical hyphae possibly indicates delocalised, sub-apical exocytosis, previously reported in hyphae of S. ferax (Bachewich and Heath, 1998),. In Fucoid algae (Corellou et al., 2000) showed the tyrosine kinase inhibitor; herbimycin A inhibited the germination of zygotes. Furthermore, genistein prevented germination but had no effect on growth of germinated zygotes and embryos. However, (Corellou et al., 2000) found that tyrphostin 25 had no inhibitory effect on germination at ranges of 5- 10 ?M, which is a lower concentration than used in this study which may account for the difference in results. Genistein was not used in the study of growth rates because the solubility of genistein required a final DMSO concentration of more than 1 %(v/v). At this concentration it shows toxicity to A. bisexualis. Furthermore, using genistein as an inhibitor in living organisms may be not useful since genistein itself is a phytoestrogen compound in plants (Dixon and Ferreira, 2002). Moreover, at low doses genistein promotes hyphal growth in the oomycete Phytophthora sojae (Connolly et al., 1999). Inhibition by tyrphostins has been reported for the cell cycle progression of S. cerevisiae (Fujimura, 1997). The mechanism by which tyrphostin blocks the yeast cell cycle remains unclear, but it is possible that tyrphostin inhibits a kinase which phosphorylates a protein kinase responsible for cell cycle pathways. At 10 and 50 ?M, tyrphostin 25 completely blocks carrot somatic embryogenesis (Barizza et al., 1999). Taken together, the data in this chapter illustrates that tip growth of A. bisexualis may utilise tyrosine phosphorylation in signalling mechanisms. The perturbation of this pathway may result in the reduction of hyphal polarity establishment. Tyrosine kinase inhibitors significantly reduce tyrosine kinase activity in vitro, suggesting that tyrosine like-kinases may be present in cell fractions derived from A. bisexualis. However, kinase activity by serine/threonine kinases can not be rule out as the latter kinases are found in diverse multicellular organisms. Chapter 4 : The role of actin and actin-binding proteins in tip growth of A. bisexualis 4.1 Introduction Eukaryotic cells have the ability to organise directed movement to migrate, feed, divide or drive the internal transport of material. The direction of movement is mediated in different ways in cells and is associated with dissipation of energy. Cytoskeletal proteins such as microtubules and actin filaments are vital for these functions by converting energy derived from ATP hydrolysis into directed movement. This chapter firstly addresses the essential background of actin. The various functions of actin will be extensively addressed in other organisms such as plants and fungi for comparison with oomycetes. Actin-binding proteins as well as signalling molecules, which are involved in the organisation of actin, will be included. 4.1.1 Actin cytoskeleton Actin is an ubiquitous 43-kDa adenine nucleotide-binding protein involved in a wide range of fundamental processes in eukaryotic cells, including cell motility, cytokinesis, vesicle transport and the establishment and maintenance of cell morphology (Vorobiev et al., 2003). Actin has no enzyme activity, and thus its participation in such cellular processes resides in its ability to provide a dynamic filamentous scaffold upon which many regulatory and motor proteins interact. Actin exists in two different forms of protein structure; a globular or monomeric form (G-actin) and a filamentous form (F- actin). Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 111 4.1.2 Actin structure The crystal structure of actin has been described in a number of organisms including the yeast S. cerevisiae (Vorobiev et al., 2003). Actin is highly conserved among organisms and thus it is likely that in the oomycetes it has a similar structure to that in other organisms. Actin in A. bisexualis contains 376 amino acid residues and has a molecular weight of approximately 43-kDa (Bhattacharya et al., 1992). The sequence identity of actin from A. bisexualis compared to other organisms is shown in Table 4.1. The full comparative protein sequence is shown in appendix 2. Table 4.1. Sequence similarity (%) between different actins from different organisms. Actin P. infestans N. crassa S. cerevisiae Dictyostelium ?-actin of human muscle A. bisexualis 98 % 82% 81% 87% 84% Each G-actin subunit is composed of four subdomains; subdomain 1 (residues 1-32, 70- 144, and 338-375), subdomain 2 (residues 33-69), subdomain 3 (residues 145-180 and 270-337), and subdomain 4 (residues 181-269) (Vorobiev et al., 2003) (Error! Reference source not found..). Consistent with the high level of sequence conservation, the overall structural similarity of A. bisexualis and other organisms is due to small deviations between the less conserved structures. The most variable region of the actin molecule appears to be the DNase I loop (residues 35-53) in subdomain 2. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 112 The DNase I loop is likely to reflect the plasticity of this segment altering conformation in response to nucleotide and polymerisation states (Vorobiev et al., 2003). The four subdomains are primarily held together and stabilised by salt bridges and hydrogen bonds between the phosphate groups of a bound nucleotide (i.e. ATP or ADP) and its associated divalent cation which are localised in the central position of actin molecule. The actin subunit is also distinctly polar, that is subdomain 2 has significantly less and subdomain 1 significantly has more mass than the other two subdomains (Steinmetz et al., 1997). The highest conservation is found in the interior of the protein and in extensive parts of the two lower subdomains 1 and 3. The core appears to be an ancient nucleotide-binding pocket and clearly is informative in elucidating common properties, such as the conformations of open and closed states coupled to interdomain motion around hinge regions or the mechanism of nucleotide hydrolysis (Troys et al., 1999). The formation of F-actin occurs by actin-actin contact between G- actin molecules. The F-actin filament can be described as consisting of either two right- handed helices of a relative long pitch or a single left-handed helix of a shallow pitch. The region contributing to longitudinal F-actin contacts are located in subdomain 3 (residues 166- 169, 286-289, 322-325), subdomain 4 (residues 202-204, 243-245), subdomain 1 (residues 375) and in subdomain 2 (residues 40-45) (Holmes et al., 1990). At the physiological ionic strength, in the presence of ATP, F-actin coexist with G-actin, this G- actin polymerises itself into polar, helical filaments in which subunits are connected by a 167? rotation and 2.7 nm axial rise (Pantaloni et al., 2001). Asymmetric subunits assemble into intrinsically polar F-actin filaments by always adding onto the filament ends in the same orientation with respect to the filament axis (Kim et al., 2000). This filament polarity can be visualised by stoichiometric binding of myosin S-1 to F-actin in vivo, thereby yielding decorated filament with an arrowhead-like appearance (Steinmetz et al., 1997). Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 113 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 114 Using the fluorescence signal of actin pyrenated at Cys374, the time course for polymerising G-actin monomers into F-actin filaments is distinctly sigmoidal. It can be divided into at least three distinct phases. The lag phase, monomer activation and nucleus formation predominate and this process strongly depends on the type of divalent cation (Ca 2+ or Mg 2+ ). After a significant number of nuclei have been formed, rapid subunit addition ensues, and the polymerisation reaction progresses into the elongation phase during which the relationship of total polymer versus time is approximate linear. The extent of filament elongation strongly depends on the exact polymerisation conditions. Finally at the steady state the monomer and polymer concentration remain invariant and the monomer concentration equals the critical concentration for polymerisation (Steinmetz et al., 1997). However, subunit exchange between the polymer and the monomer pools continues by a phenomenon called ?treadmilling?. In the treadmilling cycle of F-actin at steady state (Figure 4.1.), all rates are equal to the rate-limiting step. The cycle comprises disassociation of ADP-actin from what is referred to as the pointed end, ATP exchange for bound ADP on G-actin, followed by association of ATP-G-actin to what is referred to as the barbed ends. The rates of nucleotide exchange, which depend on the concentration of ADP-G-actin and ATP-G- actin can not be the rate-limiting step in the treadmilling cycle. Therefore, the rate- limiting step in the treadmilling cycle is the rate of subunit dissociation from the pointed ends. Hence, factors affecting the rate of depolymerisation from the pointed ends are expected to affect the turnover of actin filaments (Carlier and Pantaloni, 1997). Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 115 Figure 4.1. Treadmilling of actin filaments in the steady state in the presence of ATP (Carlier et al., 2003). Polymerisation Depolymerisation Pi release Pi ATPADP ATP ADP Barbed ends Pointed ends Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 116 4.1.3 Organisation of actin dynamic in living cells The mechanism underlying dynamic changes in actin organisation have been the subject of much interest. A major property of actin is its ability to self-assemble in helical, polar, dynamic filaments. The controlled assembly of actin filaments in response to appropriate stimuli generates cell protrusions called lamellipodia, filopodia and pseudopodia (Carlier et al., 2003). The extension of lamellipodia is considered as the paradigm of so-called ?actin-based motile processes?. Observations of the forward movement of the leading edge of motile cells and of the associated dynamics of actin filaments in the extending lamellipodia has revealed two points of interest (Carlier et al., 2003). Firstly, filaments with their barbed ends pointing toward the leading edge were constantly generated and growing at the plasma membrane during movement, while depolymerising occurs at the rear of the lamellipodium. Lamellipodium extension therefore results from the steady- state cycle of actin assembly, the flux of depolymerised actin subunits being used to feed the growth of barbed ends formed at specific sites at the leading edge, in a rapid treadmilling process (Chen et al., 2000). The treadmilling process occurring in vivo, involves proteins referred to as actin binding proteins which regulate the treadmilling of actin filaments. Some of these actin binding proteins are considered in more depth below. 