Molecular mechanisms of kinesins that control microtubule and actin polymerization dynamics

微管和肌动蛋白细丝形成动态网络，组织细胞核和细胞质，感知和影响细胞形状。例如，微管的生长和收缩建立了正确的有丝分裂纺锤体的大小和形态，从而实现了准确的染色体分离。肌动蛋白细丝在更高级的结构中快速组装和捆绑在一起，如收缩环，以调节细胞分裂。在这两种情况下，都需要对它们的聚合态进行精确的时间和空间控制，但它们的晶格结构和力学性质的巨大差异需要不同的调节模式。我们的研究计划提供了对几种不同微管解聚运动蛋白的分子作用的机械理解，以及一种促进肌动蛋白聚合的独特运动蛋白。我们发现，Kinesin-13家族酶的单体结合并促进串联微管蛋白二聚体的外弯，以削弱微管晶格，并催化微管蛋白从微管末端快速脱离。我们还表明，Kinesin-13的微管弯曲力不需要来自ATP水解的能量。相反，在肌动蛋白-微管蛋白复合体分离后，ATP被水解。利用体外生化研究、全内反射荧光(TIRF)显微镜、X射线结晶学和小角X射线散射(SAXS)技术，我们现在将确定分离的Kinesin-13-微管蛋白复合体解离的生化和结构基础，以便Kinesin-13可以催化去除额外的微管蛋白。这些相同的方法将被用来理解Kinesin-8和Kinesin-14家族的成员如何利用ATP来催化微管末端的微管蛋白解离。我们还将利用帮助我们捕获Kinesin-13-微管蛋白复合体的分子策略，捕获并阐明Kinesin-8-和Kinesin-14催化的微管解聚的中间态结构。为了将这一研究主题扩展到Kinesin领域的新领域，我们将研究Kinesin-3马达蛋白中的一个独特模块刺激肌动蛋白聚合和捆绑的机制。我们将使用蛋白质截断研究和化学生物学方法来确定该Kinesin的肌动蛋白结合界面，然后应用X射线结晶学和电子显微镜(Cryo-EM)分别确定与肌动蛋白亚单位和肌动蛋白聚合物结合的Kinesin-3复合体的高分辨结构。这些研究将为激动素这种意想不到的活性提供分子解释，并可能指导其他杂交动蛋白的设计。这些研究最终将导致对微管和肌动蛋白为基础的细胞超结构的尺寸是如何动态调节的更好的机械性理解。通过这一研究计划培训的人员将获得受到学术界和工业界追捧的生化和结构生物学研究方法方面的专业知识。

Microtubules and actin filaments form dynamic networks to organize the nucleus and cytoplasm, and to sense and influence cell shape. For example, microtubules grow and shrink to establish the correct size and morphology of the mitotic spindle apparatus for accurate chromosome segregation. Actin filaments are rapidly assembled and bundled together within higher-order structures like the contractile ring to mediate cell division. In both cases, precise temporal and spatial control of their polymeric state is required, but the large differences in their lattice structure and mechanical properties necessitate differential modes of regulation. Our research program is providing a mechanistic understanding of the molecular actions of several different microtubule-depolymerizing kinesins, and a unique kinesin that promotes actin polymerization. We showed that monomers of kinesin-13 family enzymes bind and promote outward bending of tandem tubulin dimers in order to weaken the microtubule lattice and catalyze rapid tubulin detachment from microtubule ends. We also showed that the tubulin-bending force of kinesin-13 does not require energy from ATP hydrolysis. Instead, ATP is hydrolyzed after the kinesin-tubulin complex detaches. Using in vitro biochemical studies, Total Internal Reflection Fluorescence (TIRF) microscopy, X-ray crystallography and small-angle X-ray scattering (SAXS) techniques, we will now determine the biochemical and structural basis for dissociation of the detached kinesin-13-tubulin complex, so that kinesin-13 can catalyze removal of additional tubulins. These same methods will be applied to understand how members of the kinesin-8 and kinesin-14 families employ ATP to catalyze tubulin dissociation from microtubule ends. We will also trap and elucidate the structures of intermediate states of kinesin-8- and kinesin-14-catalyzed microtubule depolymerization by using the molecular strategies that helped us trap the kinesin-13-tubulin complex. To extend this research theme into a new realm for the kinesin field, we will investigate the mechanism by which a unique module in a kinesin-3 motor protein stimulates actin polymerization and bundling. We will use protein truncation studies and chemical biology methods to identify the actin binding interfaces of this kinesin, and then apply X-ray crystallography and electron microscopy (cryo-EM) to determine high-resolution structures of kinesin-3 complexes bound to actin subunits and actin polymers, respectively. These studies will provide a molecular explanation for this unexpected activity of a kinesin, and could guide other hybrid kinesin designs. These studies will ultimately lead to a better mechanistic understanding of how the dimensions of microtubule- and actin-based cellular superstructures are dynamically regulated. Personnel trained by this research program will acquire expertise in biochemical and structural biology research methods that are sought-after by academia and industry.