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Exploring the effect of microtubule domains and various microtubule associated proteins on cellulose synthase complex velocity for determining anisotropic growth in Arabidopsis thaliana Mulvihill, Adam

Abstract

Previous research with anisotropy mutants, any1 and mor1-1, deficient in cellulose production and microtubule disruption respectively, linked the velocity of the cellulose synthase complex (CSC) to the crystalline structure of microfibrils in the wall for maintaining anisotropy during rapid elongation at elevated temperatures. CSCs have been visualized as being functional inside and outside of these microtubule domains and, in mor1-1, it was observed that increased CSC tracking outside of microtubule domains may be the means by which anisotropy fails due to correlations with increased CSC velocity and crystallinity. We hypothesized based on these findings that microtubule association with the plasma membrane, spatial organization, and polymerization status may have a significant effect on CSC velocity and anisotropy in a variety of backgrounds and/or drug treatments. We explored the concurrent visualization of YFP-CESA6 labeled cellulose synthase complexes with RFP-TUB6 labeled microtubules with variable angle total internal reflectance fluorescence (near-TIRF) microscopy. Not all genotypes had the proper RFP-TUB6 constructs, limiting our assessment to the same degree as previous research. This allowed for a more comprehensive analysis of CSC movement and velocity due to its increased resolution at specific optical sections. By comparing the results obtained from live-cell imaging with near-TIRF microscopy between a number of anisotropy-compromised mutants, such as any1, mor1-1, bot1, and oryzalin-treated seedlings, and microtubule-associated protein (MAP) genotypes with either enhanced (RIC1-OX) or non-specific anisotropy defects (clasp-1), we were able to obtain a more accurate depiction of the extent of microtubule influence on CSC activity. From these results, we were able to consider potential effects of microtubules on plasma membrane domains on CSC velocity during rapid elongation, as well as to discern potential new roles and effects of the MOR1 protein. Analysis of oryzalin-treated seedlings compared to the severely impaired double mutant, any1/mor1-1, provided us with an awareness that the mechanisms controlling CSC activity are complex. With this understanding, our exploration into the effect of several MAPs on CSC velocity highlighted that microtubule polymerization and organization do not hold equal weight, and that a potential standard exists for CSC velocity and wall crystallinity, as exemplified in wild type and RIC1-OX lines.

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