Myosin-II Dynamics and Cytokinesis

描述(由申请人提供)：肌球蛋白-II双极粗丝的形成在细胞中受到高度调控，是胞质分裂所必需的，是收缩环的关键组成部分。这一应用的长期目标是从分子方面了解肌球蛋白-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、维持和溶解的空间和时间控制。盘状芽孢杆菌在细胞质分裂的研究中具有许多优势，并将被用作模式系统。该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？已知的三种肌球蛋白-II重链激酶在细胞周期中是如何组织的，这种组织与含有肌球蛋白-II的收缩环的动力学有什么关系？在含肌球蛋白-II的收缩环的建立、维持和溶解过程中，还有哪些其他必需的蛋白质，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在肌球蛋白-II尾部区域产生定向突变，以测试对粗丝组装调节机制的具体假设。这些突变的肌球蛋白-II将被转化为网柄菌肌球蛋白-II零细胞，以测试在悬浮液中胞质分裂的挽救。肌球蛋白II尾部的片段将在体外通过生化和生物物理方法进行分析，以检查构象状态下依赖磷酸化的变化，并表征组装过程的动力学和热力学。将使用几种方法来识别在肌球蛋白II依赖的胞质分裂中起关键作用的蛋白质。CDNA互补将确定肌球蛋白-II受损的网柄基菌突变株的抑制子。直接与肌球蛋白-II尾部结构域结合的蛋白质将通过亲和柱层析进行追踪。最后，我们将寻找与其他生物的胞质分裂相关基因同源的网柄菌基因。所有已确定的新蛋白质都将在体外和体内进行表征。在活细胞中，通过荧光标记和使用计算机连接的微光成像进行体内可视化，将跟踪收缩环蛋白质和参与其形成的蛋白质的空间和时间组织。全内反射荧光显微镜可以在细胞膜下面的细胞皮质中显示单个肌球蛋白II双极粗丝。这种方法将允许肌球蛋白-II和其他荧光标记的胞质分裂相关蛋白的双重可视化。

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.