UBC Theses and Dissertations
TOG domain-tubulin interactions and the function of the Arabidopsis Microtubule Organization 1 protein Zhang, Yi
Microtubules are indispensible cellular components involved in multiple core processes such as cell division and intra cellular trafficking. Elaborate regulatory mechanisms are required to direct the precise functioning of these highly dynamic structures. Microtubule-Associated Proteins (MAPs) often perform regulatory functions in controlling microtubule dynamics. Distortion of the function of Arabidopsis Microtubule Organization protein1 (MOR1) leads to various developmental defects. However, the mechanism through which MOR1 regulates microtubule function is poorly understood. We have hypothesized that each TOG domain of MOR1 physically binds with tubulin dimers to directly regulate their addition and removal from the microtubule polymer. To address this hypothesis, we aimed to identify interaction sites between the MOR1 protein and tubulins through a genetic interaction strategy. Three left-handed twisting mor1 mutants were each crossed with sixteen right-handed twisting tubulin mutants, and pronounced genetic interactions were detected by observing non-additive and allele-specific phenotypes. Notably, tubulin point mutations in the interface between the β-tubulin at the plus end of the microtubules and the α-tubulin of the incoming dimer generated the most synergistic phenotypes when combined with the mor1 alleles. Live cell imaging of microtubules confirmed that allele-specific variations in growth phenotypes were correlated with altered microtubule dynamics. The MOR1-tubulin interaction model was further tested through the characterization of a new mor1 allele in the TOG3 domain of MOR1, mor1-11, which was determined to have a semi-dominant propyzamide-dependent right-handed twisting phenotype and microtubule organization and dynamics were found to be altered by propyzamide treatment. However, the distinct right-handed twisting phenotype, and to some extent the altered microtubule organization is lost at higher temperature, suggesting that the intrinsic increase in microtubule dynamics at 31°C overrides mor1-11’s effect. The tua6C²¹³Y single mutant, which was reported to have similar right-handed twisting upon propyzamide treatment, continues to twist in a right-handed manner at 31°C and mor1-11tua6C²¹³Y double mutants are indistinguishable from tua6C²¹³Y single mutants on propyzamide at either 21°C or 31°C. Together, these observations suggest that tubulin is more likely than MOR1 to be a direct target of propyzamide and that the motif identified by the mor1-11 mutation could play a key role in the interactions on MOR1 with α-tubulin.
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