1. Actin depolymerising factor (ADF)/cofilin (AC) AC proteins are an essential group of actin-binding proteins (18 kDa) that are ubiquitous among eukaryotes (Carlier et al., 2003). Their highly complex regulation allows them to modulate with spatial and temporal precision the turnover of filaments that is needed for many actin based process in non-muscle cells. AC accelerates pointed-end depolymerisation, which is the rate limiting step in the treadmilling cycle. A new steady state of actin assembly is established in the presence of AC, in which a high steady state concentration of monomeric ATP-G-actin supports fast barbed end growth to balance AC induced fast pointed depolymerisation (Pantaloni et al., 2001). Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 117 AC binds to ADP-actin with higher affinity than ADP-Pi-actin or ATP-actin, suggesting that the change in conformation of F-actin that follows Pi release enhances AC binding (Chen et al., 2000). The most important finding that came from electron microscopy studies was the discovery that AC changes the twists of actin filaments when it binds which indicates that the AC family has a unique property (Bamburg et al., 1999). Normally F-actin has a helical twist that results in a crossover every 35 nm (Figure 4.2. top). Actin filaments bound with AC have a shorter actin crossover every 27 nm, although the rise per subunit remains the same (Figure 4.2. bottom). The consequence of this AC induced helical twist of about -5 degree per subunit is weakening of the lateral actin-actin contact in the filament (Bamburg et al., 1999). The AC-induced twist also eliminates the phalloidin-binding site on F-actin so that AC-saturated F-actin does not stain with fluorescent phalloidin (Chen et al., 2000). Although AC binding results in dramatic changes in actin filament structure, at low resolution the conformation of the actin subunit itself appears largely unchanged. All AC proteins show in vitro pH dependence in their depolymerising activity due to an increase in critical concentration for the AC-actin complex as pH increases. This enhanced depolymerisation is most pronounced in vitro between pH 7 and 7.5 (Bamburg et al., 1999). Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 118 Figure 4.2. ADF/Cofillin on actin filament Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 119 ACs from most animals, plants and protists are regulated by phosphorylation on a highly conserved serine residue but this phosphorylation does not appear in yeast and D. discoideum AC (Bamburg et al., 1999). In vertebrates, serine 3 is phosphorylated by Lim kinase. In plants, however, Ca 2+ -stimulated protein kinase activity has been identified that phosphorylates ADF on serine 6, suggesting that plants have evolved a different kinase for ADF regulation (Allwood et al., 2001). 2. Arp2/3 complex The Arp2/3 complex is composed of two Arps (Actin-related proteins), Arp2 and Arp3 and five unique proteins (p40, p35, p21, p18 and p14), all with an apparent stoichiometry of 1:1 with each other (Millard et al., 2004). The Arp2/3 complex has been localised to the junctions of the Y-shaped actin filament branch seen in the cortical actin net work at the leading edge of motile cells (Wear et al., 2000). The Arp2/3 complex nucleates actin polymerisation in vitro resulting in the formation of Y-shaped branches on the sides of existing filaments (Error! Reference source not found..). Immunocolocalisation of Arp2/3 in the lamellipodium, microscopic observation, and polymerisation studies show that when activated by WASP (Wiskott - Aldrich syndrome) proteins, Arp2/3 multiplies the filaments by branching them. Actin filaments form a dendritic Y-branched array in the lamellipodium; daughter branches grow at 70? angle from the mother branch. Arp2/3 is located at the branching points, and the barbed ends of the two branches face the leading edge of the lamellipodium (Pantaloni et al., 2001). Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 120 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 121 3. WASP family proteins WASP is a 502 amino acid proline-rich protein expressed exclusively in haematopoietic cells. An isoform was later isolated from brain and called N-WASP, although it is in fact widely expressed (Millard et al., 2004). There are currently thought to be five proteins belonging to the WASP family: namely WASP, N-WASP, Scar/WAVE1, Scar/WAVE2, and Scar/WAVE3 (Weaver et al., 2003). Conserved features of WASP are the carboxy-terminal effecter domain-commonly referred to as the ?verprolin homology, cofilin homology and acidic? or VCA domain (Wear et al., 2000). The VCA domain binds monomeric actin and this binding, which has been localised to the V-region (verprolin homology), is essential for stimulation of Arp2/3 nucleation activity. The N-terminal of WASP-family proteins is more divergent among family members. Each possesses a proline-rich region adjacent to the WCA domain that acts as a binding site for various SH3 domain-containing proteins (Millard et al., 2004). In the full-length WASP protein, binding of actin and Arp2/3 complex to VCA is prevented due to an auto-inhibited fold of WASP. Binding of PIP2, Cdc42-GTP and Grb2 to their respective targets on WASP promotes a structural change of WASP, exposing the VCA domain for association with G-actin and Arp2/3 complex (Carlier et al., 2003). 4. Profilin Profilin specifically interacts with ATP-G-actin. A thus formed profilin-actin complex productively associates with barbed ends exclusively (Carlier et al., 2003). Profilin has been shown to enhance the extent of Cdc42-induced Arp2/3 complex nucleation, both in neutrophil extracts and in vitro with purified proteins (Yang et al., 2000). To be most effective, profilin had to simultaneously bind poly-L-proline and monomeric actin. Profilin binding to Cdc42/PIP 2 -activated WASP may result in complete activation by further relieving the inhibitory interaction between the VAC domain and the amino terminus (Wear et al., 2000). Furthermore, the close proximity of this profilin-bound actin subunit to the site of the Arp2/3-WASP interaction may contribute to the nucleation of actin polymerisation. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 122 4.1.4 Actin and components of focal adhesion proteins In animal cells, large number of proteins found in focal adhesions and cell junction sites and these proteins associate with F-actin, suggesting that these proteins may act at different stages of cell adhesion and junction assembly. For example talin and vinculin may be involved in focal adhesion formation, whereas ?-actinin may be more important in maintaining or stabilising microfilament attachment in mature focal adhesions. Talin is a 235-kDa protein of focal adhesions, which can be cleaved into a 47-kDa N- terminal globular head domain and a 190-kDa rod domain. Talin functions by interacting with particular structural and regulatory proteins of focal adhesions. The N-terminal domain has been shown to bind to focal adhesion kinases, polyphosphoinositides and integrins. Likewise, the C-terminal domain binds also to the integrin cytoplasmic domain and, in addition, to vinculin and to actin filaments. At pH 6.4 and low ionic strength talin could extensively crosslink actin filament into ordered bundles (Schmidt et al., 1999). The absence of talin and vinculin protein sequences in Arabidopsis may suggest that plants, possibly fungi and oomycete do not required these proteins for maintaining cell shape. 4.1.5 Actin and cell polarity In S. cerevisiae, actin is absolutely required for polarised morphogenesis. During bud growth, actin cables extend from the mother cell into the bud and actin patches localise to growth sites on the bud surface. In yeast, two distinct multi-protein complexes, the polarisome and the Arp2/3 complex function down stream of the Cdc42 GTPase module to direct the localised assembly of actin filaments at polarisation sites. The polarisome regulates the formation of linear, unbranched actin filaments. The key component of polarisome is the formin Bni1p, which binds to the barbed ends of actin filaments and nucleates microfilament assembly. In yeast, the regulation of actin patch formation is driven by the WASP homologue Las17p/Bee1p, Arp2/3 and the class I myosin Myo3p (Harris and Momany, 2004). The polarisome seems likely to direct the formation of actin cable at polar growth sites in filamentous fungi as well. Most Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 123 polarisome components as well as Arp2/3 proteins are conserved in filamentous fungi (Palmieri and Haarer, 1998). Taken together, these observations suggest that signals emanating from Cdc42 GTPase modules trigger local organisation of actin filaments via similar mechanism in filamentous fungi and yeast. 