Spatiotemporal Regulation Of Microtubule Initiation And The Role Of End-Binding 1 On Organization Of The Cortical Microtubule Cytoskeleton In Arabidopsis Thaliana

The microtubule cytoskeleton is a dynamic structure that is organized into different configurations that perform vital cellular functions such as cell morphogenesis, intracellular transport and cell division. Microtubule organization is shaped by nucleation, dynamics and interactions between microtubules. First imaged a half-century ago by Ledbetter and Porter (1963), microtubules are found beneath the plasma membrane of plant cells during interphase. As these cortical microtubules (CMTs) become more ordered, bundling together, cell expansion occurs perpendicularly to the net orientation of the CMT array. Disruption of the ordering of CMTs, through mutations or drug applications, leads to abnormal growth and development. However, the mechanisms for how plants create, maintain and change specific array patterns remains poorly understood. In plants, one end (the plus-end) of a microtubule is more dynamic that the other end (the minus-end). Specific interactions occur between CMTs when the growing plus-end of one CMT encounters another CMT in its path. These interactions can result in CMTs crossing over, disassembling, severing or forming bundles. In combination, these outcomes are vital for array organization. These interactions are regulated by a variety of microtubule-associated proteins (MAPs). Some MAPs have been shown to regulate dynamics through stabilization or destabilization of individual CMTs, while others are important for the initiation, severing or bundling of CMTs. My thesis work explores how specific activities that regulate the behavior of individual microtubules impact CMT array organization. In budding yeast and animal cells, microtubule-organizing centers (MTOCs) nucleate and tether microtubules to create radial arrays. In contrast, plants lack MTOCs and consequently the organization of the CMT array occurs in the absence of a centralized organizing mechanism. Instead, new CMTs originate from gamma-tubulin containing microtubule nucleation complexes that are dispersed throughout the cell cortex. Associating with preexisting CMTs, these complexes initiate new CMTs in multiple configurations. In a portion of my thesis, I examine the role of CMT nucleation in array organization by analyzing patterns of nucleation in wild-type and mutant plants. Using novel dual fluorescent marker lines and live-cell imaging, I found that the relative ratio of branch-form to parallel-form nucleation is a hallmark of transverse and longitudinal CMT arrays. In addition, I found that biased CMT growth from cell edges plays a pivotal role in orienting and reorienting the CMT array with respect to cell geometry. Analysis of the patterns of CMT nucleation of several twisted-growth mutants indicate that branch-form nucleation plays an important role in defining the overall orientation of the CMT array in addition to facilitating array reorientation. These findings support the hypothesis that regulation of the ratio of branch-form to parallel-form nucleation may represent a general mechanism for defining the organization and orientation of the CMT array. A specialized class of MAPs specifically binds to and tracks with growing microtubule plus-ends and performs much of the regulation of microtubules. Among these, End-binding 1 (EB1) proteins are highly evolutionarily conserved and have been shown to interact with other known plus-end tracking proteins. Necessary for the recruitment of other proteins to the growing microtubule plus-end, EB1 is thought to form the core of a dynamic complex of proteins. The Arabidopsis genome encodes three EB1 proteins (called EB1a, EB1b and EB1c) that are expressed throughout the plant. In a second portion of my thesis, I report a dominant-negative approach to investigate the function of EB1 in plants. I find that different expression levels of GFP-EB1bC (GFP fused to the C-terminal protein interaction domain of EB1b) in both wild-type and eb1 triple mutant plants leads to dose-dependent defects such as shorter roots, aberrant lobing of leaf pavement cells and delayed leaf emergence as compared to untransformed plants. These phenotypes are associated with altered cell expansion and division and indicate an important role for EB1 in plant growth and development. My thesis research addresses the contribution of individual CMT activities to array organization and on plant growth and development using an interdisciplinary approach that scales from single molecules to whole plants