Assembly of Cytoskeletal Proteins into the Cleavage Furrow

桑格细胞分裂是一个基本的细胞过程，其正确的执行对多细胞生物的正常发育至关重要。它需要肌动蛋白/肌球蛋白收缩系统的短暂组装，即卵裂沟，它具有肌肉细胞中的肌原纤维和非肌肉细胞中的应力纤维的一些特性。这些组合中的每一种都是由肌动蛋白、肌凝蛋白和相关蛋白聚集到细胞膜上的位点形成的，在那里它们被组织成可收缩的单元，形成附着在细胞膜上的附着物。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子介导的。这三种收缩系统中的每一种都是动态的，能够响应各种信号进行可逆的组装和拆卸，但解理沟的独特之处在于其收缩性和拆卸性的紧密耦合。拟议研究的长期目标是了解负责卵裂沟的特定位点组装和功能的因素。该建议的具体目的是：1，验证卵裂沟中的肌动蛋白丝通过类似于细胞间期肌动蛋白-肌球蛋白结构的机制附着在膜上的假设；2、检查肌凝蛋白磷酸化在肌凝蛋白向前庭募集中的作用，并验证肌动蛋白和肌凝蛋白可以相互独立募集的假设；3、检测分裂细胞中肌动蛋白的结合位点；4、确定在组织培养细胞和心肌细胞分裂过程中卵裂环的组装和收缩过程中钙水平的波动是否明显。许多显微成像方法将用于用各种探针微注射的活细胞，以分析裂解环主要成分分子的分布。所有提出的实验都是为了确定一些参数，这些参数负责细胞中最基本的过程之一：细胞质分裂的正常运作。这个项目关注的是单个细胞分裂成两个细胞的机制。在动物细胞中，我们已经知道这个过程包括在细胞赤道（或者，如果不是赤道，那么在定义细胞分裂平面的位置）组装细胞骨架细丝环。这些细丝附着在细胞膜上。然后，该环发生机械收缩，导致该平面上的细胞膜收缩。随着环的继续收缩，收缩变得越来越大，直到细胞将自己挤压成两个子细胞。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。这项工作意义重大，原因有很多。首先，细胞分裂是生命最基本的过程之一，更好地了解这一机制将有助于开发体外和体内方法，以便为各种生物医学和非医学生物技术目的操纵这一过程。其次，该过程是高度调控的，特定的生物大分子聚合物组装/拆卸和化学机械能量转导的一个很好的例子。对这一过程的更好理解将允许工程师和材料科学家直接将其作为生物分子“智能”材料或间接通过仿生纳米制造工程加以利用。＊＊＊

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. ***