4.1.6 Actin and tip growth Actin is thought to play a critical role in regulating tip growth. Tip morphogenesis involves the control of tip expansion and localisation of exocytosis of vesicles which contribute both the plasma membrane and cell wall precursors. Tip growth is thought to be driven by the coordination of turgor pressure and a gradient of regulated yielding the cell wall that generates the hyphal shape. However, tip expansion in oomycetes such as A. bisexualis and S. ferax may be more complicated. Oomycetes fail to regulate turgor pressure and can grow in the absence of measurable turgor. This suggests that a protrusive force other than turgor pressure may be present under certain conditions. This force has been suggested to be similar to that of amoeboid pseudopodia protrusion (as described above) and thus based on actin (Heath and Steinberg, 1999). In oomycetes, the organisation of the apical F-actin correlates with apical extensibility, being most concentrated at the extreme tip where the wall is weakest, F- actin patches are prominent in the sub-apical zones. However, this arrangement may not apply to all hyphae as an actin depleted zone has recently been described in the apical region of A. bisexualis (Yu et al., 2004). A similar actin depleted region has also shown in pollen tubes (Vidali and Hepler, 2001) A role for actin in tip growth is suggested as the actin distribution is growing and non- growing tips displays different pattern. Furthermore a new tip formation in branches and spore germination is characterised by the accumulation of apical actin (Bachewich and Heath, 1998). In yeast, cortical actin patches may be involved in several processes in the cell. Cortical patches are though to be cytoplasmic actin cable anchoring points to the cell membrane (Doyle and Botstein, 1996). In polarised, budding yeast, actin patches are localised to sites of polarised cell surface and actin cables are aligned along the mother- bud axis (Boldogh et al., 2001) Also supporting a role in tip growth is the observation Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 124 that disruption of actin with cytochalasins or latrunculins leads to ballooning of tips in pollen tube, oomycete, and fungi (Gupta and Heath, 1997; Gibbon et al., 1999). In this chapter a possible role for the proteins studied in the previous two chapters in directing actin dynamics was investigated. Tyrphostin was found to affect F-actin organisation in a manner that could not be simply accounted for by a slower growth rate. A study was also made looking for the presence of the actin binding protein, talin which is a component of adhesions in animal cells. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 125 4.2 Materials and Methods A. bisexualis was cultured as described in previous chapters. Alexa 488 phalloidin was purchased from Molecular Probes. Monoclonal mouse IgG1 anti-chicken gizzard talin was purchased from Sigma. Latrunculin B was purchased from Biomol, Research Labs, Inc. 4.2.1 The effect of tyrphostins and TPA on actin staining Hyphae from a growing culture of A. bisexualis were cut 1 cm behind their growing tips and allowed to recover for 60 mins in liquid PYG. To investigate the effect of tyrosine kinase inhibitors and tetrapentyl ammonium chloride (TPA) on F-actin, hyphae were incubated with tyrphostin 63 or TPA for 20 mins. Hyphae were then fixed for 30 mins in 50 mM PIPES buffer pH 6.8 containing 4% formaldehyde and 0.5 % methylglyoxal and then rinsed twice in 50 mM PIPES buffer pH 6.8. Fixed hyphae were stained for actin with Alexa 488 Phalloidin for 30 mins at room temperature and washed twice with buffer for 15 mins each time. Prior to microscopic examination, 0.1 % w/v p-phenylenediamine was added to all samples for preventing photo bleaching. Samples were examined immediately after staining using an MRC1024 confocal microscope (Bio-Rad, Mississauga, Ontario). 4.2.2 Immunocytochemistry of talin-like protein in A. bisexualis A. bisexualis: Five day old mycelial sheets were excised and allowed to grow in PYG media for 60 mins. To investigate the effect of latrunculin B (Lat B) on talin-like protein, some mycelial sheets were incubated with Lat B for 20 mins. Mycelial sheets were fixed in 4% formaldehyde, 0.5 % methylglyoxal in saline buffer containing 60 mM PIPES pH 6.8, 2 mM EGTA, 2 mM MgCl 2 , 137 mM NaCl, and 0.268 mM KCl for 45 mins at room temperature. Fixed mycelial sheets were rinsed for 5 times for 5 mins with the washing solution (0.05% v/v Tween-20 in saline) then the cell wall was digested for 15 mins in 10 mg/ml Driselase (Sigma) and membranes were permeabilised in 0.1% (v/v) Triton X-100 (Biorad) for 20 mins. The mycelial sheets were again rinsed in the washing solution 2x5 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 126 mins and blocked for 30-45 mins in blocking solution containing 2.5 % (w/v) skim milk powder and 5% (w/v) BSA (Sigma) in saline and washed 4x5 mins after blocking. The mycelial sheets were incubated overnight with a 1:100 dilution of mouse monoclonal IgG 1 antibody raised against chicken gizzard talin, at 4 ?C. The mycelial sheets were rinsed 5x5 mins and incubated with a 1:1000 dilution of the secondary antibody, Alexa TM 488 goat anti-mouse IgG (H+L) conjugate. The mycelial sheets were then washed extensively with the washing solution before addition of anti-fading solution and visualised by epifluorescence microscopy (Bio-Rad, Mississauga, Ontario). 4.2.3 Immunoblotting of talin-like protein in A. bisexualis and S. cerevisiae A. bisexualis: Forty plates of A. bisexualis grown on PYG overlaid with cellophane were cut 1 cm from the margin of mycelia and frozen with liquid nitrogen, then ground. The powder was then re-suspended in buffer containing 50 mM Tris-HCl pH 6.2, 10 mM NaF, 7 mM EDTA, 2 mM DTT, 20 % glycerol, 7 mM ?-mercapthoethanol, 2 mM PMSF, 250 mM sucrose and centrifuged at 10,000 g for 45 mins. The cytosolic fraction was then precipitated on ice by the addition of trichloroacetic acid (TCA) to a final concentration 10% for 30 mins. After centrifugation the precipitates were mixed with 5x Laemmli buffer, they were heated for 5 mins in a boiling water bath. Proteins were separated on a 5% stacking gel and 12% separating polyacrylamide gel. After SDS/PAGE, proteins were transferred to nitrocellulose membrane in a buffer containing 15.6 mM Tris-Base and 120 mM glycine and the transfer ran at 30 V at 4 ?C overnight. Transferred proteins were stained with 0.1% w/v Ponceau red to check homogeneity of the transfer. The nitrocellulose blots were then blocked in 10 mM PBS containing 5 % BSA and 0.05 % Tween-20 for 2 hours at room temperature then washed with Tris- Buffer saline (TBS) 2 x 20 mins. The nitrocellulose membrane was incubated 4 ?C overnight with a 1:250 dilution of mouse monoclonal IgG 1 antibody raised against chicken gizzard talin which was used as primary antibody. The membrane was visualised by Western Breeze. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 127 S. cerevisiae: Yeast cells (strain SY1129(F131)) were streaked onto a YPD agar plate and incubated overnight at 30 ?C, an isolated colony was selected and cultured in 10 ml YPD media overnight at 30 ?C. Then, 10 ml of the overnight culture was poured into 1 L YPD media and further incubated at 30 ?C overnight on an orbital shaker. Cells were centrifuged at 5,000 g for 30 mins and the pellet was washed twice with ice-cold 40 mM PBS pH 7.0. The pellet was then re-suspended with buffer as described for A. bisexualis and vortexed with pre-chilled ice cold glass beads for 20 mins (1 min vortex and 1 min on ice) and further sonicated for 30 mins on ice. Lysed cells were then centrifuged at 10,000 g for 30 mins. Electrophoresis and immunoblotting was carried out as similar for A. bisexualis. 4.2.4 Co-localisation of actin and talin-like protein in A. bisexualis and S. cerevisiae A. bisexualis: Prior to experiments, Five day old mycelial sheets were excised and allowed to grow in PYG media for 60 mins. Mycelial sheets were fixed in 4% formaldehyde, 0.5 % methylglyoxal in saline buffer containing 60 mM PIPES pH 6.8, 2 mM EGTA, 2 mM MgCl 2 , 137 mM NaCl, and 0.268 mM KCl for 45 mins at room temperature. Fixed mycelial sheets were rinsed for 5x 5 mins with the washing solution ( 0.05% v/v Tween-20 in saline) then the cell wall was digested for 15 mins in 10 mg/ml Driselase (Sigma) and membranes were permeabilised in 0.1% (v/v) Triton X-100 (Biorad) for 20 mins. The mycelial sheets were again rinsed in the washing solution 2 times for 5 mins and blocked for 30-45 mins in blocking solution containing 2.5 % (w/v) skim milk powder and 5% (w/v) BSA (Sigma) in saline and washed 4 times for 5 mins after blocking. The mycelial sheets were incubated overnight with a 1:100 dilution of primary antibodies mouse monoclonal IgG 1 antibody raised against chicken gizzard talin, at 4 ?C. The mycelial sheets were rinsed 5 times for 5 mins and incubated with a 1:1000 dilution of the secondary antibody, Alexa TM 488 goat anti-mouse IgG (H+L) conjugate. After washing twice, they were then incubated with Texas Red- Phalloidin for 45 mins at room temperature in the dark. Hyphae were then washed extensively with washing solution. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 128 Before examination, anti-fading solution (containing 0.1 % (w/v) of p- phenylenediamine dihydrochloride dissolved in nanopure water) was added to prevent photo bleaching. S. cerevisiae: Yeast cells were cultured as previously described. Yeast cells were fixed in fixing solution as previously described for A. bisexualis. After washing with saline, fixed cells were incubated in KS solution (containing 100 mM KPO 4 buffer pH 7.0, 1.2 M sorbitol) at 4?C overnight. The cell wall was then digested with 10 mg/ml Driselase in KS solution containing 0.5 % ?-mercapthoethanol for 30 mins at room temperature. Cells were centrifuged and re-suspended in 0.5 % v/v Triton-X in 100 mM KPO 4 buffer pH 7.0 and incubated at room temperature for 20 mins. Cells were washed and fixed to slides at 45?C for 30 mins. The slide was then washed 3 times and blocked with blocking solution ( containing 1% w/v BSA, 0.05 % v/v Tween-20 and 40 mM PBS pH 7.0) for 45 mins at room temperature, then slides were washed 5 time for 5 mins and incubated with primary antibodies and secondary antibodies as previously described. Anti-fading solution was added prior to visualisation under the microscope. