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。

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.