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

细胞迁移在胚胎发育、组织动态平衡、伤口愈合和免疫反应中起着重要作用。异常的迁移行为也会导致严重的疾病，如自身免疫综合征和癌症转移。肌动蛋白聚合在细胞的膜脂前沿，为细胞迁移提供动力。有两种主要类型的肌动蛋白核因子驱动肌动蛋白聚合，Forin和Arp2/3。 目前的模型假设Arp2/3在前沿驱动分枝的片状脂丝网络的组装，而Forin负责丝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Arp2/3介导的肌动蛋白组装作为板脂突起的驱动因素，尽管我们的实验室和其他实验室的初步实验证据表明，Forin和Arp2/3在细胞突起中共存，具有不同的分子和功能特性。因此，福尔明和Arp2/3在体内的时空配位以及它们的相互作用在细胞迁移中的作用尚未被解决。 该项目的中心假设是细胞调节Forin和Arp2/3的活性，以优化不同机械环境中的迁移效率。我的工作模型表明，这些不同的成核子在突起事件中发挥着特定的功能--Forin负责启动，Arp2/3负责加强组装，防止膜张力增加。这项研究的目标是建立Forin和Arp2/3的这种协调机制。我计划使用计算数据建模支持的定量成像方法，使我们能够去卷积这两个不同的肌动蛋白成核模块的动力学。我计划通过单分子成像结合肌动蛋白的定量荧光散斑显微镜来测量Forin和Arp2/3的激活，并确定它们的时空关系。为了证明Arp2/3介导的肌动蛋白组装的增强是机械响应的，我将研究不同底物硬度和粘附性的力学环境如何影响Arp2/3的激活。最后，我建议使用高分辨率牵引力显微镜建立突出力输出与Forin和Arp2/3活性之间的协调之间的关系。

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.