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 129 4.3 Results 4.3.1 The effect of tyrphostins on the F-actin in A. bisexualis To assess whether inhibition of tyrosine phosphorylation affects the F-actin cytoskeleton, hyphae were incubated with tyrphostin 63 for 20 mins, fixed in a formaldehyde/methylglyoxal combination fixative and stained with Alexa phalloidin. Growing hyphae that had not been exposed to tyrphostin showed an apical actin cap with or without an actin depleted zone (Yu et al., 2004) and a sub-apical array of peripheral plaques and diffuse filaments (Error! Reference source not found.. A). Hyphae that had been grown in the presence of 0.5 and 1 mM tyrphostin 63 showed a significant difference in actin organisation. In these hyphae, the apical cap was less predominant and the plaques moved to a position that was much closer to the tip. The transition in organisation from filamentous actin to plaques moving away from the tip was noticeably sharper. The transition effect appeared to be dose-dependent with a sharper transition with 1 mM tyrphostin 63 (Error! Reference source not found.. C) compared to 0.5 mM tyrphostin 63 (Error! Reference source not found.. B). Hyphae that had been exposed to tyrphostin and then returned to normal growth media prior to fixation showed the F-actin pattern typical of growing hyphae (Error! Reference source not found.. D). To assess whether the observed effects of tyrphostin 63 on the cytoskeleton was not simply a consequence of slower growth rates, hyphae were treated with tetrapentyl ammonium chloride (TPA). TPA is a K + channel blocker that has previously been used to slow growth rates in the oomycete S. ferax (Kaminskyj et al., 1992). For this comparison TPA was added at a concentration of 2 mM that slowed tip growth the same amount as 1 mM tyrphostin 63 (growth at these concentrations was 25% of that control hyphae). Both TPA and tyrphostin 63 shortened the F-actin cap and decreased the intensity of staining at the tips of hyphae (Error! Reference source not found.. A-C). In a representative experiment the measured distance (mean ? SD) from the tips to the first actin plaques were 41 ? 5 ?m (control, n= 5 hyphae), 15 ? 5 ?m (tyrphostin 63, n= 6 hyphae), and 17 ? 5 ?m (TPA, n= 8 hyphae). The differences between the tyrphostin 63 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 130 and TPA-treated hyphae and the control hyphae were statistically significant (P<0.05; t test). There was no significant difference between tyrphostin 63 and TPA-treated hyphae (P>0.05; t test). With tyrphostin 63, however, the transition from the cap to plaques was much sharper than in TPA-treated hyphae. Hyphal morphology was also affected by the compounds, TPA gave tips with a bulbous shape (Error! Reference source not found.. B), while tyrphostin 63 eliminated the long taper (Error! Reference source not found.. C) that is characteristic of A. bisexualis (Error! Reference source not found.. A). 4.3.2 Immunocytochemistry of talin-like protein in A. bisexualis hyphae To assess the presence of the ABP talin in A. bisexualis hyphae were treated with an anti-talin antibody. The latter protein is a component of focal adhesions and acts by stabilising ECM-integrin-actin links and promoting signalling downstream of active integrins. Staining of talin-like protein was mainly distributed in sub-apical regions (Error! Reference source not found.. D) with less staining in the apical region of hyphae (Error! Reference source not found.. B). By counting each patch of immunofluorescence signal of the putative talin-like proteins in hyphae, from 0-10 ?m behind the tip there was approximately 4.75 ? 1.75 patches (n=20); or 19 % of total fluorescence signal and from 10-40 ?m behind the tip approximately 20.8 ? 3.9 patches (n=20); or 81 % of total fluorescence signal. The distribution of immunofluorescence signals of talin-like proteins in apical and sub-apical region are significantly different (P< 0.05; t test). Moreover, staining does not appear specifically to be localised at the peripheral region of hyphae. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 131 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 132 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 133 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 134 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 135 To further investigate whether the disruption of F-actin by Lat B affects the distribution of the talin-like protein staining in hyphae of A. bisexualis, hyphae were treated with 1?M of Lat B for 20 mins prior to fixation and then stained with the anti- talin antibody. Hyphae treated with Lat B showed swollen tips (Error! Reference source not found.. A), the distribution of talin-like protein staining was mainly located in the peripheral region of hyphae and the majority of immunofluorescence signal was located mainly (20.25 ? 6.8 (n=8); 64 %) in sub-apical regions (Error! Reference source not found.. B). The distribution of immunofluorescence signal patches of talin-like protein in hyphae treated with Lat B, 0-10 ?m from the tip was approximately 11 ? 4.75 (n=8); or 36 % of fluorescence signal. The number of stained patches was significantly increased (P<0.05; t test) in the apical region in comparison with non-treated hyphae However, there was no significantly difference (P>0.05; t test) in the distribution of talin-like protein in the region 10-40 ?m from the tip in non-treated versus treated hyphae. The presented confocal image shows staining of talin-like proteins are mainly located in the peripheral region (Error! Reference source not found.. D). 4.3.3 Immunoblotting of talin-like protein in A. bisexualis hyphae In an attempt to identify the proteins responsible for the staining in hyphae, immunoblotting on mycelial extracts was carried out using the same antibody as above. There are three bands, with approximate molecular weights of 49, 42, and 35 kDa that were present in the supernatant at 10,000 g (Error! Reference source not found.. lane A). For comparative purposes, immunoblotting was also carried out on S. cerevisiae extracts. Only one very weak band with a molecular weight of about 37 kDa was present (Error! Reference source not found.. lane B). Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 136 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 137 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 138 4.3.4 Co-localisation of talin-like protein and F-actin in A. bisexualis hyphae and S. cerevisiae To determine whether this talin-like protein is associated with F-actin in hyphae of A. bisexualis, hyphae were co-stained with the anti-talin antibody and Texas Red phalloidin. Total numbers of Immunofluorescent patches of talin-like protein and the numbers of talin-like protein patches that co-localised with F-actin were counted from hyphal tips to a distance 40 ?m back from the tips. Numbers of talin-like protein patches not associated with F-actin were calculated. The talin-like protein in hyphae of A. bisexualis does not appear to co-localise with F-actin. Numbers of total talin-like protein and unassociated talin-like protein were not significantly different (P>0.05; t test, Table 4.2.). As previously suggested, the talin-like protein was more predominant in the sub-apical region of A. bisexualis (Error! Reference source not found..). For comparative purposes, an investigation of the talin-like protein was carried out in S. cerevisiae (Error! Reference source not found..). Immunofluorescence of talin-like protein was distributed throughout yeast cells. The total numbers of immunofluorescence patches of talin-like protein were counted from 20 yeast cells, which are given approximately 7.45 ? 3.3 or 100 % of distribution of fluorescence patches (Table 4.3.). The number of those that were not associated with F-actin was approximately 6.05 ? 2.8 or 81%. The total number of talin-like protein was not significantly (P>0.05; t test) different from those of unbound talin-like protein. This data suggests that as in A. bisexualis the talin-like protein in yeast also does not co-localise with F-actin. Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 139 Table 4.2. The distribution of co-localised talin-like proteins with F-actin in hyphae of A. bisexualis. Total number of fluorescence spots of talin-like protein and spots which were co-localised with actin were counted. The number of unbound talin-like proteins were obtained by subtracting the numbers of fluorescence spots of bound talin-like protein with F-actin from the total number of fluorescence spots of talin-like protein. The region from the tip to 40 ?m behind tip was examined. Total fluorescence patches of talin-like protein 19 ? 5.2 (n=15 hyphae) 100 % Fluorescence patches of unbound of talin-like protein with F-actin 18.2 ? 5.3 (n=15 hyphae) 95% Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 140 Table 4.3. The distribution of immunofluorescence of talin-like proteins in S. cerevisiae. Numbers represent the immunofluorescence patches of talin-like proteins in yeast cells. Total fluorescence patches of talin-like protein 7.45 ? 3.3 (n= 20 cells) 100% Fluorescence patches of unbound of talin-like protein with F-actin 6.05 ? 