Dissecting the mechanistic contributions of Coronin 1B and Coronin 1C to directed cell migration.

摘要 有效的细胞迁移依赖于肌动蛋白-细胞骨架的动态重塑，并在细胞迁移中起着重要作用。 许多生理事件，包括组织形态发生、伤口愈合和炎症反应。在这些 在某些情况下，细胞通常通过感测化学、物理、机械或电线索而被引导向靶迁移 身边为了促进细胞迁移，肌动蛋白必须以高度稳定的方式经历快速的组装和拆卸循环。 协调的方式。I型冠蛋白是一类保守的肌动蛋白结合蛋白，已被证明可调节这一过程 通过调节细胞前缘的分支肌动蛋白网络的周转来控制细胞迁移。最近的研究表明， I型冠蛋白，冠蛋白1C（Coro 1C）介导细胞迁移期间膜突起的形成;而 结构相似的I型冠蛋白Coronin 1B（Coro 1B）调节细胞内分支肌动蛋白网络的分解， 这些突起。然而，尽管有这些结果，我们对Coro 1B和Coro 1C对肌动蛋白的功能的理解 动力学，特别是定向细胞迁移仍然不完整。因此，本提案的总体目标是 将阐明Coro 1B和Coro 1C在分支稳定性和去分支过程中的机理贡献。 定向迁移。我假设Coro 1B和Coro 1C通过调节肌动蛋白的功能来调节肌动蛋白的分支动力学 各种肌动蛋白调节剂，包括1）Arp 2/3复合物，2）cofilin和3）Rac 1在定向细胞迁移过程中。测试 基于这一假设，我建立了一个来源于Coro 1B基因敲除（KO）的成纤维细胞系，即Coro 1CFL/FL小鼠， 用可降解的Coro 1B-GFP表达构建体拯救。Cre介导的重组后，外源Coro 1B- GFP和内源性Coro 1C被删除，以创建配对的无效细胞系，以测试冠蛋白在板状伪足中的作用。 动力学和细胞运动性。在目的1中，我将利用空细胞来描述Coro 1B和Coro 1C的机制作用 使用光遗传学控制的Coro 1B和Coro 1C拯救构建体对板状伪足动力学的影响。在目标2中，我将利用 匹配对和微加工技术，以评估Coro 1B和Coro 1C在趋触性中的功能作用， 趋化性，两种不同的和临床相关的定向迁移形式。在所有光遗传学和迁移实验中 提出，细胞反应将被测量为细胞形态、细胞行为和细胞骨架结构的变化， organization.这些研究将为Coro 1B和Coro 1C在调节肌动蛋白中的作用提供基本的见解。 在定向细胞迁移过程中的细胞骨架，以及提供优秀的培训机会，以促进我的职业生涯 在生物医学科学中。

Abstract Effective cell migration is dependent on dynamic remodeling of the actin-cytoskeleton, and plays an essential role in many physiological events, including tissue morphogenesis, wound healing and inflammatory responses. During these events, cells are typically directed to migrate towards targets by sensing chemical, physical, mechanical or electrical cues around them. To facilitate cell migration, actin must undergo rapid cycles of assembly and disassembly in a highly- coordinated manner. Type I coronins, a conserved class of actin binding proteins, have been shown to regulate the process of cell migration by modulating the turnover of the branched actin network at the leading edge. Recent work suggests that the type I coronin, Coronin 1C (Coro1C) mediates the formation of membrane protrusions during cell migration; while the structurally similar type I coronin Coronin 1B (Coro1B) regulates the disassembly of branched actin networks within these protrusions. However, despite these results our understanding of the function of Coro1B and Coro1C to actin dynamics and specifically in directed cell migration remains incomplete. Therefore, the overall objective of this proposal will be to elucidate the mechanistic contributions of Coro1B and Coro1C in branch stability and de-branching during directed migration. I hypothesize that Coro1B and Coro1C regulate branched actin dynamics by modulating the functions of various actin-regulators, including 1) the Arp2/3 complex, 2) cofilin and 3) Rac1 during directed cell migration. To test this hypothesis, I have established a fibroblast cell line derived from Coro1B knockout (KO), Coro1CFL/FL mice that is rescued with a flox-able Coro1B-GFP expression construct. Upon Cre-mediated recombination, both exogenous Coro1B- GFP and endogenous Coro1C are deleted to create a matched-pair null cell line to test the role of coronins in lamellipodial dynamics and cell motility. In Aim 1, I will utilize the null cells to delineate the mechanistic role of Coro1B and Coro1C on lamellipodia dynamics using optogenetically controlled Coro1B and Coro1C rescue constructs. In Aim 2, I will utilize the matched-pair and a microfabrication technique to assess the functional roles of Coro1B and Coro1C in haptotaxis and chemotaxis, two distinct and clinically relevant forms of directed migration. In all optogenetic and migration experiments proposed, cellular responses will be measured as changes to cell morphology, cell behavior, and cytoskeletal structure and organization. These studies will provide fundamental insights into the role of Coro1B and Coro1C in regulating the actin cytoskeleton during directed cell migration, as well as providing outstanding training opportunities to advance my career in biomedical science.