ACTIN-MYOSIN INTERACTIONS IN CELL MOTILITY

本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， 运动力耦合ATP水解，（2）表征 肌球蛋白-1的细胞器运动机制和（3）确定 控制肌球蛋白积累的机制及其 在胞质分裂期间激活分裂沟。 达成 为实现这些目标，我们建议开展以下研究： 项目1，肌动球蛋白中间体的电子显微镜 周期 我们将使用一种新型的快速混合/停流快速- 冷冻装置，以捕获肌球蛋白中间体， 在肌动球蛋白ATP酶循环中与肌动蛋白丝结合。 后 冷冻断裂，这些中间体的结构将是 通过电子显微镜直接研究， 产生力和运动的变化。 项目2. 肌球蛋白II的组装和功能。 表征 肌球蛋白组装和功能中的分子事件 纤维在非肌肉细胞，我们将使用各种生物物理 建立装配机制的方法及其调控 其他蛋白质。 这将包括单克隆抗体的作用。 抗体对组装、ATP酶活性的影响以及 能动性 该分子的cDNA将在细菌中表达 为了精确地绘制40多个单克隆抗体的结合位点， 抗体，为生产新的 单克隆抗体和产生通过缺失修饰的肌球蛋白 或体外点突变用于功能的生化测试。 项目3。 肌球蛋白-1与细胞膜的相互作用。 创新实施 定量测定来表征肌球蛋白-1与 分离的细胞器的膜，并测试重组的 细胞膜上有沿着肌动蛋白丝移动的能力。 到 建立肌球蛋白-1的膜结合区，我们将测试 在细菌中表达的蛋白质的明确定义的片段， 克隆cDNA的能力，以结合到膜。 我们还将 用肌球蛋白-1进行肌动蛋白运动性的体外测定， 确立2类重链的调控功能 激酶。 项目4。 胞质分裂的调节。 我们将生产单克隆 肌球蛋白轻链磷酸化位点的抗体 并利用它们来绘制肌球蛋白 在细胞分裂过程中磷酸化。 在平行实验中， 将用荧光染料修饰分离的肌球蛋白轻链， 当它们被显微注射到活细胞中（在那里它们将 与肌球蛋白重链重组），我们可以跟踪 细胞分裂时的肌球蛋白。 通过修改这些 在蛋白质上具有硫代磷酸的荧光轻链 激酶C位点或肌球蛋白轻链激酶位点，我们将 确定这些修改之一或两者是否影响 胞质分裂时肌球蛋白进入分裂沟的运动。

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.