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 许多生理事件，包括组织形态发生、伤口愈合和炎症反应。在此期间 事件中，细胞通常通过感知化学、物理、机械或电气线索而被指示向目标迁移 在他们周围。为了促进细胞迁移，肌动蛋白必须经历快速的组装和拆解循环，在高度... 协调一致的方式。I型冠状蛋白是一类保守的肌动蛋白结合蛋白，已被证明调节这一过程。 通过调节前沿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型柯罗宁，柯罗宁1C(Coro1C)在细胞迁移过程中介导膜突起的形成；而 结构上类似的I型柯罗宁1B(Coro1B)调节体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Coro1B和Coro1C对肌动蛋白功能的理解 动力学，特别是定向的细胞迁移仍然不完整。因此，这项提案的总体目标是 将阐明Coro1B和Coro1C在分支稳定和去分支过程中的作用机制 定向迁移。我假设Coro1B和Coro1C通过调节功能来调节分支肌动蛋白的动力学 不同的肌动蛋白调节因子，包括1)Arp2/3复合体，2)cofilin和3)细胞定向迁移过程中的rac1。为了测试 在这一假设下，我建立了一种来源于Coro1B基因敲除(KO)的Coro1CFL/FL小鼠的成纤维细胞系，即 用可漂浮的Coro1B-GFP表达载体拯救。在Cre介导的重组过程中，外源Coro1B- GFP和内源性Coro1C被删除，以创建匹配的空细胞系，以测试冠状体素在片脂中的作用 动力学和细胞运动性。在目标1中，我将利用零细胞来描述Coro1B和Coro1C的机械作用 利用光基因控制的Coro1B和Coro1C救援构建物研究片状脂虫动力学。在《目标2》中，我将利用 配对和微制造技术评估Coro1B和Coro1C在触觉和运动中的功能作用 趋化性，两种截然不同且临床相关的定向迁移形式。在所有的光遗传和迁移实验中 建议，细胞反应将通过细胞形态、细胞行为和细胞骨架结构的变化来衡量 组织。这些研究将对Coro1B和Coro1C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，以及提供出色的培训机会来推进我的职业生涯 在生物医学科学方面。

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.