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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本项目的目的是获得关于网柄柄细胞分裂沟皮质中肌动蛋白-肌球蛋白-II网络的三维结构信息，该网络在沟向内的不同阶段。在我们的工作中，我们一直在开发细胞质分裂的定量框架，以了解控制细胞形状变化动态的分子机制。肌球蛋白II和肌动蛋白是这一过程的中心。许多教科书上的细胞质分裂模型调用了一条肌动蛋白细丝的圆周带，肌球蛋白-II粗丝排列得像钱包里的细绳一样收缩。虽然这个组织对于某些生物体来说可能是正确的，但有几条证据对此提出了质疑，认为这是一个普遍的框架。首先，肌球蛋白-II突变的Dictyostelius细胞进行有丝分裂的速度几乎与野生型细胞一样快。其次，Dictyostelialmyosin-II被动态地调节并组装成粗丝，以至于很难概念化简单的肌节样收缩机制；一些哺乳动物的非肌肉肌球蛋白-II也有类似的调节。第三，在哺乳动物和网柄苔藓细胞中，不能清晰地检测到肌动蛋白细丝的清晰环。高分辨率的结构信息将使我们能够相当程度地改进我们目前的网柄苔藓细胞质分裂模型。这个项目的最初目标是确定肌动蛋白细丝和肌球蛋白-II粗细丝在沟入水的不同阶段的取向。我们已经准备好了细胞，通过快速跳入冰冻的带有附着细胞的镀金EM网格，并将这些细胞成像为冷冻-水合状态下的整体支架和冷冻-替代后的塑料切片。

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.