UBC Theses and Dissertations
Mapping the microtubule landscape : the role of microtubule-associated proteins during cell division and expansion in Arabidopsis thaliana Halat, Laryssa
The microtubule cytoskeleton is a filamentous network that reinforces cell shape, aids in vesicle transport, and enables cells to divide. In plant cells, the cortical microtubule array lies directly beneath the plasma membrane and is an intermediary between the cell exterior and the cytoplasmic environment. The dynamic flux of microtubules is largely attributed to an assortment of microtubule-associated proteins (MAPs) that bind to the microtubule polymer and promote growth, shrinkage, or rescue of a depolymerizing filament. In Arabidopsis thaliana, the MAP MICROTUBULE ORGANIZATION 1 (MOR1) is a microtubule polymerase essential for plant survival, but how this protein interacts with the microtubule to promote rapid assembly is unclear. Another important MAP is CYTOPLASMIC LINKER ASSOCIATED PROTEIN (CLASP), which prevents microtubule depolymerization and sustains hormone flux by tethering endosomes near the cell surface in root cells. A central question is how CLASP integrates these functions to regulate root meristem growth. In this thesis, I investigated the cellular and molecular roles of MOR1 and CLASP in plant development. By devising an imaging procedure using fluorescence recovery after photobleaching, I visualized fluorescent MOR1 and found that the degree of turnover on the microtubule plus end was correlated to microtubule growth rate. To study the behaviour of CLASP in roots, I used live-cell imaging to observe that cells in the clasp-1 null mutant spent longer in mitosis, and the formation of CLASP-dependent microtubule bundles occurred during G1 and persisted throughout S phase. I found that CLASP was controlled by brassinosteroid hormone signalling, and excess brassinosteroid reduced CLASP expression, protein levels, and changed microtubule organization. This was further demonstrated in a brassinosteroid-insensitive version of CLASP, which showed reduced root meristem growth despite increased CLASP fluorescent protein. Analysis of dark-grown root meristems revealed that a translational checkpoint reduces CLASP protein levels, possibly to inhibit root growth under conditions when hypocotyl expansion is required. Finally, I explored the evolution of the CLASP-SNX1 interaction through a series of BLAST searches and found that this sequence is specific to land plants. This thesis has furthered our understanding of microtubule dynamics and the interplay of MAPs and hormones in root development.
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