Identification and visualisation of actin-binding proteins in Arabidopsis thaliana and tobacco BY2 cells
Thesis DisciplinePlant Biotechnology
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
The cytoskeleton is a remarkable system of filaments that helps in the organisation and functioning of living cells. In plant cells, this cytoskeleton comprises actin microfilaments and microtubules that polymerise from actin and tubulin respectively. While these proteins are highly conserved in eukaryotes, the plant cytoskeleton performs many plant-specific functions. The organisation and functions of the cytoskeleton are determined by a plethora of accessory proteins (actin-binding proteins, microtubule-associated proteins) that link the cytoskeletal filaments to other cell components and to each other. While there is extensive data for the subcellular localisation of actin-binding proteins with actin microfilaments in animal cells, surprisingly few experiments of this type have previously been tried in plants, and the subcellular localisation of most plant actin-binding proteins remains unknown. Such information is important in assessing functions of these proteins to give a better understanding of the actin cytoskeleton. In this study, an attempt was made to visualise the association of actin microfilaments and actin-binding proteins. A range of antibodies raised against various plant and animal actin-binding proteins were screened in two model systems for plant cytoskeleton research, the root of Arabidopsis thaliana and in whole cells of the tobacco BY2 liquid cell culture. Further, because previous data in the localisation of the actin-binding protein tropomyosin have suggested that the localisation of this actin-biding protein with the finer cortical actin microfilaments in Arabidopsis roots might not be discerned due to high cytoplasmic background, immunolabelling experiments were also conducted on plasma membrane ghosts generated from tobacco BY2 from which any non-specific cytoplasmic labelling could be washed away. There experiments gave some preliminary suggestions for the association of the actin-binding proteins to the actin cytoskeleton in plant cells. The most intriguing observations were obtained with antibodies against the β-subunit of capping protein which colocalised with larger microfilament bundles in tobacco BY2 cells. No colocalisation was observed on membrane ghosts on which these bundles are not well retained. However, the previous experiments in which there were suggestions of tropomyosin-related proteins associated with fine cortical microfilaments in Arabidopsis could not be replicated. As no cytoskeletal localisation was observed in either Arabidopsis or tobacco with antibodies raised against known actin-bundling proteins from Arabidopsis such as villin and fimbrin, it is speculated that the labelling protocols, currently optimised for visualising the actin cytoskeleton, might not to be modified to allow visualisation of actin-binding proteins in plant cells.