Role of a Minor Ubiquitous Beta-Tubulin in Microtubule Assembly

微管是真核细胞纳米级结构的重要特征。这些细胞骨架元素赋予细胞形状和运动性。它们是亚细胞结构的组织者，是纳米级分子马达将亚细胞细胞器（包括染色体和囊泡）以主动定向的方式从细胞的一个地方移动到另一个地方的轨迹，也是可运动的细胞附属物（纤毛和鞭毛）的关键组成部分。微管是由一类叫做微管蛋白的独特蛋白质组成的。微管蛋白有α -和β -亚型。脊椎动物产生6-7种不同的β -微管蛋白基因产物，这些基因在物种间具有良好的保守性，并在不同组织中表达差异。这种独特的β -微管蛋白基因产物的存在导致了不同的微管蛋白具有不同功能的假设。然而，迄今为止的大多数数据表明，不同的同型在微管组装的功能上是可互换的。然而，与这些早期的研究相反，最近的研究表明，V类β -微管蛋白，通常是一种低丰度但广泛分布的蛋白质，在细胞中过度表达时具有破坏微管和抑制细胞增殖的强大能力。本研究项目的目标是确定正常（低）细胞水平的β -5-微管蛋白的自然功能。实验的目的是询问低丰度的β -5-微管蛋白是否对细胞增殖是必要的，并阐明β -5-微管蛋白影响微管组装的机制。抑制性rna将用于减少或消除β -5-微管蛋白的表达，并检查对细胞生长和细胞周期进展的影响。由于在过度表达β -5-微管蛋白的细胞中，微管组装被显著破坏，因此假设β -5-微管蛋白被细胞用来保持微管处于动态状态。这个想法将通过使用荧光标记的微管结合蛋白GFP-MAP4来测试活细胞中过度或不足表达β -5微管蛋白的微管的动态方面（生长和收缩）。最后，β -5微管蛋白的定点诱变将用于鉴定对蛋白质对微管动力学的影响至关重要的结构元件。将关键残基映射到已发表的最丰富的β -微管蛋白异构体的晶体结构将为亚基相互作用提供重要线索，这些相互作用可能受到β -5微管蛋白掺入微管的影响。知识价值：这些研究将为微管组装中尚未充分研究和潜在关键的β -5微管蛋白亚基提供重要的新信息。了解β -5微管蛋白在调节微管的组装和拆卸中的可能参与，可能是揭示细胞通过改变其组成微管蛋白的组成来调节微管动态功能的先前未被认识的机制的第一步。更广泛的影响：该项目将作为培养博士后和研究生的工具。此外，关于细胞如何调节微管组装的新基础知识和理解可能会通过各种不同的手段为社会带来尚未预料到的利益，例如，自组装纳米结构的仿生工程或作物植物的基因工程（因为微管是某些类型的农业除草剂的主要目标）。

Microtubules are critical nanoscale structural features of eukaryotic cells. These cytoskeletal elements confer shape and motility to the cells. They serve as organizers of subcellular structure, as tracks along which nanoscale molecular motors move subcellular organelles (including chromosomes and vesicles) in actively directed fashion from one place in the cell to another, and as key components of motile cellular appendages (cilia and flagella). Microtubules are made up of a unique class of proteins called tubulins. Tubulins come in alpha- and beta- isoforms. Vertebrates produce 6-7 distinct beta-tubulin gene products that are well conserved across species and are differentially expressed among various tissues. The existence of such distinct beta-tubulin gene products has led to the hypothesis that different tubulins subserve different functions. However, most of the data to date suggest that the different isotypes are functionally interchangeable for microtubule assembly. In contrast to these earlier studies, however, recent work has demonstrated that class V beta-tubulin, which is normally a low abundance but widely distributed protein, has a potent ability to disrupt microtubules and inhibit cell proliferation when overexpressed in cells. The goal of this research project is to determine the natural function of normal (low) cellular levels of beta-5-tubulin. The experiments are directed toward asking whether a low abundance of beta-5-tubulin is necessary for cell proliferation and toward elucidation of the mechanism by which beta-5-tubulin affects microtubule assembly. Inhibitory RNAs will be used to reduce or eliminate beta-5-tubulin expression, and the effects on cell growth and cell cycle progression will be examined. Because microtubule assembly is dramatically disrupted in cells that overexpress beta-5-tubulin, it is hypothesized that beta-5-tubulin is used by cells to keep the microtubules in a dynamic state. This idea will be tested by using a fluorescently-marked microtubule-binding protein, GFP-MAP4, to measure the dynamic aspects (growth and shrinkage) of microtubules in living cells that over- or under-express beta-5-tubulin. Finally, site-directed mutagenesis of beta-5-tubulin will be used to identify structural elements that are critical for the protein's effect on microtubule dynamics. Mapping the critical residues to the published crystal structure of the most abundant beta- tubulin isoform will provide important clues about the subunit interactions that are likely to be impacted by incorporation of the beta-5-tubulin into microtubules. Intellectual Merit:These studies will provide important new information about an understudied and potentially critical beta-5-tubulin subunit for microtubule assembly. Understanding the putative involvement of beta-5-tubulin in modulating the assembly and disassembly of microtubules may be the first step in uncovering a previously unappreciated mechanism that cells use to regulate the dynamic function of microtubules by altering the composition of their constituent tubulin proteins. Broader Impacts:The project will serve as a vehicle for training postdoctoral and graduate students. In addition, new fundamental knowledge and understanding of how cells regulate microtubule assembly may lead to as-yet unanticipated benefits to society through a variety of disparate means, e.g., biomimetic engineering of self-assembling nanostructures or genetic engineering of crop plants (since microtubules are the primary target of certain types of agricultural herbicides).