RhoA GEF Regulation by Mechanotransduction

细胞以多种方式对机械力做出反应。最近的研究表明，机械力 应用于细胞黏附分子可以影响Rho家族GTP酶的活性，从而 影响细胞骨架的组织、细胞的黏附、迁移等许多细胞 活动。该项目旨在阐明机械力的信号通路。 在许多情况下会影响Rho GTP酶，特别是RhoA。在这个项目的第一部分， 我们将专注于迁移细胞并使用shRNA敲除策略来识别GEF 负责前沿的RhoA激活。利用生物传感器检测RhoA和相关的GEF 由Hahin和Sondeic开发，结合Danuser和Sondeic的多路传输成像策略 I-laiin，我们将确定全球环境基金的激活是否发生在前沿，作为整合素的结果。 接合或通过机械力。我们的初步研究表明，机械张力对 钙粘素还激活RhoA，在项目的第二部分，与Hall合作，我们的目标是 确定所涉及的GEF和信号通路。使用磁性镊子和涂有 E-钙粘蛋白的胞外结构域，我们将探索力量如何导致 钙粘附素介导的粘连。该项目的最后一部分考察了机械张力如何 施加到细胞表面会影响细胞核中的RhoA信号。根据我们的初步工作 证明拉伸分离的核可以激活RhoA，我们将把RhoA生物传感器对准 原子核，这样我们就可以检查当机械力施加到 细胞表面。我们将筛选负责的GEF，然后检查 RhoA在核内的激活，检查核僵硬和对基因表达的影响。

Cells respond to mechanical force in many ways. Recent work has shown that mechanical force applied to cell adhesion molecules can affect the activities of Rho family GTPases, thereby influencing the organization of the cytoskeleton, cell adhesion, migration and many other cellular activities. This project is aimed at elucidating the signaling pathways by which mechanical force affects Rho GTPases, particularly RhoA, under a number of situations. In the first part of this project, we will focus on migrating cells and use shRNA knockdown strategies to identify the GEFs responsible for RhoA activation at the leading edge. Using biosensors for RhoA and relevant GEFs developed by Hahin and Sondeic, combined with the multiplexing imaging strategies of Danuser and i-laiin, we will determine whether GEF activation occurs at the leading edge as a result of integrin engagement or by mechanical force. Our preliminary studies show that mechanical tension on cadherins also activates RhoA and in the second part of the project, working with Hall, we aim to identify the GEFs and signaling pathways involved. Using magnetic tweezers and beads coated with the extracellular domain of E-cadherin we will explore how force leads to the strengthening of cadherin-mediated adhesions. The final section of the project examines how mechanical tension applied to the cell surface affects RhoA signaling in the nucleus. Based on our preliminary work showing that stretching isolated nuclei activates RhoA, we will target the RhoA biosensor to the nucleus so that we can examine RhoA activity in this compartment as mechanical force is applied to the cell surface. We will screen for the GEFs responsible and then examine the consequences of RhoA activation within the nucleus, examining nuclear stiffening and effects on gene expression.