Non-mammalian Microtubules and Microtubule-Associated Proteins

非哺乳动物微管和微管相关蛋白

• 批准号: RGPIN-2020-04876
• 负责人: Brouhard, Gary
• 金额: $ 3.64万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739852
• 关键词: Non; mammalian; Microtubules; Microtubule; Associated

In biology, diversity is our strength: comparisons across diverse species are a source of many critical insights. Such comparisons can distinguish fundamental mechanisms, which are conserved across great evolutionary distances, from divergent mechanisms, which point to speciation and specialized function. An example of a fundamental mechanism is the dynamic instability of microtubules: in all eukaryotes, microtubules grow by polymerization, switch to shrinkage in "catastrophes," and switch back to growth in "rescues." The ab-tubulin dimer itself is highly conserved. Also conserved is the 13 protofilament (pf) lattice, although there is more divergence in microtubule structure than is generally appreciated. Microtubules also appear to diverge in the conformational changes that accompany GTP hydrolysis, wherein some lattices undergo a "compaction" and others do not. Therefore, we lack a universally-applicable explanation of dynamic instability that distinguishes conserved and divergent phenomena. Aim 1. Investigate the dynamic instability of microtubules from non-mammalian model organisms Our ability to purify tubulin from any eukaryotic source opens up a broad range of organisms that were previously intractable to biophysical study. We can purify tubulin from two major model organisms, namely the nematode worm C. elegans and the fruit fly D. melanogaster. By reconstituting dynamic instability from these eukaryotes, we will learn how tubulin has diverged over evolutionary distances, discovering what is conserved and what is divergent. Aim 2. Determine the evolutionarily-conserved response to GTP hydrolysis Tubulin is a GTPase, and when GTP is hydrolyzed, mammalian microtubules undergo a large-scale "compaction" of their lattices. This compaction was thought to explain how GTP hydrolysis destabilizing the polymer. But microtubules from yeast (S. pombe) and C. elegans do not appear to undergo compaction. By solving the cryo-EM structures of Drosophila microtubules, we will determine if there is an evolutionarily-conserved response to GTP hydrolysis. Aim 3. Investigate the co-evolution of microtubule-associated proteins in divergent species. Cells control their microtubules using microtubule-associated proteins (MAPs), such as microtubule polymerases, depolymerases, and end-binding proteins. We hypothesize that MAPs may have co-evolved to be responsive to the unique structure and dynamics of their cognate microtubules. We will test these ideas by expressing and purifying candidate MAPs from C. elegans and Drosophila and reconstituting their interactions with cognate microtubules. Overall, these experiments will define the fundamental mechanism of microtubule dynamic instability by determining which features are conserved across evolution and which are divergent.

在生物学中，多样性是我们的优势：不同物种之间的比较是许多重要见解的来源。这种比较可以区分基本机制，这是保守的跨越很大的进化距离，从不同的机制，这指向物种形成和专门的功能。基本机制的一个例子是微管的动态不稳定性：在所有真核生物中，微管通过聚合生长，在“灾难”中切换到收缩，在“拯救”中切换回生长。“ab-微管蛋白二聚体本身是高度保守的。同样保守的是13原丝（pf）晶格，虽然有更多的分歧，微管结构比一般理解。微管也出现分歧，伴随GTP水解的构象变化，其中一些晶格进行“压实”和其他不。因此，我们缺乏一个普遍适用的解释动力学不稳定性，区分保守和发散现象。目标1。研究来自非哺乳动物模式生物的微管的动态不稳定性我们从任何真核生物来源纯化微管蛋白的能力开辟了以前难以生物物理研究的广泛生物。我们可以从两种主要的模式生物中纯化微管蛋白，即线虫C。elegans和果蝇D.黑腹菌通过重建这些真核生物的动态不稳定性，我们将了解微管蛋白如何在进化距离上分化，发现什么是保守的，什么是分歧的。目标2.微管蛋白是一种GTP酶，当GTP水解时，哺乳动物微管的晶格会发生大规模的“压缩”。这种压实被认为解释了GTP水解如何使聚合物不稳定。但酵母中的微管（S. pombe）和C.秀丽线虫似乎不经历致密化。通过解决果蝇微管的cryo-EM结构，我们将确定是否有一个进化保守的响应GTP水解。目标3.研究微管相关蛋白在不同物种中的协同进化。细胞使用微管相关蛋白（MAP）控制其微管，如微管聚合酶、解聚酶和末端结合蛋白。我们推测，地图可能已经共同进化，以响应其同源微管的独特结构和动力学。我们将通过表达和纯化来自C的候选MAP来测试这些想法。elegans和Drosophila，并重建它们与同源微管的相互作用。总的来说，这些实验将通过确定哪些特征在进化过程中是保守的，哪些是发散的，来定义微管动态不稳定性的基本机制。