Assembly of Cytoskeletal Proteins into the Cleavage Furrow

这个项目的重点是负责动物细胞分裂的收缩装置。目的是了解肌动蛋白和肌球蛋白是如何组装成形成卵裂沟的收缩单元的，并确定这些单元是如何附着在细胞膜上的。将肌动蛋白、肌球蛋白、肌肽和血管蛋白的荧光类似物注射到活细胞中，并分析它们与卵裂沟的结合情况。将测量肌动蛋白和肌球蛋白在沟槽中组装和拆卸的动力学，并引入特定的抑制剂来验证这两种蛋白质是同时招募但彼此独立的假设。在有丝分裂期间，荧光肌动蛋白和肌球蛋白类似物的短期掺入将被测量，以确定肌动蛋白和肌球蛋白是否优先掺入赤道皮层。talin和vinculin的抑制剂将被引入有丝分裂细胞，以验证这些蛋白质在将肌动蛋白丝附着在卵裂沟膜上的作用。肌动蛋白和肌凝蛋白类似物的光活化和光漂白将用于确定是否存在肌动蛋白和肌凝蛋白从两极到赤道皮层的皮层流动以形成卵裂沟。所提出的实验的意义在于，它们有可能描述肌动蛋白和肌球蛋白是如何组装成收缩装置的，而收缩装置对于细胞分裂这一最基本的生物过程之一是必不可少的。

The focus of this project is on the contractile apparatus responsible for cytokinesis in animal cells. The goals are to understand how actin and myosin are assembled into the contractile units that form the cleavage furrow, and to determine how these units are attached to the cell membrane. Fluorescent analogues of actin, myosin, talin, and vinculin will be injected into living cells and their incorporation into cleavage furrows analyzed. The kinetics of assembly and disassembly of actin and myosin in the furrow will be measured and specific inhibitors introduced to test the hypothesis that the two proteins are recruited co-temporaneously but independently of one another. Short term incorporation of fluorescent actin and myosin analogues during mitosis will be measured to determine if actin and myosin are incorporated preferentially into the equatorial cortex. Inhibitors of talin and vinculin will be introduced into mitotic cells to test the idea that these proteins play a role in attaching actin filaments to the cleavage furrow membrane. Photoactivation and photobleaching of actin and myosin analogues will be used to determine if there is a cortical flow of actin and myosin from the poles to the equatorial cortex to form the cleavage furrow. The significance of the proposed experiments lies in their potential for describing how actin and myosin are assembled into the contractile apparatus that is essential for one of the most basic of biological processes: cell division.