ORGANIZATION OF ACTIN-MYOSIN-II NETWORK IN CLEAVAGE FURROW OF DICTYOSTELIUM CELL

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本项目的目的是获得三维结构信息的肌动蛋白-肌球蛋白-II网络的组织中的裂沟皮层的Dictyosteophytes细胞在不同阶段的沟内陷。在我们的工作中，我们一直在开发一个定量的胞质分裂的框架，以了解控制细胞形状变化的动态的分子机制。肌球蛋白-II和肌动蛋白是这个过程的核心。许多教科书模型的胞质分裂调用肌动蛋白丝与肌球蛋白-II粗丝排列，使分裂沟收缩像一个钱包字符串环带。虽然这种组织对于某些生物体来说可能是正确的，但一些证据对这一通用框架提出了挑战。首先，肌球蛋白-II突变的网骨藻细胞经历有丝分裂细胞分裂几乎一样快的野生型细胞。第二，Dictyosteoblasts myosin-II是动力学调谐和组装成粗丝的方式，使其难以概念化一个简单的肌节样收缩机制，一些哺乳动物的非肌肉myosin-II类似的调谐。第三，在哺乳动物和网骨藻细胞中，不能清楚地检测到肌动蛋白丝的清晰环。高分辨率的结构信息将使我们能够大大完善我们目前的模型胞质分裂在网柄藻。本项目的最初目标是确定在沟内陷的各个阶段肌动蛋白丝和肌球蛋白-II粗丝的方向。我们已经准备了细胞，通过快速冷冻formvar涂层的金EM网格与附着的细胞，并成像这些断层重建作为整个安装在冷冻水化状态和冷冻置换后的塑料部分。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this project is to acquire 3D structural information about the organization of the actin-myosin-II network in the cleavage furrow cortex of Dictyostelium cells at different stages f furrow ingression. In our work, we have been developing a quantitative framework for cytokinesis in order to understand the molecular mechanisms that govern the dynamics of cell shape change. Myosin-II and actin are central to this process. Many textbook models of cytokinesis invoke a circumferential belt of actin filaments with the myosin-II thick filaments arranged so that the cleavage furrow is constricted like a purse string. While this organization is likely to be correct for some organisms, several lines of evidence challenge this as a general framework. First, myosin-II mutant Dictyostelium cells undergo mitotic cell division nearly as fast as wild-type cells. Second, Dictyostelium myosin-II is kinetically tuned and assembled into thick filaments in such a way as to make it difficult to conceptualize a simple sarcomeric-like contraction mechanism; some mammalian nonmuscle myosin-IIs are similarly tuned. Third, in mammalian and Dictyostelium, cells, a clear ring of actin filaments is not clearly detectable. High resolution structural information would allow us to considerably refine our current models for cytokinesis in Dictyostelium. The initial goal of this project is to determine the orientation of actin filaments and myosin-II thick filaments at various stages of furrow ingression. We have prepared cells by rapidly plunge freezing formvar-coated gold EM grids with attached cells and are imaging these for tomographic reconstruction both as whole mounts in the frozen-hydrated state and as plastic sections after freeze-substitution.