Specification of actomyosin function in the cell

描述（由申请人提供）：该提案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，使其适应其特定的作用？目前，我们的重点是肌球蛋白-I是如何被定向到内吞作用的网站（肌动蛋白补丁），肌球蛋白-II如何在收缩环在胞质分裂过程中的功能，以及肌球蛋白-V是如何指定的细胞内运输沿着肌动蛋白电缆。裂变酵母是目前我们的选择模型，由于其易于处理的遗传学，明确的肌动蛋白结构，我们的能力，测定肌球蛋白-I，-II，和-V的功能，在体内和体外的方法。从长远来看，我们的目标是了解在人类细胞中肌球蛋白运动功能的机制。 我们推测，肌动蛋白轨道的组成起着至关重要的作用，在指导肌球蛋白电机，以其适当的肌动蛋白结构。目的1将采用延时落射荧光显微镜和生化分析相结合，以评估肌动蛋白丝交联剂（fimtrin和transgelin）在控制原肌球蛋白（Tm）和肌球蛋白-I在肌动蛋白补丁功能的贡献。目的2研究Tm、α-辅肌动蛋白和肌球蛋白-II尾在肌动球蛋白环功能中的作用。将使用体外肌球蛋白珠测定来确定Tm在环组装期间促进肌球蛋白II运动的机制。将使用延时epi荧光和激光扫描共聚焦显微镜沿着体外研究，以检查在环收缩和重塑过程中肌动蛋白丝通过α-辅肌动蛋白交联的作用。尾部引导肌球蛋白II自组装成集合体（有利于环组装）的能力也将使用分析性超离心和电子显微镜进行测试。目的3试图了解肌动蛋白轨道如何调节肌球蛋白-V沿沿着电缆的运输。将采用肌球蛋白珠测定和通过全内反射荧光（TIRF）显微镜对Myo 52 p运动性进行体内跟踪，以确定Tm和平行丝束如何调节肌球蛋白-V运动功能和细胞内运动性。 我们的研究集中在高度保守的肌动蛋白结合蛋白的作用，并将对人类癌症中的肌动蛋白功能产生影响。肌动蛋白结构的变化促进细胞转化，因为肌动球蛋白应力纤维为促进细胞运动和转移的更动态的网络让路。细胞增殖依赖于肌动球蛋白环来驱动分裂。在人类细胞中发现的Tm的>40种同种型被认为在指定不同的肌动球蛋白结构中起关键作用。然而，这个想法一直难以测试，因为它还不知道哪些Tm异构体调节这种复杂的非肌肉细胞中的特定肌球蛋白异构体。我们使用的裂变酵母（1 Tm异构体和5肌球蛋白总数）将克服这种复杂性，并提供新的和新颖的见解肌动球蛋白结构的规格。

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.