Molecular mechanisms of kinesins that control microtubule and actin polymerization dynamics

控制微管和肌动蛋白聚合动力学的驱动蛋白的分子机制

• 批准号: RGPIN-2019-05924
• 负责人: Allingham, John
• 金额: $ 3.64万
• 依托单位: Queen's 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=738597
• 关键词: Molecular; mechanisms; kinesins; control; microtubule

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.

微管和肌动蛋白丝形成动态网络来组织细胞核和细胞质，并感知和影响细胞形状。例如，微管生长和收缩以建立正确的有丝分裂纺锤体装置的大小和形态，从而实现准确的染色体分离。肌动蛋白丝迅速组装并捆绑在一起，形成更高级的结构，如收缩环，以介导细胞分裂。在这两种情况下，需要精确的时间和空间控制其聚合状态，但它们的晶格结构和机械性能的巨大差异需要不同的调节模式。我们的研究计划是提供一个机制的理解几种不同的微管解聚驱动蛋白的分子作用，和一个独特的驱动蛋白，促进肌动蛋白聚合。我们发现驱动蛋白-13家族酶的单体结合并促进串联微管蛋白二聚体的向外弯曲，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，驱动蛋白-13的微管弯曲力不需要ATP水解的能量。相反，ATP在驱动蛋白-微管蛋白复合物分离后水解。使用体外生物化学研究，全内反射荧光（TIRF）显微镜，X射线晶体学和小角X射线散射（SAXS）技术，我们现在将确定分离的驱动蛋白-13-微管蛋白复合物解离的生物化学和结构基础，使驱动蛋白-13可以催化去除额外的微管蛋白。这些相同的方法将被应用于理解驱动蛋白-8和驱动蛋白-14家族的成员如何利用ATP催化微管蛋白从微管末端解离。我们还将捕获和阐明驱动蛋白-8-和驱动蛋白-14-催化微管解聚的中间状态的结构，通过使用分子策略，帮助我们捕获驱动蛋白-13-微管蛋白复合物。为了将这一研究主题扩展到驱动蛋白领域的一个新领域，我们将研究驱动蛋白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短研究和化学生物学方法来确定这种驱动蛋白的肌动蛋白结合界面，然后应用X射线晶体学和电子显微镜（cryo-EM）来确定驱动蛋白3复合物结合肌动蛋白亚基和肌动蛋白聚合物的高分辨率结构。这些研究将为驱动蛋白的这种意想不到的活性提供分子解释，并可以指导其他混合驱动蛋白的设计。这些研究将最终导致更好地了解微管和肌动蛋白为基础的细胞上层结构的尺寸是如何动态调节的机制。通过本研究计划培训的人员将获得学术界和工业界所追求的生物化学和结构生物学研究方法的专业知识。