Extracellular matrix sensing in cellular signaling, migration and wound repair

项目概要/摘要 整合素和其他粘附蛋白的人类基因突变引起一系列自身免疫性疾病， 很可能导致伴随许多广泛的人类疾病的慢性炎症。但我们的 对连接整合素功能和免疫活化的分子机制的认识仍然不完全。 这项研究计划的长期目标是通过揭示新的调控机制来解决这一知识缺口。 细胞外基质（ECM）传感和巨噬细胞活化之间的联系。的总体目标 这个提议是为了评估ECM传感（我们称之为“触敏”）如何调节巨噬细胞激活 在分子和细胞水平上，并将这种理解转化为体内伤口愈合研究。的 该提议的中心假设是触敏通过整合素Arp 2/3调节巨噬细胞活化 与免疫受体的依赖性串扰。申请人实验室生成的强有力的初步数据支持 这一假设，以及证明所提出的研究在申请人手中是可行的。的 拟议研究计划的基本原理是，获得对触觉传感的机械见解将使我们能够 开始了解其在慢性人类疾病状态中的作用，这反过来可能揭示其潜在的药理学作用。 目标的该假设将在三个具体目标的背景下进一步测试：1）定义整合素启动的 在触觉感知期间激活Arp 2/3的信号; 2）确定触觉感知通路如何调谐 炎症性巨噬细胞活化; 3）确定创伤如何需要巨噬细胞触敏 体内分辨率。这些目标将主要通过机械解剖巨噬细胞行为来实现， 定制设计的微流体腔室，并使用活体内技术将这些体外发现转化为体内环境， 多光子显微术这些研究将采用功能丧失的方法（基因无效，shRNA 敲除、抑制剂洗入），其在申请人手中已经建立并且有效。最后， 申请人的实验室将采用仔细，严格的图像分析方法来研究细胞迁移，肌动蛋白 在体外和体内，在原代巨噬细胞中的动力学和亚细胞蛋白定位， 许多细胞外的线索，包括ECM梯度。该方法是创新的，因为它适用于机械 在体外产生的观察结果，以指导体内免疫细胞迁移和活性的观察。的 申请人的实验室为此目的所采用的方法代表了显著的技术进步 有可能阐明细胞粘附和巨噬细胞活化之间的联系。拟议 研究计划是重要的，因为这些目标的完成将确定巨噬细胞的机制细节， 触觉传感，并将分配一个在体内的功能给它。重大的概念和技术进步 在这项研究中发挥的作用将对多个领域产生重大影响，包括方向性 迁移、TLR 4信号传导和创伤修复。

PROJECT SUMMARY/ABSTRACT Human genetic mutations in integrins and other adhesion proteins cause a range of autoimmune diseases and likely contribute to the chronic inflammation that accompanies many widespread human maladies. However, our knowledge of the molecular mechanisms that link integrin function and immune activation remain incomplete. The long-term goal of this research program is to address this knowledge gap by revealing novel regulatory connections between extracellular matrix (ECM) sensing and macrophage activation. The overall objective of this proposal is to evaluate how ECM sensing (which we refer to as ‘haptosensing’) tunes macrophage activation at the molecular and cellular levels, and to translate this understanding to in vivo wound healing studies. The central hypothesis of this proposal is that haptosensing regulates macrophage activation via integrin-Arp2/3 dependent crosstalk with immune receptors. Strong preliminary data generated by the applicant’s lab supports this hypothesis, as well as demonstrating that the proposed studies are feasible in the applicant’s hands. The rationale for the proposed research plan is that gaining mechanistic insight into haptosensing will allow us to begin understanding its role in chronic human disease states, which may in turn reveal potential pharmacological targets. The hypothesis will be tested further in the context of three specific aims: 1) Define the integrin-initiated signals that activate Arp2/3 during haptosensing; 2) Determine how the haptosensing pathway tunes inflammatory macrophage activation; 3) Determine how macrophage haptosensing is required for wound resolution in vivo. These aims will primarily be pursued by mechanistically dissecting macrophage behavior in custom-designed microfluidic chambers and translating these in vitro findings to in vivo settings using intravital multiphoton microscopy. These studies will employ loss of function approaches (genetic nulls, shRNA knockdown, inhibitor wash-in), which are already established and are effective in the applicant’s hands. Finally, the applicant’s lab will employ careful, rigorous image analysis approaches to study cell migration, actin dynamics and subcellular protein localization in primary macrophages, in vitro and in vivo, responding to numerous extracellular cues, including ECM gradients. The approach is innovative, as it applies mechanistic observations generated in vitro to direct observations of immune cell migration and activity in vivo. The approaches employed to this end by the applicant’s lab represent significant technological advances that make it possible to clarify the connection between cellular adhesion and macrophage activation. The proposed research program is significant as completion of these aims will identify the mechanistic details of macrophage haptosensing and will assign an in vivo function to it. The significant conceptual and technological advances brought to bear in this research study will have significant impact across multiple fields including directional migration, TLR4 signaling and wound repair.