Extracellular matrix sensing in cellular signaling, migration and wound repair

项目摘要/摘要 人类整合素和其他黏附蛋白的基因突变会导致一系列自身免疫性疾病和 可能会导致伴随着许多广泛传播的人类疾病的慢性炎症。然而，我们的 对整合素功能和免疫激活之间的分子机制的了解还不完全。 这项研究计划的长期目标是通过揭示新的监管措施来解决这一知识鸿沟 细胞外基质(ECM)感知和巨噬细胞激活之间的联系。总的目标是 这项建议是为了评估细胞外基质感觉(我们称之为触觉感觉)如何调节巨噬细胞的激活。 在分子和细胞水平上，并将这一理解转化为体内伤口愈合研究。这个 这一提议的中心假设是触觉通过整合素-Arp2/3调节巨噬细胞的激活 与免疫受体的依赖串扰。申请者实验室生成的强大初步数据支持 这一假设，以及证明拟议的研究在申请人手中是可行的。这个 提议的研究计划的基本原理是，获得对触觉的机械性洞察将使我们能够 开始了解它在慢性人类疾病状态中的作用，这可能反过来揭示潜在的药理作用 目标。这一假设将在三个具体目标的背景下得到进一步检验：1)定义整合素启动的 触觉感觉过程中激活Arp2/3的信号；2)决定触觉感觉通路如何调节 炎性巨噬细胞激活；3)确定伤口需要巨噬细胞触觉 体内分辨率。这些目标主要是通过机械地解剖巨噬细胞的行为来实现的 定制设计的微流体室，并将这些体外研究结果转化为体内环境 多光子显微镜。这些研究将采用功能丧失方法(遗传缺失、shRNA 击倒、抑制剂冲入)，这些规则已经确立并在申请人手中有效。最后， 申请者的实验室将使用仔细、严格的图像分析方法来研究细胞迁移、肌动蛋白 体外和体内原代巨噬细胞的动力学和亚细胞蛋白定位 大量的细胞外信号，包括细胞外基质梯度。这种方法是创新的，因为它应用了机械论 在体外产生的观察结果直接观察免疫细胞在体内的迁移和活动。这个 申请人的实验室为此目的采用的方法代表了重大的技术进步，使 阐明细胞黏附和巨噬细胞激活之间的联系是可能的。建议数 研究计划意义重大，因为完成这些目标将确定巨噬细胞的机制细节 触觉，并将赋予它体内的功能。重大的概念和技术进步 这项研究的实施将对包括方向性在内的多个领域产生重大影响 迁移、TLR4信号和创伤修复。

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.