Spatiotemporal Coordination of Formin and Arp2/3 in Epithelial Cell Migration

描述（由申请人提供）：细胞迁移在胚胎发育、组织稳态、伤口愈合和免疫应答中起重要作用。异常的迁移行为也可能导致严重的疾病，如自身免疫综合征和癌症转移。肌动蛋白聚合在细胞的板状伪足前缘为细胞迁移提供了力的产生。有两种主要类型的驱动肌动蛋白聚合的肌动蛋白成核剂，Arp和Arp 2/3。目前的模型假设，Arp 2/3驱动的分支lamellipodial丝网络在前缘的组装，而natural是负责丝状伪足和应力纤维的成核。大多数现有的细胞迁移模型完全集中在Arp 2/3介导的肌动蛋白组装作为板状伪足突起的驱动程序，尽管我们实验室和其他实验室的初步实验证据表明，Arp 2/3与细胞突起中不同的分子和功能特性共存。因此，时空协调的Arp和Arp 2/3在体内以及它们的串扰的功能方面的细胞迁移尚未解决。该项目的中心假设是，细胞调节Arp和Arp 2/3的活性，以优化在不同机械环境中的迁移效率。我的工作模型表明，这些不同的成核剂在突出事件中发挥特定的功能--Arp负责启动，Arp 2/3负责加强组装以对抗膜张力的增加。本研究的目的是建立这种协调的机制，ESTA和Arp 2/3。我计划使用定量成像方法支持的计算数据建模，这将使我们能够去卷积这两个不同的肌动蛋白成核模块的动态。我计划通过单分子成像结合肌动蛋白的定量荧光斑点显微镜来测量Arp和Arp 2/3的激活，并确定它们的时空关系。为了证明Arp 2/3介导的肌动蛋白组装的增强是机械响应的，我将研究不同刚度和基底刚度的机械环境如何影响Arp 2/3激活。最后，我建议使用高分辨率牵引力显微镜建立突出力输出和协调之间的关系？Arp 2/3活动。

DESCRIPTION (provided by applicant): Cell migration plays important roles in embryonic development, tissue homeostasis, wound healing, and immune response. Abnormal migratory behaviors can also lead to serious diseases such as autoimmune syndrome and cancer metastasis. Actin polymerization at the lamellipodial leading edge of the cell provides force generation for cell migration. There are two major types of actin nucleators which drive actin polymerization, formin and Arp2/3. Current models postulate that Arp2/3 drives the assembly of a branched lamellipodial filament network at the leading edge while formin is responsible for the nucleation of filopodia and stress fibers. Most existing models of cell migration entirely focus on Arp2/3-mediated actin assembly as the driver of lamellipodial protrusions, despite initial experimental evidence from our lab and others that formin and Arp2/3 coexist with distinct molecular and functional properties in cell protrusions. Therefore, the spatiotemporal coordination of formin and Arp2/3 in vivo as well as the function of their cross-talk in terms of cell migration has not been addressed. The central hypothesis in this project is that cells modulate the activities of formin and Arp2/3 to optimize migration efficiency in different mechanical environments. My working model suggests that these different nucleators serve specific functions in a protrusion event - formin is responsible for initiation and Arp2/3 for reinforcement of assembly against increasing membrane tension. The goal of this research is to establish the mechanism of such coordination of formin and Arp2/3. I plan to use quantitative imaging approaches supported by computational data modeling that will allow us to deconvolve the dynamics of these two different actin nucleation modules. I plan to measure formin and Arp2/3 activation by single molecule imaging in conjunction with quantitative fluorescent speckle microscopy of actin and determine their spatiotemporal relationships. In order to show that the reinforcement of Arp2/3-mediated actin assembly is mechano-responsive, I will investigate how the mechanical environment of different stiffness and adhesiveness of substrates affects Arp2/3 activation. Finally, I propose to establish relationships between the protrusion force output and the coordination between formin and Arp2/3 activities using high-resolution traction force microscopy.