Non-mammalian Microtubules and Microtubule-Associated Proteins

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。这样的比较可以区分基本机制和分化机制，前者在漫长的进化过程中是保守的，后者指向物种形成和特化功能。一个基本机制的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”中转为收缩，在“救援”中转为增长。抗体-微管蛋白二聚体本身是高度保守的。同样保守的还有13个原丝（pf）晶格，尽管微管结构的差异比通常认为的要大。伴随GTP水解的构象变化中，微管也出现了分歧，其中一些晶格经历了“压实”，而另一些则没有。因此，我们缺乏一个普遍适用的动力不稳定性的解释，以区分保守和发散现象。目的1。我们能够从任何真核生物源中纯化微管蛋白，这为以前难以进行生物物理研究的生物开辟了广阔的领域。我们可以从线虫和果蝇两种主要的模式生物中纯化微管蛋白。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在进化距离上是如何分化的，发现什么是保守的，什么是分化的。目标2。确定对GTP水解的进化保守反应微管蛋白是一种GTP酶，当GTP被水解时，哺乳动物微管的晶格会发生大规模的“压实”。这种压实被认为解释了GTP水解如何破坏聚合物的稳定。但酵母（S. pombe）和秀丽隐杆线虫的微管似乎不会被压实。通过解决果蝇微管的低温电镜结构，我们将确定是否存在对GTP水解的进化保守反应。目标3。研究不同物种中微管相关蛋白的共同进化。细胞利用微管相关蛋白（MAPs）控制它们的微管，如微管聚合酶、解聚合酶和末端结合蛋白。我们假设map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秀丽隐杆线虫和果蝇的候选map，并重建它们与同源微管的相互作用来验证这些想法。总的来说，这些实验将通过确定哪些特征在进化中是保守的，哪些是发散的来定义微管动态不稳定性的基本机制。

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.