Non-mammalian Microtubules and Microtubule-Associated Proteins

在生物学中，多样性是我们的优势：跨不同物种的比较是许多关键见解的来源。这样的比较可以将跨越进化距离的基本机制与指向物种形成和专门化功能的差异机制区分开来。一个基本机制的例子是微管的动态不稳定：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高度保守。13原细丝(PF)晶格也是保守的，尽管在微管结构中有比通常认为的更多的分歧。微管似乎也在伴随着GTP水解的构象变化中出现分歧，其中一些晶格经历了“压缩”，而另一些则没有。因此，我们缺乏一种普遍适用的动力不稳定性的解释来区分守恒现象和发散现象。目的1.研究非哺乳动物模式生物微管的动态不稳定性我们从任何真核来源中纯化微管蛋白的能力开辟了许多以前难以进行生物物理研究的生物。我们可以从两种主要的模式生物，即线虫线虫和果蝇D黑腹果蝇中纯化微管蛋白。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是如何在进化距离上分化的，发现哪些是保守的，哪些是分歧的。目的2.确定GTP水解酶对微管蛋白在进化上的保守反应，当GTP被水解时，哺乳动物的微管发生大规模的格子“压缩”。这种压实作用被认为可以解释GTP的水解性如何破坏聚合物的稳定性。但酵母(S.pombe)和线虫的微管似乎不会发生压缩。通过解决果蝇微管的冷冻-EM结构，我们将确定是否存在对GTP水解的进化保守反应。目的3.研究微管相关蛋白在不同物种中的共同进化。细胞使用微管相关蛋白(MAPs)控制其微管，如微管聚合酶、解聚酶和末端结合蛋白。我们假设，MAP可能已经共同进化为对其同源微管的独特结构和动力学做出反应。我们将通过表达和纯化线虫和果蝇的候选图谱，并重建它们与同源微管的相互作用来测试这些想法。总体而言，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。

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.