Assembly of Cytoskeletal Proteins into the Cleavage Furrow

小桑格9319041 胞质分裂是一个基本的细胞过程，其正确的执行是多细胞生物正常发育所必需的。 它需要肌动蛋白/肌球蛋白收缩系统的短暂组装，即卵裂沟，其具有肌细胞中肌原纤维和非肌细胞中应力纤维的一些特性。 这些集合体中的每一种都是通过将肌动蛋白、肌球蛋白和相关蛋白募集到细胞膜上的位点而形成的，在细胞膜上，它们被组织成与膜形成附着的收缩单位。 在每个系统中，收缩都需要ATP，至少在肌肉中，收缩是由钙离子等因素介导的。 三个收缩系统中的每一个都是动态的，并且能够响应于各种信号可逆地组装和拆卸，但是卵裂沟在其收缩性和拆卸的紧密耦合中是独特的。 拟议的研究的长期目标是了解的因素，负责特定地点的组装和功能的卵裂沟。 本研究的具体目的是：1、验证以下假设：卵裂沟中的肌动蛋白丝是通过类似于细胞用于其间期肌动蛋白-肌球蛋白结构的机制附着在膜上的; 2,研究肌球蛋白磷酸化在肌球蛋白向前额募集中的作用，并验证肌动蛋白和肌球蛋白可以独立募集的假说。另一个; 3，检查肌动蛋白在裂解细胞中的掺入位点;和4，确定在分裂的组织培养细胞和心肌细胞中裂解环的组装和收缩期间钙水平的波动是否明显。 许多显微镜成像方法将用于活细胞，这些活细胞已经显微注射了各种探针，以分析裂解环的主要组分分子的分布。 所有提议的实验都是 旨在定义一些负责细胞中最基本过程之一的正常功能的参数：胞质分裂。 这个项目的重点是一个细胞从字面上分裂成两个的机制。 在动物细胞中，我们已经知道，这一过程涉及在细胞赤道（如果不是赤道，则在确定细胞分裂平面的位置）组装一圈细胞骨架丝。 这些细丝附着在细胞膜上。 然后发生该环的机械收缩，这导致该平面处的细胞膜收缩。 随着环的继续收缩，收缩变得越来越大，直到细胞将自己捏成两个子细胞。 该项目旨在详细了解收缩丝附着在膜上的机制，环的蛋白质构建块被招募到环的位置的机制，以及细胞调节这一过程的生化机制。 这项工作的重要性有许多原因。 首先，细胞分裂是生命最基本的过程之一，更好地理解这一机制将有助于开发用于各种生物技术目的（生物医学和非医学）的体外和体内方法。 第二，该过程是高度调节的，特定的生物大分子聚合物组装/拆卸和化学机械能量转换的一个很好的例子。 更好地理解这一过程将允许工程师和材料科学家利用它，无论是直接作为生物分子“智能”材料，还是间接通过仿生纳米纤维工程。 ***

9319041 Sanger Cytokinesis is a fundamental cellular process whose corrrect execution is essential for the normal development of muticellular organisms. It requires the transitory assembly of an actin/myosin contractile system, the cleavage furrow, that shares some of the properties of myofibrils in muscle cells and of stress fibers in non-muscle cells. Each of these assemblages is formed by the recruitment of actin, myosin and associated proteins to sites on the cell membrane where they become organized into contractile units that form attachments to the membrane. In each system, contraction requires ATP, and in muscle, at least, contraction is mediated by factors such as calcium ions. Each of the three contractile systems is dynamic and capable of reversible assembly and disassembly in response to a variety of signals, but the cleavage furrow is unique in the close coupling of its contractility and disassembly. The long term goals of the proposed research are to understand the factors responsible for the site-specific assembly and functioning of the cleavage furrow. The specific aims of this proposal are: 1, to test the hypothesis that the actin filaments in the cleavage furrow are attached to the membrane by a mechanism similar to that used by the cell for its interphase actin-myosin structures; 2, to examine the role of myosin phosphorylation in recruitment of myosin to the forrow and test the hypotehsis that actin and myosin can be recruited independently of one another; 3, to examine the sites of incorporation of actin in cleaving cells; and 4, to determine if fluctuations in calcium levels are evident during the assembly and contraction of the cleavage ring in dividing tissue culture cells and cardiomyocytes. A number of microscopic imaging methods will be used with living cells that have been microinjected with various probes to analyze the distribution of the major component molecules of the cleavage ring. All of the proposed experiments are designed to define some of the parameters responsible for the proper functioning of one of the most basic processes in cells: cytokinesis. %%% This project focuses on the mechanisms by which a single cell literally divides itself into two. In animal cells, we already know that this process involves the assembly of a ring of cytoskeletal filaments at the equator of the cell (or, if not equatorial, then at a position which defines the plane of cell division). These filaments are attached to the cell membrane. A mechanical contraction of that ring then occurs, which results in a constriction of the cell membrane at that plane. As the contraction of the ring continues, the constriction becomes greater and greater until the cell pinches itself off into two daughter cells. This project aims to learn in detail the mechanism by which the contractile filaments are attached to the membrane, the mechanism by which the protein building blocks of the ring are recruited to the site of the ring, and the biochemical mechanism whereby the cell regulates this process. This work is significant for many reasons. First, cell division is one of the most fundamental processes of life, and a better understanding of the mechanism would permit the development of in vitro and in vivo methods for manipulating the process for various biotechnological ends, both biomedical and non-medical. Second, the process is an excellent example of highly regulated, specific biomacromolecular polymer assembly/disassembly and chemomechanical energy transduction. A better understanding of this process would permit its exploitation by engineers and materials scientists, either directly as a biomolecular "smart" material or indirectly through biomimetic nanofabrication engineering. ***