A study of M0R1/GEM1 and kinl-like kinesins arabidopsis

Abstract

Microtubules perform essential functions in eukaryotic cells and, with other cytoskeletal elements, are involved in diverse cellular processes. Plant cells construct four microtubule arrays; There are two cortical arrays, the interphase cortical array, and the preprophase band, the mitotic spindle and the cytokinetic phragmoplast. The control and rearrangement of these arrays through the cell cycle is coordinated by Microtubule Associated Proteins or MAPs. These proteins have diverse functions including anchoring & crosslinking microtubules or otherwise regulating microtubule formation and destruction within the cell. MOR1/GEM1 is the Arabidopsis member of the conserved XMAP215/TOGp family of proteins which are microtubule stablising MAPs and promote microtubule polymerization in vitro. In many organisms such stabilization is opposed by Kinl catastrophic kinesins, which depolymerise and destabilize microtubules. In addition to the further characterization of MOR1/GEM1, here in this thesis, two putative KinI kinesins are identified in the Arabidopsis genome, a subfamily of kinesins previously uncharacterized in plants, AtCMK1 & AtCMK2. Immunolocalisation shows that MOR1/GEM1 associates with all plant microtubule arrays throughout the cell cycle and Is found to concentrate at the plus end of microtubules In the spindle next to chromosomes and the midline of the phragmoplast where oppositely orientated microtubules overlap. Furthermore, consistent with this localization, we show that a C-terminal fragment of MOR1/GEM1 which Is absent in the pollen cytokinesis mutant, gem1 can bind microtubules and as such we propose that the defects in the phragmoplast In gem1 mutant is a result of the reduction of the MOR1/GEM1 protein to bind microtubules. Further studies indicate that unlike its budding yeast homologue Stu2, MOR1/GEM1 does not for dimers.Immunolocalisation shows that AtCMK2 associates with all the plant microtubule arrays throughout the cell cycle, particularly the metaphase spindle. However in our experiments, AtCMKI does not, but rather locates to structures within the cytoplasm, such as golgi vesicles or organelles. Here we also show that AtCMK kinesins form homodimers and do not physically Interact with MOR1/GEM1 suggesting that these factors may be genetic interactors instead. Over-expression of AtCMK2 as a GPP fusion protein results in the disruption of microtubules, leaving short microtubule fragments and tubulin oligomers or aggregates, suggesting that AtCMK2 Is a true Arabidopsis catastrophic kinesin. In addition, GUS promoter fusions show that the expression patterns of these two kinesins are very different.