RhoA GEF Regulation by Mechanotransduction

RhoA GEF 通过机械转导进行调节

• 批准号: 9346615
• 负责人: Keith Burridge
• 金额: $ 15.44万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9346615
• 关键词: Adherens Junction; Adhesions; Affect; Binding; Biosensor; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cell Nucleus; Cell Surface Receptors; Cell surface; Cells; Collaborations; Coupled; Cytoskeletal Modeling; Cytoskeleton; E-Cadherin; Elements; Extracellular Domain; Extracellular Matrix; Family; GRLF1 gene; Gene Expression; Generations; Genes; Genetic Transcription; Growth; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Image; Imaging Device; Integrins; Kinetics; Lead; Magnetism; Measurement; Measures; Mechanics; Mediating; Modeling; Movement; Nuclear; Nuclear Envelope; Pathway interactions; Pattern; Population; Proteins; Recruitment Activity; Regulation; Role; Signal Pathway; Signal Transduction; Site; Stretching; Surface; Testing; Work; base; cell behavior; cell motility; direct application; knock-down; magnetic beads; mechanical force; mechanotransduction; migration; response; rho; rho GTP-Binding Proteins; small hairpin RNA; spatiotemporal; time interval

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.

细胞以多种方式对机械力作出反应。最近的研究表明，机械力 应用于细胞粘附分子可以影响Rho家族GTP酶的活性，从而 影响细胞骨架的组织，细胞粘附，迁移和许多其他细胞 活动该项目旨在阐明机械力 在许多情况下影响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GEFs。 负责前缘的RhoA激活。使用RhoA和相关GEF的生物传感器 由Hahin和Sondeic开发，结合Danuser的多路复用成像策略， 因此，我们将确定GEF激活是否由于整合素而发生在前沿 接合或通过机械力。我们的初步研究表明， 钙粘蛋白也激活RhoA，在项目的第二部分，与霍尔合作，我们的目标是 识别GEF和相关的信号通路。使用磁性镊子和涂有 E-钙粘蛋白的细胞外结构域，我们将探讨如何力导致加强 钙粘蛋白介导的粘连。该项目的最后一部分研究了机械张力如何 施加到细胞表面影响核中的RhoA信号传导。根据我们的初步工作 显示拉伸分离的核激活RhoA，我们将RhoA生物传感器靶向 核，以便我们可以检查RhoA活性在这个隔室作为机械力施加到 细胞表面。我们将筛选负责的全球环境基金，然后研究 核内RhoA激活，检查核硬化和对基因表达的影响。