Myosin-II Dynamics and Cytokinesis

描述（由申请方提供）：肌球蛋白II双极粗丝形成在细胞中受到高度调节，是胞质分裂所需的，是收缩环的关键组分。本申请的长期目标是从分子水平了解肌球蛋白II双极粗丝组装的调节，并阐明细胞分裂过程中收缩环形成、维持和溶解的时空控制。D.盘状突在胞质分裂研究中具有许多优势，将被用作模型系统。研究计划旨在回答以下问题。肌球蛋白II重链磷酸化如何在分子水平上控制肌球蛋白II双极粗丝的组装？三种已知的肌球蛋白-II重链激酶在细胞周期中是如何组织的，这种组织与肌球蛋白-II收缩环的动力学有何关系？在含肌球蛋白II的收缩环的建立、维持和溶解中涉及的其他必需蛋白质是什么？它们在细胞分裂过程中的细胞组织和动力学是什么？分子遗传学方法将被用来创建定向突变的肌球蛋白-II尾部结构域，以测试特定的假设的机制，厚丝组装的调节。将这些突变肌球蛋白II转化为网骨藻肌球蛋白II无效细胞，以测试悬浮液中胞质分裂的拯救。肌球蛋白-II尾部的片段将在体外通过生物化学和生物物理方法进行分析，以检查构象状态中的磷酸化依赖性变化，并表征组装过程的动力学和热力学。几种方法将被用来确定蛋白质是肌球蛋白II依赖性胞质分裂的关键球员。cDNA互补将确定抑制肌球蛋白-II受损的突变株的网柄藻。直接结合肌球蛋白-II尾部结构域的蛋白质将通过亲和柱层析进行追踪。 最后，将寻找与其他生物中的网囊藻相关基因同源的基因。 所有识别出的新蛋白质都将在体外和体内进行表征。 收缩环的蛋白质和参与其形成的蛋白质的空间和时间组织将在活细胞中通过荧光标记和使用计算机连接的低光水平成像的体内可视化来进行。 全内反射荧光显微镜允许可视化的单肌球蛋白-II双极粗丝细胞皮质下的细胞膜。 这种方法将允许双重可视化的肌球蛋白-II和其他荧光标记的肌球蛋白相关蛋白质。

DESCRIPTION (provided by applicant): Myosin-II bipolar thick filament formation is highly regulated in cells and is required for cytokinesis as a key component of the contractile ring. The long-term objective of this application is to understand in molecular terms the regulation of myosin-II bipolar thick filament assembly, and to elucidate the spatial and temporal control of contractile ring formation, maintenance, and dissolution during cell division. D. discoideum has a number of advantages for the study of cytokinesis, and will be used as the model system. The research plan is designed to answer the following questions. How does myosin-II heavy chain phosphorylation control the assembly of myosin-II bipolar thick filaments at the molecular level? How are the three known myosin-II heavy chain kinases organized during the cell cycle, and how does that organization relate to the dynamics of the myosin-IIcontaining contractile ring? What are the other essential proteins involved in the establishment, maintenance and dissolution of the myosin-II-containing contractile ring, and what are their cellular organizations and dynamics during cell division? Molecular genetic approaches will be used to create directed mutations in the myosin-II tail domain, to test specific hypotheses of the mechanism of regulation of thick filament assembly. These mutant myosin-IIs will be transformed into Dictyostelium myosin-II null cells to test for rescue of cytokinesis in suspension. Fragments of the myosin-II tail will be analyzed in vitro by biochemical and biophysical methods to examine phosphorylation-dependent changes in conformational states, and to characterize the kinetics and thermodynamics of the assembly process. Several approaches will be used to identify proteins that are crucial players in myosin-II-dependent cytokinesis. cDNA complementation will define suppressors of myosin-II -impaired mutant strains of Dictyostelium. Proteins that bind directly to the myosin-II tail domain will be pursued by affinity column chromatography. Finally, Dictyostelium genes homologous to cytokinesis-related genes in other organisms will be searched for. All identified new proteins will be characterized in vitro and in vivo. The spatial and temporal organization of proteins of the contractile ring and of proteins involved in its formation will be followed in live cells by fluorescent tagging ans visualization in vivo using computer-linked, low-light-level imaging. Total internal reflection fluorescence microscopy allows visualization of single myosin-II bipolar thick filaments in the cell cortex just beneath the cell membrane. This method will allow dual visualization of myosin-II and other fluorescent-labeled cytokinesis-related proteins.