Collaborative Research: Cytokinetic Furrow Specification in Sea Urchin Embryos

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。知识价值。动物细胞通过在细胞赤道周围组装一个肌动蛋白和肌球蛋白的收缩环来进行自我分裂，在有丝分裂结束时，这个收缩环收缩了复制染色体组之间的细胞表面。经典实验表明，细胞“知道”在哪里构建收缩环，因为有丝分裂装置的微管（对染色体进行分类的细胞机器）将空间线索传递到细胞表面。相关空间信号的性质，以及将它们传递到细胞表面的机制，一直是难以捉摸和有争议的。最近的研究表明，信号蛋白Rho是有丝分裂装置和分裂过程中收缩蛋白向细胞赤道募集之间的关键生理联系。分子遗传学研究表明，微管相关的Rho调节因子参与细胞分裂的控制，这支持了一个被广泛支持的假设：沿着微管运动的分子马达将细胞质深处的事件信息带到细胞表面，从而以某种方式创造了Rho活动的空间模式，有利于在正确的地点和时间招募肌动蛋白和肌凝蛋白，形成收缩环。该项目将测试细胞微管不同亚群与Rho活性之间的因果关系，阐明微管的几何形状和行为如何与Rho调节因子协同识别细胞表面的分裂平面，并将寻求解释细胞在分裂过程中如何快速响应扰动。可行的假设是，一群微管——从有丝分裂装置的每一极向细胞表面辐射的紫外光——限制了染色体分离后从细胞深处的另一群微管——有丝分裂装置的中间区——释放出来的扩散信号。该项目将测试这样一种假设，即任何一种种群都足以定位Rho激活，但它们之间的协同作用使细胞分裂准确而精确。细胞分裂的准确性和精确性对细胞和它们组成的有机体的生命至关重要。在基因组分割中，即使是很小的错误也会造成不可挽回的灾难。本项目以海胆胚胎为模型系统，利用高空间和时间分辨率的活细胞荧光成像来测量正常细胞和实验扰动细胞的细胞内动力学。这样，这些研究将解决长期以来关于细胞分裂机制的争论。许多关于细胞分裂的经典结果来自对海胆卵和类似胚胎细胞的研究，但经典工作的一些重要结论似乎与最近关于细胞分裂的分子遗传学研究不一致。通过使用分子探针重新检查海胆胚胎中的细胞分裂，这项研究将显示大的胚胎细胞是否遵循与小的体细胞不同的规则，或者是否所有类型的动物细胞都适应一种共同的机制来适应它们生物学的不同需求。更广泛的影响。该项目将培养研究生和本科生在最先进的活细胞成像和高分辨率共聚焦显微镜。同时，该研究将通过开发广泛应用的荧光蛋白探针来可视化亚细胞组织和动力学，从而推动该领域的技术发展。该项目还包括一个主要的教育部分。该研究小组包括一名活跃的公立高中科学教师，他们将准备高分辨率的胚胎正常细胞行为的延时胶片，表达对关键细胞成分（如微管、肌动蛋白丝和染色体）的荧光探针。这些影片将与适合高中和本科细胞生物学课程课堂使用的教学材料一起进行注释和分发。此外，主要研究人员通过网站、公开讲座、学校外展和博物馆展览，积极参与将科学研究带入公共领域。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual merit. Animal cells divide themselves by assembling a contractile ring of actin and myosin around the cell equator, which constricts the cell surface between duplicated chromosome sets at the end of mitosis. Classical experiments suggest that cells "know" where to build the contractile ring because microtubules of the mitotic apparatus, the cellular machine that sorts chromosomes, convey spatial cues to the cell surface. The nature of the relevant spatial cues, and the mechanism that conveys them to the cell surface, have been elusive and controversial. Recent research shows that the signaling protein Rho is a key physiological link between the mitotic apparatus and recruitment of contractile proteins to the cell equator during division. Molecular genetic studies implicate microtubule-associated Rho regulators in the control of cell division, supporting a widely-favored hypothesis: that molecular motors traveling along microtubules bring information about events deep in the cytoplasm to the cell surface, thereby somehow creating a spatial pattern of Rho activity that favors actin and myosin recruitment at the right place and time to form the contractile ring. This project will test the causal relationship between distinct subsets of cellular microtubules and Rho activity, elucidate how microtubule geometry and behavior collaborate with Rho regulators to identify the division plane on the cell surface, and will seek to explain how cells rapidly respond to perturbations during division. The working hypothesis is that one population of microtubules -- the asters, which radiate toward the cell surface from each pole of the mitotic apparatus -- confine a diffusible signal that is released, after chromosome segregation, from another population of microtubules -- the midzone of the mitotic apparatus -- deep in the cell. The project will test the hypothesis that either population suffices to localize Rho activation, but that synergy between them makes cell division accurate and precise. Accuracy and precision in cell division are fundamentally important to the lives of cells and the organisms they compose. Even small errors in genome partitioning can be irrevocably disastrous. This project relies on sea urchin embryos as a model system, using fluorescent imaging of live cells at high spatial and temporal resolution to measure intracellular dynamics in normal and experimentally-perturbed cells. In so doing, the studies will resolve long-standing debates about the mechanism of cell division. Many classical results on cell division derive from studies of sea urchin eggs and similar embryonic cells, but several significant conclusions from classical work seem to disagree with recent work on the molecular genetics of cell division. By re-examining cell division in the sea urchin embryo using molecular probes, this research will show whether large embryonic cells follow different rules than small somatic cells, or whether animal cells of all types adapt a common mechanism to the diverse demands of their biology.Broader impacts. This project will train graduate and undergraduate students in state-of-the-art live-cell imaging and high-resolution confocal microscopy. At the same time the research will advance technique development in this area by developing widely-applicable fluorescent protein probes for visualizing subcellular organization and dynamics. The project also includes a major educational component. The research group, which includes an active public high school science teacher, will prepare high-resolution time-lapse films of normal cell behavior in embryos expressing fluorescent probes for key cellular constituents such as microtubules, actin filaments, and chromosomes. Such films will be annotated and disseminated along with didactic materials appropriate for classroom use in high-school and undergraduate cell biology curriculum. In addition, the principal investigators are actively involved in bringing scientific research into the public sphere through websites, public lectures and school outreach, and museum exhibitions.