Specification of actomyosin function in the cell

描述(由申请人提供)：该建议解决了细胞骨架领域的一个基本问题：细胞中不同的肌动蛋白结构如何招募适当的肌动蛋白结合蛋白，使其适应给定的角色？目前，我们主要集中在肌球蛋白-I是如何被导向内吞作用的部位(肌动蛋白补丁)，肌球蛋白-II在胞质分裂过程中如何在收缩环上发挥作用，以及肌球蛋白-V是如何被指定用于肌动蛋白电缆的细胞内运输的。裂解酵母目前是我们选择的模型，因为它易于处理的遗传学，明确定义的肌动蛋白结构，以及我们使用体内和体外方法检测肌球蛋白-I、-II和-V功能的能力。从长远来看，我们的目标是了解人类细胞中肌球蛋白运动功能的具体机制。我们推测，肌动蛋白轨迹的组成在将肌球蛋白马达引导到其适当的肌动蛋白结构中起着至关重要的作用。目的1将结合延时荧光显微镜和生化分析来评估肌动蛋白细丝交联物(纤毛蛋白和转明胶)在肌动蛋白斑块控制原肌球蛋白(TM)和肌球蛋白-I功能中的作用。目的2研究TM、α-肌动蛋白和肌球蛋白-II在肌动球蛋白环功能中的作用。在体外，肌球蛋白微珠分析将被用来确定TM在环组装过程中促进肌球蛋白II运动的机制。延时荧光和激光扫描共聚焦显微镜将与体外研究一起使用，以检查肌动蛋白细丝由α-肌动蛋白交联素在环收缩和重塑过程中的作用。尾巴引导肌球蛋白II自组装成整体(有利于环组装)的能力也将使用分析性超速离心法和电子显微镜进行测试。目标3试图了解肌动蛋白轨迹如何调节肌球蛋白-V沿电缆的运输。肌球蛋白微球分析和全内反射荧光显微镜(TIRF)对Myo52p运动的活体跟踪将被用来确定TM和平行细丝束如何调节肌球蛋白V运动功能和细胞内运动。我们的研究集中在高度保守的肌动蛋白结合蛋白的作用上，并将对肌动蛋白在人类癌症中的功能产生影响。肌动蛋白结构的变化促进了细胞的转化，因为肌动蛋白应激纤维为更动态的网络让路，促进了细胞的运动和转移。细胞的增殖依赖于肌动球蛋白环来促进分裂。在人类细胞中发现的TM的&gt；40亚型被认为在确定不同的肌动球蛋白结构方面发挥了关键作用。然而，这一想法很难测试，因为目前还不知道在这种复杂的非肌肉细胞中，哪些TM亚型调节哪些特定的肌球蛋白亚型。我们使用裂解酵母(有1个TM异构体和5个肌球蛋白)将克服这种复杂性，并为肌动球蛋白结构的规范提供新的和新的见解。

DESCRIPTION (provided by applicant): The proposal tackles a fundamental question in the cytoskeleton field: How do different actin structures in the cell recruit the appropriate actin-binding proteins that adapt them for their given roles? Presently, we are focused on how myosin-I is directed to sites of endocytosis (actin patches), how myosin-II functions at contractile rings during cytokinesis, and how myosin-V is specified for intracellular transport along actin cables. Fission yeast is currently our model of choice owing to its tractable genetics, well-defined actin architecture, and our ability to assay myosin-I, -II, and -V function using both in vivo and in vitro approaches. In the long-term we aim to understand mechanisms specifying myosin motor function in human cells. We hypothesize that the composition of the actin track plays a crucial role in directing myosin motors to their appropriate actin structures. Aim 1 will employ a combination of time-lapse epi-fluorescence microscopy and biochemical assays to assess the contribution of actin filament cross-linkers (fimbrin and transgelin) in controlling tropomyosin (Tm) and myosin-I function at actin patches. Aim 2 will study the role of Tm, a-actinin, and the myosin-II tail in actomyosin ring function. In vitro myosin-bead assays will be used to define the mechanism by which Tm promotes myosin-II motility during ring assembly. Time-lapse epi- fluorescence and laser-scanning confocal microscopy will be used along with in vitro studies to examine the role of actin filament cross-linking by a-actinin during ring constriction and remodeling. The ability of the tail to direct the self-assembly of myosin-II into ensembles (that favor ring assembly) will also be tested using analytical ultracentrifugation and electron microscopy. Aim 3 seeks to understand how the actin track regulates myosin-V transport along cables. Myosin-bead assays and in vivo tracking of Myo52p motility by total internal reflection fluorescence (TIRF) microscopy will be employed to determine how Tm and parallel filament bundling regulate myosin-V motor function and intracellular motility. Our studies focus on the role of highly conserved actin-binding proteins and will have implications for actin function in human cancers. Changes in actin structure facilitates cell transformation, as actomyosin stress fibers make way for a more dynamic network facilitating cell motility and metastasis. Cell proliferation relies on actomyosin rings to power division. The >40 isoforms of Tm found in human cells are believed to play a key role in specifying different actomyosin structures. However, this idea has been difficult to test because it is not yet known which Tm isoforms regulate which specific myosin isoforms in such complex, non-muscle cells. Our use of fission yeast (with 1 Tm isoform and 5 myosins total) will overcome this complexity and provide new and novel insights into the specification of actomyosin structures.