2.8 (n= 20 cells) 81% Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 141 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 142 Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 143 4.4 Discussion The actin cytoskeleton has been implicated in the development and maintenance of polarised growth as disruption of actin and actin associated molecules result in loss of cell development. In yeast, three essential elements are required for polarised growth: cortical tag, a set of proteins that localises to the cell pole, believed to mark sites of polarised cell growth; GTPases, which are though to direct cytoskeletal regulatory components to growth sites; and the cytoskeleton, which appear to direct secretary vesicles to the growth site (Casamayor and Snyder, 2002). In oomycete hyphal tips however, such elements are still largely unknown but there are several lines of evidence that indicate that actin plays a central role in tip growth. Oomycete hyphal tips contain two populations of F-actin with different organisation. The cortical population consists of a fibrillar cap in the apex, and cables and plaques sub-apically, while the central population is diffuse and less organised (Jackson and Heath, 1993a) The presented results show that the organisation of F-actin in hyphae of A. bisexualis was disturbed by tyrphostin 63 which also results in a reduction of tip growth. In hyphae treated with the inhibitor, the transition between the F-actin cap and sub-apical plaques was diminished with actin plaques locating close to the tips of hyphae. The tyrphostin 63 affect on F-actin was dose-dependent and reversible. Tyrphostin 63 does not appear to affect actin in the same way as latrunculin B, an actin polymerising agent (Error! Reference source not found..). Latrunculin disruption of actin causes tips to balloon with the ballooning extending sub-apically in the oomycete S. ferax (Gupta and Heath, 1997). While the exact inhibitory target of tyrphostin 63 A. bisexualis is unknown, previous chapters suggest a kinase capable of phosphorylating tyrosine residues. This may affect F-actin via actin binding proteins. Several actin binding proteins (ABPs) involved in actin polymerisation are regulated by phosphorylation. There is as yet no report of actin binding proteins in A. bisexualis. However, both P. infestans and P. sojae have actin depolymerising factor and Arp2/3, which is analogous to those found in Lilium longiflorum, Arabidopsis thaliana and Zea mays (http://www.pfgd.org). This may suggest that tyrphostin may affect such processes via ABPs (Chen et al., 2000; Foissner et al., 2002). Alternatively, tyrphostin may block tyrosine phosphorylation of F-actin itself. Tyrosine phosphorylation of F-actin has been Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 144 found in leaflet movement in Mimosa (Kameyama et al., 2000). Interestingly, a single tyrosine residue at position 53 in D. discoideum (equivalent to position 54 of A. bisexualis), becomes tyrosine phosphorylated in amoeboid actin and phosphorylation of this site contributes to the binding of DNase to actin (Jungbluth et al., 1995). Furthermore, a change of the tyrosine phosphorylation state of actin results in the cell- shape changes in D. discoideum amoeboid (Gauthier et al., 1997). Also, tyrphostin may affect protein kinases and affect the phosphorylation state of myosin or of actin-cross linking proteins, which regulate tension within actin cytoskeleton (Grabski et al., 1998). Other tyrosine kinase inhibitors such as genistein have been shown to disrupt actin organisation and axis formation in Pelvetia zygotes (Corellou et al., 2000). Disruption of F-actin in A. bisexualis by tyrphostin 63 and TPA gave different tip morphologies and this may suggest that the inhibitors are likely to affect different targets. Hyphae treated with TPA showed a bulbous shape while those treated with tyrphostin 63 give a blunt shape. TPA was used as an inhibitor to block K + channels and reduce the growth rate of oomycete S. ferax (Garrill et al., 1993). The K + channels are inhibited by TPA, which inhibits tip extension transiently. Because the K + movement is into the cell, the K + permeable channels may function in maintaining intracellular osmolarity during the volume increase due to tip extension (Lew, 1998). These data suggest that the cytoskeletal rearrangements that occur upon exposure to tyrphostin are unlikely to arise simply because of a slower growth rate. In animal cells, tyrphostins have a strong affect on actin organisation. Specific tyrphostins selectively disrupt either cell-cell junctions or cell-substrate attachment and many disrupt both. Disruption of elements by tyrphostins suggests that a certain steady- state level of tyrosine phosphorylation is required for the maintenance of these structures within the cell (Farooki et al., 1998). In animal cells, structural components of focal adhesion and cell junctions are composed of several anchoring proteins that include vinculin, ?-actinin, ?-catenin, ?-catenin, and talin (Brunton et al., 2004). The latter protein is a component of focal adhesions and acts by stabilising ECM-integrin-actin links and promoting signalling downstream of active integrins. Nevertheless, there is currently no evidence for the existence of talin-like proteins in plants, fungi and oomycetes. In fact protein sequences for talin is absent in the Arabidopsis genome Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 145 database (Hussey et al., 2002). In spite of this, this study also looked for the presence of talin or talin-like protein(s) in hyphae of A. bisexualis and any association with F-actin. The immunocytochemistry and immunoblotting results suggest that A. bisexualis has proteins that are antigenically related to talin. The distribution of this talin-like protein is predominantly in the sub-apical region (10-40 ?m from the tip). However, these proteins do not co-localise with F-actin. The monoclonal anti-talin antibody is raised against an epitope located on the 190 kDa fragment talin of chicken gizzard, which corresponds to an antigen with a molecular weight of approximately 38 kDa (Otey et al., 1990). Nonetheless, this antibody does not bind to 47 kDa fragment of talin. Immunoblotting strongly shows three polypeptide bands at molecular weight 35, 42, and 49 kDa are antigenically bound to the anti-talin antibody. Interestingly, these polypeptides present in A. bisexualis are much close to the 38 kDa antigen used to raise the antibody. S. cerevisiae also contains talin-like protein staining throughout the cells. However, immunoblotting showed only a weak peptide band, in contrast with those from A. bisexualis, with a molecular weight of approximately 37 kDa. The polypeptides found in A. bisexualis and S. cerevisiae have molecular weights somewhat smaller than that of intact talin and this may be proteins with epitopic similarity to talin rather than being talin itself. Although, there is no evidence to support the presence of actual talin in oomycetes, at least one protein found in S. cerevisiae shares a conserved sequence with talin in animal cells. The best characterised actin-binding site in animal talin ABS3 (residues 2345- 2541) is located close to the C-terminal region and is highly conserved across species. It contains a so- called I/LWEQ module that is found in actin-binding proteins such as Sla2p in S. cerevisiae (Lee et al., 2004). This C-terminal conserved sequence tagged with GFP has recently been useful as a molecular tool for studying F-actin in plants (Ketelaar et al., 2004). However, there is no protein sequence having similarity to Sla2p existence in the P. infestans and P. sojae protein databases. The above raises the question of whether tip growing cells require anchoring proteins to maintain cell morphology and cell development. In the plant cell membrane, an enrichment of heterotrimeric G-protein, along with several unidentified anchored proteins Chapter 4: The role of actin and actin-binding proteins in tip growth of A. bisexualis 146 as well as arabinogalactan proteins (AGPs) have been found (Wasteneys and Galway, 2003). In oomycetes, the cortical F-actin is believed to be anchored to the plasma membrane and cell wall (Kaminskyj and Heath, 1995; Bachewich and Heath, 1997). However, the relative ease with which the apical cortical F-actin contracted back to form aligned bundles in the first 7 ?m of the hyphae upon plasmolysis suggest that this plasma membrane associated F-actin may not be tightly anchored through to the wall. On the other hand, the central F-actin may be more strongly anchored, via the plasma membrane, to the wall in the apex. The tightly anchored actin in the very apex could also assist in restricting growth-related components to the extreme tip (Bachewich and Heath, 1997). The presence of talin-like protein in A. bisexualis is mainly in the sub-apical region, where cables and plaques of F-actin predominate. Actin plaques present in the sub-apical region associated with the plasma membrane may be required for maintaining hyphal shape of the sub-apical region while tip growth take place at the apex of hyphae. It seems likely that talin-like proteins in A. bisexualis may be mediating linking between actin plaques and the plasma membrane, however, this is contradictory with results that talin- like proteins do not co-localise with F-actin in the sub-apical region. The disruption of F- actin by latrunculin B also disrupts the localisation of talin-like protein in hyphae. There is more staining of talin-like protein present at the apex of hyphae, this would suggest that talin-like proteins are presence in the cytoplasm and this also may suggest that the distribution of talin-like protein may require actin. Taken together, this chapter suggests that the tyrosine kinase inhibitor tyrphostin affects F-actin organisation that subsequently leads to slow tip growth of A. bisexualis. Also there are proteins epitopic similarities to animal talin present in A. bisexualis. These proteins however do not associate with F-actin. Chapter 5 : Conclusions Tip growth in fungal and oomycete hyphae and pollen tubes and root hairs of plants requires the localised synthesis and expansion of the apical plasma membrane (PM) and cell wall. The complexity of tip growth suggests a high degree of regulation that is likely to arise via bidirectional interplay between the cell wall and cytoplasm, although the exact mechanism by which such communication might occur is unknown. However, there have been suggestions that proteins with functional similarity to the integrins of the metazoa might be involved in this process. The data presented suggests that proteins with epitopic similarity to the human ?4 integrin subunit are present at the tips of hyphae of the oomycete Achlya bisexualis. In view of their presence at wall-membrane attachment sites, these are referred to these as integrin-like proteins rather than integrin-cross-reactive proteins. Immunoblotting identified two proteins that may be responsible for this staining, a membrane associated protein of 85 kDa and a cytosolic protein of 65 kDa. These proteins co- localised with F- actin and with anti-phosphotyrosine antibodies, a finding that would be consistent with the integrin-like proteins functioning in a signaling pathway, between the cell wall and cytoplasm, which involves tyrosine phosphorylation. Inhibition of phosphorylation with tyrphostins slows tip growth suggesting that such a pathway may be important for tip growth. In the animal literature the ?4 subunit has aroused interest because of its roles