Characterizing the role of proteins in modulating microtubule dynamics and cell morphogenesis in Arabidopsis thaliana

Microtubules are dynamic polymers that are important for the growth and development of plant cells. Because of their vital role, the dynamics and organization of microtubules need to be tightly modulated by other proteins. The major focus on this dissertation is to elucidate and characterize the role of specific proteins (ARK1, NEK6, MOR1) that are responsible for orchestrating the dynamics and organization of microtubules in the model plant, Arabidopsis thaliana. The motor protein, ARK1, was previously shown to play an important role in root hair morphogenesis but had an unknown role in modulating microtubule dynamics. Moreover, evidence showed that ARK1 physically interacts with the NEK6 kinase, although for an undetermined reason. Based on my data, I determined that ARK1 functions as a plus-end tracking protein that has a specific role in promoting the depolymerization of microtubules. I also discovered that ARK1 has a secondary microtubule-binding domain in addition to the motor domain, which is the canonical microtubule-binding domain. I noted, however, that this secondary microtubule-binding domain is not essential for ARK1’s ability to induce microtubule depolymerization. While NEK6 and ARK1 both modulate microtubule dynamics, I determined that neither protein requires each other for function or localization, suggesting that they operate independently from each other to control microtubule dynamics and cell elongation. In addition, I provided evidence that shows that ARK1 has a putative yet unknown role in controlling NEK6 gene expression. Finally, the microtubule-associated protein MOR1 was confirmed to be a plus-end tracking protein through live-cell imaging of MOR1 fused to a fluorescent reporter (MOR1-3xYpet). In revealing that MOR1 binds to both growing and shrinking microtubule plus ends, my data corroborated previous studies showing reduced microtubule growth and shrinkage in mor1 mutants, and confirm MOR1’s role as a microtubule polymerase. Comparing MOR1-3xYpet live-cell imaging with previous experiments using fixed samples revealed that the chemical fixation process affects the plus-ends of microtubules, stressing the importance of using non-fixed samples for the most accurate results. My dissertation thus expands our knowledge about how microtubule dynamics and organization is controlled in plant cells by three distinct proteins.Science, Faculty ofBotany, Department ofGraduat