Microfilament organization in mitosis and transformation

描述(由申请人提供)：本提案的目的是通过确定肌球蛋白II(MLC)调节轻链的磷酸化在这些过程中的具体作用，阐明细胞分裂和细胞迁移的分子机制。我们的研究集中在肌球蛋白磷酸酶靶向亚基(MYPT)和肌球蛋白轻链激酶(MLCK)控制的关键通路上。具体目的1是确定MYPT在纺锤体组装和胞质分裂中的功能(S)。MYPT被cdc2激酶磷酸化，与Polo-like kinase1(PLK1)形成复合体。MYPT的耗竭延迟了前中期/中期的转变，并导致胞质分裂失败。由于PLK1是纺锤体组装和胞质分裂所必需的激酶，我们假设MYPT-PLK1联合调节这些过程。具体目的2是确定MLCK和MYPT在细胞定向迁移中的作用，并确定控制MLCK和MYPT的上游分子。MLCK控制细胞外周MLC的磷酸化，并限制细胞迁移过程中的膜突起。将产生针对MLCK和MYPT的磷酸化特异性抗体以及磷酸化位点突变，以确定哪些上游激酶对MLCK和MYPT起关键的空间调节作用。具体目的3是确定MLC磷酸化控制皮质肌动蛋白组装和调节3D基质中细胞迁移的机制。在蛋白酶抑制剂的存在下，肿瘤细胞调整其细胞形状和迁移方式，以克服基质的物理障碍：它收缩细胞体，像阿米巴一样移动。将进行分析，以确定在收缩介导的肿瘤细胞变形体运动过程中MLC磷酸化发生的时间和地点，并确定哪些上游分子对收缩环的组装和功能是必不可少的。这些研究将提供新的见解，不仅对正常细胞如何分裂和移动，而且还涉及癌症病理生理学的两个基本方面，即细胞分裂控制的丧失和转移。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the molecular mechanisms of cell division and cell migration, by defining the specific roles of phosphorylation of the regulatory light chain of myosin II (MLC) in these processes. Our studies focus on a critical pathway controlled by myosin phosphatase targeting subunit (MYPT), and myosin light chain kinase (MLCK). Specific aim 1 is to determine the function(s) of MYPT in spindle assembly and cytokinesis. MYPT is phosphorylated by cdc2 kinase, resulting in complex formation with polo-like kinase 1 (PLK1). MYPT depletion delays the prometaphase/metaphase transition and causes cytokinesis failure. Because PLK1 is an essential kinase for spindle assembly, as well as cytokinesis, we hypothesize that the MYPT-PLK1 association regulates these processes. Specific aim 2 is to determine the roles of MLCK and MYPT in directed cell migration, and to identify the upstream molecules that control MLCK and MYPT. MLCK controls MLC phosphorylation at the cell periphery and restricts membrane protrusions during cell migration. Phospho-specific antibodies against MLCK and MYPT, as well as phosphorylation-site mutants will be generated to determine which upstream kinases provide critical spatial regulation to MLCK and MYPT. Specific aim 3 is to determine the mechanism by which MLC phosphorylation controls the assembly of cortical actin and regulates cell migration in a 3D matrix. In the presence of protease inhibitors, a tumor cell adapts its cell shape and mode of migration to overcome the physical barrier of the matrix: it constricts its cell body and moves like an amoeba. Analyses will be performed to determine when and where MLC phosphorylation occurs during constriction-mediated amoeboid movement of tumor cells, and to identify which upstream molecules are essential for the assembly and function of the constriction ring. These studies will provide new insights, not only into how normal cells divide and move, but also into two fundamental aspects of cancer pathophysiology, the loss of cell division control and metastasis.