in cell adhesion in hemidesmosomes, in the stabilisation of protrusions that are rich in actin, in the mediation of force necessary for cell movement and in signaling mechanisms that stimulate cell migration and invasion (Mercurio and Rabinovitz, 2001). Chapter 5: Conclusions 148 These processes and their underlying molecules and mechanisms may be relevant to tip growth as one model of this process describes the apical cytoplasm as being protruded like a pseudopodium (Heath and Steinberg, 1999). The description of a molecule that is antigenically related to the ?4 subunit is consistent with such a model and is especially pertinent in a growing walled cell, in that such a molecule may be capable of providing structural support and providing a means for cell migration. The presence of ?4 integrin- like molecules is not unprecedented in non-metazoan cells as there have been suggestions of such proteins in prokaryotes (May and Ponting, 1999). In animals, the ?4 subunit differs from other integrins in that, in its adhesive capacity in hemidesmosomes, it binds to laminin and intermediate filaments. These connections can change however, as cell migration is possible irrespective of any specific matrix environment (O'Connor et al., 1998) and there have been reports of linkages with the actin cytoskeleton (Rabinovitz and Mercurio, 1997). Thus, the demonstration of a ?4- integrin like protein that is associated with F-actin is not without precedent. The ?4 integrin is also structurally distinct from other integrins with a much larger cytoplasmic domain of about 1000 amino acid residues compared to the 40-50 residues of other subunits (Hynes, 2002). The antibody used in the present study was a monoclonal that was raised against the human ?4 subunit. In the absence of sequence data for A. bisexualis, by comparing the nucleotide sequence of the human ?4 integrin with the published genome of the related oomycete Phytophthora sojae (http://www.pfgd.org), a short sequence of 30 nucleotides was found over which there is 97% identity. While it is unsure if this sequence represents the epitopic site of the ?4 integrin-like protein, it is interesting to note that similarly sized sequences of ten amino acids, that have high identity to the human ?1 integrin, have been found in a number of cells reported to be devoid of true integrins (Nagpal and Quatrano, 1999). In other tip growing cells, integrin-like proteins have been located predominantly at the cell apex (Kaminskyj and Heath, 1995; Sun et al., 2000; Degousee et al., 2002). It should be noted that these are referred to as integrins in some studies but due to the genome data discussed elsewhere in this thesis this term is regarded as somewhat presumptuous. Immunoblots indicate a range of sizes for the proteins responsible for this staining. Chapter 5: Conclusions 149 Kaminskyj and Heath, (1995) using an anti-?1 serum identified a 178 kDa protein from the oomycete Saprolegnia ferax, that under reducing conditions gave a band of 120 kDa. ?1 integrin-like proteins have also been identified in immunoblots of cell extracts from fungi such as Neurospora crassa (Degousee et al., 2002), Uromyces appendiculatus (Correa et al., 1996) and Candida albicans (Marcantonio and Hynes, 1988) and from a range of plant cells including Arabidopsis thaliana (Faik et al., 1998), onion (Gens et al., 1996) and tobacco (Lynch et al., 1998). These proteins range in size from 60- kDa to 110- kDa. Integrin-like proteins do not appear to be restricted to those with epitopic similarity to the ?1subunit, however, as an 84-kDa integrin ?5-like protein has been reported in Candida albicans (Santoni et al., 1998) and in plants, a 150- kDa protein has been detected in lily and Hemerocallis citrina pollen tube extracts using anti-?3 and anti- ?5 serum (Sun et al., 2000). The observation that ?4 integrin-like proteins are located at the tip in some hyphae, but are more common sub-apically in others, presents an apparent paradox if, as it is proposed, these proteins are required for growth. While all attempts were made to ensure that hyphae were growing prior to fixation, the individual hyphae within a hyphal mat will typically grow at different rates. One possible explanation for the different staining patterns could therefore be that the distribution is dependant upon growth rate. The lack of any obvious morphological differences between the two populations of hyphae, however, would argue against this, as faster growing hyphae typically have a longer taper. An alternate explanation could be that certain hyphae take slightly longer to fix and thus are prone to changes in the distribution of their proteins, although again this would likely be accompanied by a morphological change. A third possibility is that these represent areas and hyphae where branching is about to occur. Clearly, this area warrants further investigation with a definitive description of the location of these proteins unlikely until they are able to be examined in the living cell. In animal cells, integrins are capable of bidirectional signaling (Hynes, 2002). Thus, the cell is able to control ligand-integrin interactions at the extra-cellular matrix, and conversely, the ligation of integrins can affect a variety of transduction pathways. When the ?6?4 integrin dimer binds to the matrix the cytoplasmic domain of the ?4 subunit Chapter 5: Conclusions 150 becomes phosphorylated at both serine and tyrosine residues (Dans et al., 2001). Tyrosine phosphorylation occurs via a Src family kinase. This induces the recruitment of an adaptor protein Shc that upon phosphorylation activates mitogen- activated protein kinase cascades and hence cell migration. The demonstration of the co-localisation of a ?4- integrin like protein and phosphotyrosine residues is consistent with a similar mechanism operating in an oomycete. However, there are no reports of autophosphorylation of the ?4 integrin, so the demonstration of autophosphorylation may be unique to the oomycete ?4 integrin-like protein. It is also possible, however, that an associated kinase of similar molecular weight to the ?4 integrin-like protein was present in the immunoblots and was responsible for the additional phosphorylation in the presence of ATP. Many cell signaling responses occur via integrin-induced autophosphorylation of integrin associated proteins (Westhoff et al., 2004). The prevalence of tyrosine phosphorylation in oomycetes, plants and fungi is still an area of some controversy due to the absence of typical tyrosine kinase genes, or indeed any homologues of these in the genomes that have been sequenced. Despite this, there are an increasing number of reports of phosphorylated tyrosine residues and indeed tyrosine kinase activities in these organisms. These include pea (Torruella et al., 1985), Saccharomyces cerevisiae (Dailey et al., 1990), alfalfa (Duerr et al., 1993), tobacco (Zhang et al., 1996), maize (Trojanek et al., 1996), coconut (Islas-Flores et al., 1998), Mimosa (Kameyama et al., 2000), Fucus (Corellou et al., 2000) and Candida albicans (Santoni et al., 2002). This latter study is of interest in that the phosphorylated protein, CaFAK, was antigenically related to the focal adhesion kinases of vertebrates that functions in integrin-mediated signalling. In C. albicans ?v?3 and ?v?5 integrin-like proteins, upon binding to vitronectin, stimulate tyrosine phosphorylation of CaFAK (Santoni et al., 2002). Immunoblots of A. bisexualis extracts identified a number of proteins that contained phosphotyrosine residues. One of these had an approximate molecular weight of 85 kDa which is the same as the ?4 integrin-like protein that was isolated in the microsomal fraction. While it cannot be certain that these are the same protein, this is consistent with the co-localisation that we observed being due to phosphorylated tyrosine residues in the Chapter 5: Conclusions 151 ?4 integrin-like protein itself. The possibility can not be discounted, however, that co- localisation is due to the interaction of other phosphotyrosine containing proteins that interact with the ?4 integrin-like protein. Additional prominent bands of 26- kDa and 60- kDa were visible. This is thought to be the first report of phosphotyrosine containing proteins in an oomycete. Reported sizes of phosphotyrosine containing proteins in plant and fungal species include 97 and 57 kDa in pea (Torruella et al., 1985), 40-65 kDa in maize (Trojanek et al., 1996), 10-63 kDa in Catharanthus roseus (Rodriguez-Zapata and Hern?ndez-Sotomayor, 1998) and 26 kDa in Papaver rhoeas (Rudd et al., 1996). The fact that no phosphotyrosine staining was observed in cells that had been exposed to tyrphostins suggests that, in addition to possible autophosphorylation there may be some phosphorylation that is due to the activity of a tyrosine kinase. This is further supported by the observation that the Triton- X100 extracted fraction and, to a lesser extent the crude fraction, showed tyrosine phosphorylation activity that could be inhibited by tyrphostins, in both ELISA and in coupled enzyme assays. Whether this activity is due to a tyrosine kinase or a dual- specificity kinase, however, is at present unclear as (Rudrabhatla and Rajasekharan, 2004) have shown a dual specificity kinase in peanut to be sensitive to tyrphostin. Regardless of this, the observation that the kinase activity was sensitive to tyrphostins, as was tip growth, suggests that the phosphorylation may be necessary for tip growth. The tyrphostins are a group of synthetic compounds that were originally designed to bind the substrate subsite in the kinase domain of the epidermal growth factor receptor, against which tyrphostins 25, 51 and 63 have IC 50 values of 3 ?M, 0.8 ?M and 6.5 mM respectively (Levitzki, 1990). They have since been used to inhibit a number of other tyrosine kinases, with the sensitivity to a particular tyrphostin dependant upon the identity of the kinase (Levitzki and Gazit, 1995). The results in this thesis show that the kinase activity is more sensitive to tyrphostins 25 and 51 than to tyrphostin 63, which is in keeping with a number of animal tyrosine kinases. In other walled cells tyrphostins have been