ACTIN-MYOSIN INTERACTIONS IN CELL MOTILITY

这项研究计划的长期目标是：(1)建立 肌动球蛋白复合体的结构变化 与ATP水解力耦合的运动力，(2)表征 肌球蛋白-1和(3)决定细胞器运动的机制 肌球蛋白蓄积的控制机制及其研究进展 胞质分裂过程中卵裂沟的激活。要达到 为了实现这些目标，我们建议进行以下研究： 项目1，肌动球蛋白中间体的电子显微镜 周而复始。我们将使用一种新型的快速混合/停流快速除尘器。 用于捕获弱肌球蛋白中间体的冷冻装置 在肌动球蛋白ATPase循环中与肌动蛋白细丝结合。之后 冷冻破裂，这些中间体的结构将是 直接通过电子显微镜研究来确定结构 产生力量和运动的变化。 项目2.肌球蛋白-II的组装和功能。刻画 肌球蛋白组装和功能中的分子事件 在非肌肉细胞的细丝中，我们将使用各种生物物理 方法建立装配及其调控机制 通过其他蛋白质。这将包括单抗的影响。 抗体的组装、ATPase活性和体外检测 能动性。分子的cDNAs将在细菌中表达 精确绘制40多个单抗的结合位点图 抗体，为生产新的 并产生通过缺失修饰的肌球蛋白 或在体外进行点突变以进行功能的生化测试。 项目3.肌球蛋白-1与膜的相互作用。我们会带着 定量分析肌球蛋白-1与肌球蛋白结合的特性 分离细胞器的膜并测试重组的 膜具有沿肌动蛋白细丝移动的能力。至 建立肌球蛋白-1的膜结合区，我们将测试 在细菌中表达的明确的蛋白质片段 克隆的cDNA具有与膜结合的能力。我们还将 肌球蛋白-1对肌动蛋白运动性的体外检测 确立2类重链的调控功能 激活剂。 项目4.胞质分裂的调节。我们将生产单克隆体 肌球蛋白轻链磷酸化位点的抗体 并用它们绘制出肌球蛋白出现的时间和地点 在细胞分裂过程中被磷酸化。在平行实验中，我们 将用荧光染料修饰分离的肌球蛋白轻链 在它们被显微注射到活细胞中后(它们将在那里 与肌球蛋白重链重新结合)我们可以跟踪 细胞分裂过程中的肌球蛋白。通过还修改这些 蛋白质上带有硫代磷酸的荧光光链 或肌球蛋白轻链激酶位点，我们将 确定这些修改中的一项或两项是否会影响 胞质分裂时肌球蛋白进入卵裂沟的运动。

The long range goals of this research program are to (1) establish the structural changes in the actomyosin complex that produce motile force coupled to ATP hydrolysis, (2) characterize the mechanism of organelle movements by myosin-1 and (3) determine the mechanisms that control the accumulation of myosin and its activation in the cleavage furrow during cytokinesis. To reach these goals, we propose the following studies: Project 1, Electron microscopy of intermediate in the actomyosin cycle. We will use a novel rapid-mixing/stopped-flow rapid- freezing device to capture the myosin intermediates that are weakly bound to actin filaments in the actomyosin ATPase cycle. After freeze-fracturing, the structure of these intermediates will be studied directly by electron microscopy to establish the structural changes that produce force and motion. Project 2. Assembly and function of myosin-II. To characterize the molecular events in the assembly and function of myosin filaments in non-muscle cells, we will use a variety of biophysical methods to establish the mechanism of assembly and its regulation by other proteins. This will include the effects of monoclonal antibodies on assembly, ATPase activity and in vitro assays for motility. cDNA's for the molecule will be expressed in bacteria to map precisely the binding sites for more than 40 monoclonal antibodies, to provide antigens for the production of new monoclonal antibodies and to produce myosins modified by deletions or point mutations in vitro for biochemical tests for function. Project 3. Interaction of myosin-1 with membranes. We will carry out quantitative assays to characterize the binding of myosin-1 to the membranes of isolated organelles and test the reconstituted membranes for their ability to move along actin filaments. To establish the membrane binding region of the myosin-1, we will test well defined segments of the protein expressed in bacteria from cloned cDNAs for their ability to bind to membranes. We will also carry out in vitro assays of actin motility with myosin-1 to establish the regulatory functions of 2 classes of heavy chain kinases. Project 4. Regulation of cytokinesis. We will produce monoclonal antibodies to the phosphorylated sites of the myosin light chain and use them to map out the times and places where myosin is phosphorylated during cell division. In parallel experiments we will modify isolated myosin light chains with a fluorescent dye so that after they are microinjected into live cells (where they will recombine with myosin heavy chains) we can follow the movements of the myosin during cell division. By also modifying these fluorescent light chains with thiophosphate on either the protein kinase C site or the myosin light chain kinase site, we will establish whether either or both of these modifications influence the movement of the myosin into the cleavage furrow at cytokinesis.