The roles of integrin-like proteins, tyrosine phosphorylation and F-actin in hyphal tip growth
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
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 β4 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 β4 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-β4 integrin subunit antibody. Furthermore, there are a number of proteins containing phosphotyrosine residues. Immunocytochemistry shows that this anti-β4 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.