found to block the cell cycle of S. cerevisiae (Fujimura, 1997) and to inhibit a STY dual specificity kinase in peanut (Rudrabhatla and Rajasekharan, 2004). In Fucus, tyrphostin 25 was found to have no effect on either germination of embryos or the Chapter 5: Conclusions 152 elongation of rhizoids, although a number of other tyrosine kinase inhibitors affected germination and two of these had a moderate inhibitory effect on rhizoid elongation (Corellou et al., 2000). It is possible that the lack of effect of tyrphostin reflects the concentration that was used (10 ?M) as inhibition of tip growth of A. bisexualis occurs at low millimolar concentrations. The demonstration of F-actin co-localising with the ?4 integrin-like protein at wall membrane attachment sites has implications with regard to the role that these proteins may play in tip growth. Kaminskyj and Heath (1995) have suggested that a ?1 integrin-F- actin association in S. ferax might anchor the cytoplasm, thus enabling apically directed contractions, facilitate the motility of organelles and provide a means for cell signalling. The first two of these functions would depend largely on a tip high gradient of integrin- like proteins, which is only observed in approximately one-half of A. bisexualis hyphae. The demonstration of co-localised phosphorylated tyrosine residues is, however, consistent with the latter function. One consequence of such signalling could be dynamic remodelling of the F-actin cytoskeleton. Such remodelling has been invoked in models of polarised growth in higher plants and is likely to involve the activation of actin-binding proteins by a variety kinases, phosphatases and phosphoinositides (Wasteneys and Galway, 2003; Wasteneys and Yang, 2004). With this in mind, it is interesting to note the effect of tyrphostin on the F-actin cytoskeleton and the recent description of an F-actin depleted zone in the actin caps at the tips of A. bisexualis hyphae (Yu et al., 2004). This zone may represent areas of growth where vesicles accumulate prior to exocytosis at the tip and it is possible to speculate that its activity and location could be controlled by the integrin-like proteins and any associated kinase. The data presented also suggests that S. cerevisiae contains proteins with epitopic similarity to the human ?4 integrin subunit. Immunoblotting identified only one candidate polypeptide in a microsomal pellet fraction. This polypeptide has a molecular weight at approximately 45 kDa. Immunofluorescence shows that staining of this integrin-like protein is mainly at cortical sites. However, the staining of integrin-like protein does not co-localise with phosphotyrosine containing proteins, which are located throughout cells. Chapter 5: Conclusions 153 Furthermore, immunoblotting shows that several proteins from S. cerevisiae contain phosphotyrosine residues especially prominent ones have molecular weights of approximately 35, 60, and 90 kDa. In addition, using the amino acid sequence between residues 28-128 residues of the human ?4 integrin subunit and comparing proteins from S. cerevisiae database, the search showed that there eight candidate proteins containing amino acid residues which show identity to amino acid of ?4 integrin subunit. However, only one polypeptide has a molecular weight close to 45 kDa, which is shown by immunoblotting. Hem12p is 41.3 kDa and contains a transmembrane domain; this domain contains amino acid residues with 39 % identities to amino acid residues between 75-110 of the human ?4 integrin subunit. Using ELISA and a coupled enzyme assay, the crude and partial purified fractions of S. cerevisiae have tyrosine kinase activity. This kinase activity is sensitive to tyrphostin 25 and tyrphostin 51. The SDS-PAGE shows protein bands which may be responsible for the kinase activity in partial purified fraction. These protein bands have sizes of 35, 45, 50 and 93 kDa. In summary, the data of this thesis presents antigenic evidence suggesting the existence of a ?4 integrin-like protein in hyphae of the oomycete Achlya bisexualis that co-localises with phosphotyrosine residues. 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Each reaction contains - 300 ?M ATP - Tyrosine kinase buffer - 150 ?l of 1.25 ?M poly(Glu:Tyr)4:1 was coated to wells microtiter at 45 ?C overnight. - 50 ?l of crude fractions or 2 units of EGFR for the positive control. - Final volume is 250 ?l Tyr + PYK + ATP P-Tyr HRP substrate Color change at 492 nm poly(Glu:Tyr) 4:1 Anti-pTyr antibody HRP Appendices 169 Scheme 2. Tyrosine kinase activity is determined using the coupled assay. Each reaction contains: - 0.3 mM NADH - 1 mM phosphoenolpyruvate (PEP) - 20 units of pyruvate kinase (PK) - 7-10 units of lactic dehydrogenase (LDH) - Buffer - 300 ?l of phosphorylation mixture - The coupled assay was initiated by the addition of 300 ?l of the phosphorylation mixture into 700 ?l of the coupled assay mixture and then the reaction was monitored by following the reduction of NADH at 340 nm for 2-3 minutes. Phosphoenolpyruvate ADP ATP Pyruvate kinase Pyruvate NADH NAD + Lactate dehydrogenase L-Lactate Appendices 170 Appendix 2: Sequence homology of actin among organisms Note that Query is actin from A. bisexualis 1. A. bisexualis vs. P. infestans Score = 751 bits (1940), Expect = 0.0 Identities = 370/376 (98%), Positives = 373/376 (98%) Query: 1 MADDDVQALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPKHPGIMVGMDQKDAYVGDEAQ 60 MADDDVQALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPKH GIMVGMDQKDAYVGDEAQ Sbjct: 1 MADDDVQALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPKHLGIMVGMDQKDAYVGDEAQ 60 Query: 61 SKRGVLTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANRERM 120 SKRGVLTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANRERM Sbjct: 61 SKRGVLTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANRERM 120 Query: 121 TQIMFETFNVPAMYVNIQAVLSLYASGRTTGCVLDSGDGVSHTVPIYEGYALPHAIVRLD 180 TQIMFETFNVPAMYVNIQAVLSLYASGRTTGCVLDSGDGVSHTVPIYEGYALPHAIVRLD Sbjct: 121 TQIMFETFNVPAMYVNIQAVLSLYASGRTTGCVLDSGDGVSHTVPIYEGYALPHAIVRLD 180 Query: 181 LAGRDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLTYIALDFDQEMKTAAESSGLEKS 240 LAGRDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLTYIALDFDQEMKTAAESSGLEKS Sbjct: 181 LAGRDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLTYIALDFDQEMKTAAESSGLEKS 240 Query: 241 YELPDGNVLVIGNERFRTPEVLFQPALIGKEASGIHDCTFQTIMKCDVDIRKDLYCNIVL 300 YELPDGNV+VIGNERFRTPEVLFQP+LIGKEASGIH+CTFQTIMKCDVDIRKDLYCNIVL Sbjct: 241 YELPDGNVIVIGNERFRTPEVLFQPSLIGKEASGIHECTFQTIMKCDVDIRKDLYCNIVL 300 Query: 301 SGGTTMYPGISERMTKELTALAPSTMKIKVVAPPERKYSVWIGGSILSSLSTFQQMWISK 360 SGGTTMYPGI ERMTKELTALAPSTMKIKVVAPPERKYSVWIGGSI SSLSTFQQMWISK Sbjct: 301 SGGTTMYPGIGERMTKELTALAPSTMKIKVVAPPERKYSVWIGGSIQSSLSTFQQMWISK 360 Appendices 171 Query: 361 AEYDESGPSIVHRKCF 376 AEYDESGPSIVHRKCF Sbjct: 361 AEYDESGPSIVHRKCF 376 2. A. bisexualis vs. N. crassa Score = 659 bits (1699), Expect = 0.0 Identities = 308/374 (82%), Positives = 351/374 (93%) Query: 3 DDDVQALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPKHPGIMVGMDQKDAYVGDEAQSK 62 +++V ALV+DNGSGMCKAGFAGDDAPRAVFPSIVGRP+H GIM+GM QKD+YVGDEAQSK Sbjct: 2 EEEVAALVIDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHHGIMIGMGQKDSYVGDEAQSK 61 Query: 63 RGVLTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANRERMTQ 122 RG+LTL+YPIEHG+VTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAP+NPK+NRE+MTQ Sbjct: 62 RGILTLRYPIEHGVVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPINPKSNREKMTQ 121 Query: 123 IMFETFNVPAMYVNIQAVLSLYASGRTTGCVLDSGDGVSHTVPIYEGYALPHAIVRLDLA 182 I+FETFN PA YV+IQAVLSLYASGRTTG VLDSGDGV+H VPIYEG+ALPHAI R+D+A Sbjct: 122 IVFETFNAPAFYVSIQAVLSLYASGRTTGIVLDSGDGVTHVVPIYEGFALPHAIARVDMA 181 Query: 183 GRDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLTYIALDFDQEMKTAAESSGLEKSYE 242 GRDLTDY+MKIL ERGY+F+TTAEREIVRDIKEKL Y+ALDF+QE++TAA+SS LEKSYE Sbjct: 182 GRDLTDYLMKILAERGYTFSTTAEREIVRDIKEKLCYVALDFEQEIQTAAQSSSLEKSYE 241 Query: 243 LPDGNVLVIGNERFRTPEVLFQPALIGKEASGIHDCTFQTIMKCDVDIRKDLYCNIVLSG 302 LPDG V+ IGNERFR PE LFQP+++G E+ GIH TF +IMKCDVD+RKDLY NIV+SG Sbjct: 242 LPDGQVITIGNERFRAPEALFQPSVLGLESGGIHVTTFNSIMKCDVDVRKDLYGNIVMSG 301 Query: 303 GTTMYPGISERMTKELTALAPSTMKIKVVAPPERKYSVWIGGSILSSLSTFQQMWISKAE 362 GTTMYPG+S+RM KE+TALAPS+MK+K++APPERKYSVWIGGSIL+SLSTFQQMWISK E Sbjct: 302 GTTMYPGLSDRMQKEITALAPSSMKVKIIAPPERKYSVWIGGSILASLSTFQQMWISKQE 361 Query: 363 YDESGPSIVHRKCF 376 YDESGPSIVHRKCF Sbjct: 362 YDESGPSIVHRKCF 375 Appendices 172 3. A. bisexualis vs. D. discoideum (Slime mold) Score = 683 bits (1762), Expect = 0.0 Identities = 327/373 (87%), Positives = 353/373 (93%) Query: 4 DDVQALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPKHPGIMVGMDQKDAYVGDEAQSKR 63 +DVQALV+DNGSGMCKAGFAGDDAPRAVFPSIVGRP+H G+MVGM QKD+YVGDEAQSKR Sbjct: 3 EDVQALVIDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHTGVMVGMGQKDSYVGDEAQSKR 62 Query: 64 GVLTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANRERMTQI 123 G+LTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANRE+MTQI Sbjct: 63 GILTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANREKMTQI 122 Query: 124 MFETFNVPAMYVNIQAVLSLYASGRTTGCVLDSGDGVSHTVPIYEGYALPHAIVRLDLAG 183 MFETFN PAMYV IQAVLSLYASGRTTG V+DSGDGVSHTVPIYEGYALPHAI+RLDLAG Sbjct: 123 MFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVSHTVPIYEGYALPHAILRLDLAG 182 Query: 184 RDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLTYIALDFDQEMKTAAESSGLEKSYEL 243 RDLTDYMMKILTERGYSFTTTAEREIVRDIKEKL Y+ALDF+QEM TAA SS LEKSYEL Sbjct: 183 RDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLAYVALDFEQEMATAASSSALEKSYEL 242 Query: 244 PDGNVLVIGNERFRTPEVLFQPALIGKEASGIHDCTFQTIMKCDVDIRKDLYCNIVLSGG 303 PDG V+ IGNERFR PE LFQP+ +G E++GIH+ T+ +IMKCDVDIRKDLY N+VLSGG Sbjct: 243 PDGQVITIGNERFRCPEALFQPSFLGMESAGIHETTYNSIMKCDVDIRKDLYGNVVLSGG 302 Query: 304 TTMYPGISERMTKELTALAPSTMKIKVVAPPERKYSVWIGGSILSSLSTFQQMWISKAEY 363 TTM+PGI++RM KELTALAPSTMKIK++APPERKYSVWIGGSIL+SLSTFQQMWISK EY Sbjct: 303 TTMFPGIADRMNKELTALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKEEY 362 Query: 364 DESGPSIVHRKCF 376 DESGPSIVHRKCF Sbjct: 363 DESGPSIVHRKCF 375 Appendices 173 4. A. bisexualis vs ?-actin of human muscle Score = 662 bits (1707), Expect = 0.0 Identities = 315/374 (84%), Positives = 346/374 (92%) Query: 3 DDDVQALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPKHPGIMVGMDQKDAYVGDEAQSK 62 +D+ ALV DNGSG+ KAGFAGDDAPRAVFPSIVGRP+H G+MVGM QKD+YVGDEAQSK Sbjct: 4 EDETTALVCDNGSGLVKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSK 63 Query: 63 RGVLTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANRERMTQ 122 RG+LTLKYPIEHGI+TNWDDMEKIWHHTFYNELRVAPEEHP LLTEAPLNPKANRE+MTQ Sbjct: 64 RGILTLKYPIEHGIITNWDDMEKIWHHTFYNELRVAPEEHPTLLTEAPLNPKANREKMTQ 123 Query: 123 IMFETFNVPAMYVNIQAVLSLYASGRTTGCVLDSGDGVSHTVPIYEGYALPHAIVRLDLA 182 IMFETFNVPAMYV IQAVLSLYASGRTTG VLDSGDGV+H VPIYEGYALPHAI+RLDLA Sbjct: 124 IMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRLDLA 183 Query: 183 GRDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLTYIALDFDQEMKTAAESSGLEKSYE 242 GRDLTDY+MKILTERGYSF TTAEREIVRDIKEKL Y+ALDF+ EM TAA SS LEKSYE Sbjct: 184 GRDLTDYLMKILTERGYSFVTTAEREIVRDIKEKLCYVALDFENEMATAASSSSLEKSYE 243 Query: 243 LPDGNVLVIGNERFRTPEVLFQPALIGKEASGIHDCTFQTIMKCDVDIRKDLYCNIVLSG 302 LPDG V+ IGNERFR PE LFQP+ IG E++GIH+ T+ +IMKCD+DIRKDLY N V+SG Sbjct: 244 LPDGQVITIGNERFRCPETLFQPSFIGMESAGIHETTYNSIMKCDIDIRKDLYANNVMSG 303 Query: 303 GTTMYPGISERMTKELTALAPSTMKIKVVAPPERKYSVWIGGSILSSLSTFQQMWISKAE 362 GTTMYPGI++RM KE+TALAPSTMKIK++APPERKYSVWIGGSIL+SLSTFQQMWI+K E Sbjct: 304 GTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWITKQE 363 Query: 363 YDESGPSIVHRKCF 376 YDE+GPSIVHRKCF Sbjct: 364 YDEAGPSIVHRKCF 377 Appendices 174 Appendix 3: Amino acid sequences of ?4 integrin subunit of Homo sapiens 1 magprpspwa rlllaalisv slsgtlanrc kkapvkscte cvrvdkdcay ctdemfrdrr 61 cntqaellaa gcqresivvm essfqiteet qidttlrrsq mspqglrvrl rpgeerhfel 121 evfeplespv dlyilmdfsn smsddldnlk kmgqnlarvl sqltsdytig fgkfvdkvsv 181 pqtdmrpekl kepwpnsdpp fsfknvislt edvdefrnkl qgerisgnld apeggfdail 241 qtavctrdig wrpdsthllv fstesafhye adganvlagi msrnderchl dttgtytqyr 301 tqdypsvptl vrllakhnii pifavtnysy syyeklhtyf pvsslgvlqe dssnivelle 361 eafnrirsnl diraldsprg lrtevtskmf qktrtgsfhi rrgevgiyqv qlralehvdg 421 thvcqlpedq kgnihlkpsf sdglkmdagi icdvctcelq kevrsarcsf ngdfvcgqcv 481 csegwsgqtc ncstgslsdi qpclregedk pcsgrgecqc ghcvcygegr yegqfceydn 541 fqcprtsgfl cndrgrcsmg qcvcepgwtg pscdcplsna tcidsnggic ngrghcecgr 601 chchqqslyt dticeinysa ihpglcedlr scvqcqawgt gekkgrtcee cnfkvkmvde 661 lkraeevvvr csfrdedddc tysytmegdg apgpnstvlv hkkkdcppgs fwwlipllll 721 llpllallll lcwkycacck aclallpccn rghmvgfked hymlrenlma sdhldtpmlr 781 sgnlkgrdvv rwkvtnnmqr pgfathaasi nptelvpygl slrlarlcte nllkpdtrec 841 aqlrqeveen lnevyrqisg vhklqqtkfr qqpnagkkqd htivdtvlma prsakpallk 901 ltekqveqra fhdlkvapgy ytltadqdar gmvefqegve lvdvrvplfi rpedddekql 961 lveaidvpag tatlgrrlvn itiikeqard vvsfeqpefs vsrgdqvari pvirrvldgg 1021 ksqvsyrtqd gtaqgnrdyi pvegellfqp geawkelqvk llelqevdsl lrgrqvrrfh 1081 vqlsnpkfga hlgqphstti iirdpdeldr sftsqmlssq ppphgdlgap qnpnakaags 1141 rkihfnwlpp sgkpmgyrvk ywiqgdsese ahlldskvps veltnlypyc dyemkvcayg 1201 aqgegpyssl vscrthqevp sepgrlafnv vsstvtqlsw aepaetngei tayevcyglv 1261 nddnrpigpm kkvlvdnpkn rmllienlre sqpyrytvka rngagwgper eaiinlatqp 1321 krpmsipiip dipivdaqsg edydsflmys ddvlrspsgs qrpsvsddtg cgwkfepllg 1381 eeldlrrvtw rlppeliprl sassgrssda eaphgppddg gaggkggslp rsatpgppge 1441 hlvngrmdfa fpgstnslhr mtttsaaayg thlsphvphr vlstsstltr dynsltrseh 1501 shsttlprdy stltsvsshd srltagvpdt ptrlvfsalg ptslrvswqe prcerplqgy 1561 sveyqllngg elhrlnipnp aqtsvvvedl lpnhsyvfrv raqsqegwgr eregvities 1621 qvhpqsplcp lpgsaftlst psapgplvft alspdslqls werprrpngd ivgylvtcem 1681 aqgggpataf rvdgdspesr ltvpglsenv pykfkvqart tegfgpereg iitiesqdgg 1741 pfpqlgsrag lfqhplqsey ssittthtsa tepflvdgpt lgaqhleagg sltrhvtqef 1801 vsrtlttsgt lsthmdqqff qt Appendices 175 Appendix 4: Comparing sequences of amino acid at 28-128 of ?4 integrin subunit with protein sequences from S. cerevisiae database YMR178W YMR178W SGDID:S0004790, Chr XIII from 618478-619302, Length = 275 Score = 58 (25.5 bits), Expect = 1.3, P = 0.73 Identities = 17/57 (29%), Positives = 26/57 (45%) Query: 14 VRVDKDCAYCTDEMFRDRRCNTQAELLAAGCQRESIVVMESSFQITEETQIDTTLRR 70 V+V C DE+ + +T + A C I + E + +ETQI T+RR Sbjct: 2 VKVTAACIIIGDEVLNGKVVDTNSTFFAKYCFDHGIQLKEIATIGDDETQIVDTVRR 58 YBR150C TBS1 SGDID:S0000354, Chr II from 544487-541203, reverse complement, Score = 57 (25.1 bits), Expect = 1.4, Sum P(2) = 0.74 Identities = 11/34 (32%), Positives = 18/34 (52%) Query: 1 RCKKA-PVKS-CTECVRVDKDCAYCTDEMFRDRR 32 RC + P+ C C++ +KDC + E + RR Sbjct: 116 RCTEIEPISGKCRNCIKYNKDCTFHFHEELKRRR 149 Score = 42 (19.8 bits), Expect = 1.4, Sum P(2) = 0.74 Identities = 15/40 (37%), Positives = 17/40 (42%) Query: 52 MESSFQITEETQIDTT--LRRSQMSPQGLRVRLRPGEERH 89 ME SF I E I L+ S + L PGEE H Sbjct: 893 MEVSFNIFNEITIQDLNFLQFSSIPKLWENKTLEPGEEYH 932 Appendices 176 YNL267W PIK1 SGDID:S0005211, Chr XIV from 140877-144077, Score = 59 (25.8 bits), Expect = 4.9, P = 0.992 Identities = 18/58 (31%), Positives = 28/58 (48%) Query: 44 CQR-ESIVVMESSFQITEETQIDTTLRRSQMSPQGLRVRLRPGEERHFELEVFEPLES 100 CQ+ + E F I + Q+ T+ M+ + L +RLR E HF L F L++ Sbjct: 55 CQKLATFPHSELQFYIPQLVQVLVTMETESMALEDLLLRLR-AENPHFALLTFWQLQA 111 YLR098C CHA4 SGDID:S0004088, Chr XII from 339474-337528, reverse complement, Score = 57 (25.1 bits), Expect = 5.0, P = 0.993 Identities = 11/50 (22%), Positives = 24/50 (48%) Query: 8 KSCTECVRVDKDCAYCTDEMFRDRRCNTQAELLAAGCQ--RESIVVMESS 55 K C+ C++ +C + ++ R T E L + + +E + ++ SS Sbjct: 58 KPCSNCIKFRTECVFTQQDLRNKRYSTTYVEALQSQIRSLKEQLQILSSS 107 YDR272W GLO2 SGDID:S0002680, Chr IV from 1009004-1009828, Score = 52 (23.4 bits), Expect = 7.8, P = 0.9996 Identities = 25/78 (32%), Positives = 28/78 (35% Query: 11 TECVRVDKDCAYCTDEMFRDRRCNTQAELLAAGCQRESIVVMESSFQITEETQIDTTLR 69 T C D C Y D D RC T L AGC R E I I T+ Sbjct:118 TPCHTRDSICYYVKDPT-TDERCIFTGDTLFTAGCGRFFEGTGEE-MDIALNNSILETVG 175 Query: 70 RSQMSPQGLRVRLRPGEE 87 R S + R+ PG E Sbjct: 176 RQNWS----KTRVYPGHE 189 Appendices 177 YGL116W CDC20 SGDID:S0003084, Chr VII from 289812-291644, Score = 55 (24.4 bits), Expect = 8.6, P = 0.9998 Identities = 16/64 (25%), Positives = 32/64 (50%) Query:38 ELLAAGCQRESIVVM---ESSFQITEETQI-DTTLRRSQMSPQGLRVRLRPGEERHFELE 93 E++A G E+ + + E+ F++ E + + SQ+SP G + G+E + Sbjct:494 EIVATGGNPENAISVYNYETKFKVAEVVHAHEARICCSQLSPDGTTLATVGGDENLKFYK 553 Query:94 VFEP 97 +F+P Sbjct:554 IFDP 557 YLR030W YLR030W SGDID:S0004020, Chr XII from 203291-204082, Score = 51 (23.0 bits), Expect = 9.8, P = 0.99995 Identities = 11/30 (36%), Positives = 18/30 (60%) Query: 65 DTTLRRSQMSPQGLRVRLRPGEERHFELEV 94 +T+L ++ S V L+PGE+ H LE+ Sbjct: 191 NTSLAENKRSSDSF-VSLKPGEDEHSPLEI 219 Hem12p [Saccharomyces cerevisiae]; gi|283290|pir||S23471 uroporphyrinogen decarboxylase (EC 4.1.1.37) - yeast (Saccharomyces cerevisiae); gi|3767|emb|CAA45253.1|Uroporphyrinogen decarboxylase [Saccharomyces cerevisiae]; gi|416894|sp|P32347|DCUP_YEAST Uroporphyrinogen decarboxylase URO-D) (UPD); gi|4776|emb|CAA79514.1| Uroporphyrinogen decarboxylase [Saccharomyces cerevisiae]; gi|798900|emb|CAA89078.1| Hem12p [Saccharomyces cerevisiae] Length = 362 Appendices 178 Score = 60 (26.2 bits), Expect = 2.5, P = 0.91 Identities = 15/38 (39%), Positives = 22/38 (57%) Query: 75 PQ--GLRVRLRPGEERHFELEVFEPLESPVDLYILMDF 110 PQ G+RV + G+ HF EPL +P DL ++D+ Sbjct: 86 PQAMGMRVEMLEGKGPHFP----EPLRNPEDLQTVLDY 119 Appendices 179 Appendix 5: Sequences of P. sojae compared with sequences from different eukaryotic organisms Putative tyrosine kinase-like protein [Dictyostelium discoideum] Score = 94.7 bits (234), Expect = 1e-18 Identities = 52/128 (40%), Positives = 70/128 (54%), Gaps = 10/128 (7%) Frame = +2 Query 11 WGAKVSDFGLSREKSVDETMSVTGTPLWLPPEMIRGERYTEKADVYSFGIVLAELDTRKI 190 W KV+DFGLS + TM+ GTP W PE++R +RYTEKADVYSFGI+L E TR+ Sbjct 526 WKVKVADFGLSTIEQQGATMTACGTPCWTSPEVLRSQRYTEKADVYSFGIILWECATRQD 585 Query 191 PYHDIKAKGARNKKVSGSTLMHMVAYENLRPSLSKNCMDSVRDLYKRCTSDDQSVRPTFE 370 PY I ++ V E +RP K L K C +++ S RPT E Sbjct 586 PYFGIPP----------FQVIFAVGREGMRPPTPKYGPPKYIQLLKDCLNENPSQRPTME 635 Query 371 EIVQFLEN 394 + ++ LE+ Sbjct 636 QCLEILES 643 C. elegans KIN-24 protein (corresponding sequence K07F5.4) [Caenorhabditis elegans] Hypothetical protein K07F5.4 - Caenorhabditis elegans Tyrosine kinase family member (kin-24) [Caenorhabditis elegans] Length=517 Score = 66.6 bits (161), Expect = 8e-10 Identities = 37/95 (38%), Positives = 58/95 (61%), Gaps = 5/95 (5%) Query 139 LFLEAAEGLRYLHEEIKLVHSNLKSDNILVTGDSRVKLTDFGLGVLALQDQAVQDKKFQE 318 L ++AA G+RYLH++ K VH +L S N L++ D VK+ DFGL +DQ + +E Sbjct 262 LLIDAARGMRYLHDK-KCVHRDLASRNCLISFDGIVKIADFGLSKTLEKDQKAFKEALKE 320 Query 319 --LGWRAPNCVQKKPFRRPSFQDDVYSFGLCVLDV 417 L W AP C+Q++ S + DV++FG+ + +V Sbjct 321 APLAWLAPECIQRE--SEFSTKTDVWAFGVVIFEV 353 Appendices 180 Tyrosine-protein kinase Srms Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites [Homo sapiens] Length=488 Score = 59.3 bits (142), Expect = 7e-08 Identities = 34/96 (35%), Positives = 50/96 (52%), Gaps = 2/96 (2%) Frame = -2 Query 386 WTAPEVLDGRQYTPKADIYSFGVLLAQLATYECTSAEHSVMDDTEVPMLNNHKGSASGDA 207 WTAPE + R ++ K+D++SFGVLL ++ TY E +T ++ ++ A Sbjct 392 WTAPEAANYRVFSQKSDVWSFGVLLHEVFTYGQCPYEGMTNHETLQQIMRGYR--LPRPA 449 Query 206 EAPTPVRLLMFRCQAFQPEGRPTADELLEELHQIER 99 P V +LM C PE RP+ L E+LH I R Sbjct 450 ACPAEVYVLMLECWRSSPEERPSFATLREKLHAIHR 485 Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (predicted) [Rattus norvegicus] Length=507 Score = 57.0 bits (136), Expect = 3e-07 Identities = 34/98 (34%), Positives = 50/98 (51%), Gaps = 2/98 (2%) Frame = -2 Query 386 WTAPEVLDGRQYTPKADIYSFGVLLAQLATYECTSAEHSVMDDTEVPMLNNHKGSASGDA 207 WTAPE + R ++ K+D++SFG+LL ++ TY E +T + ++ A Sbjct 407 WTAPEAANYRVFSQKSDVWSFGILLYEVFTYGQCPYEGMTNHETLQQISRGYR--LPRPA 464 Query 206 EAPTPVRLLMFRCQAFQPEGRPTADELLEELHQIEREL 93 P V +LM C PE RPT L E+L+ I R L Sbjct 465 VCPAEVYMLMVECWKGSPEERPTFATLREKLNAINRRL 502 Appendices 181 Tyrosine-specific protein kinase [Mus musculus] Length=496 Score = 55.8 bits (133), Expect = 7e-07 Identities = 34/98 (34%), Positives = 50/98 (51%), Gaps = 2/98 (2%) Frame = -2 Query 386 WTAPEVLDGRQYTPKADIYSFGVLLAQLATYECTSAEHSVMDDTEVPMLNNHKGSASGDA 207 WTAPE + R ++ K+D++SFG+LL ++ TY E +T + ++ A Sbjct 396 WTAPEAANYRVFSQKSDVWSFGILLYEVFTYGQCPYEGMTNHETLQQISRGYR--LPRPA 453 Query 206 EAPTPVRLLMFRCQAFQPEGRPTADELLEELHQIEREL 93 P V +LM C PE RPT L E+L+ I R L Sbjct 454 VCPAEVYVLMVECWKGSPEERPTFAILREKLNAINRRL 491