Mechanical Regulation of Cell Shape and Migration by Actin Stress Fiber Subpopulations

摘要 定向细胞迁移在许多正常和病理过程中都很重要，包括胚胎发育、创伤 治愈和肿瘤进展。张力肌球蛋白应激纤维(SF)网络在很大程度上负责 产生收缩力量以建立极化迁移所需的细胞结构和形状。超过了 在过去的十年里，该领域将SFS分为三个亚群，每个亚群与Focus的联系不同 迁移细胞中的黏附、分子组成和定位。然而，目前还不清楚机械设备是如何 每个亚群中的单个SFs的特性有助于保持细胞的形状、张力和 迁移。此外，关于SFS的许多现有知识都是从观测中间接推断出来的 在理想的二维基质上培养的细胞的研究，这些基质不能代表体内的 组织微环境。在这份奖学金提案中，我将研究每个SFS中单个SFS的力学性能 亚群及其对维持细胞形状和迁移所产生的张力的贡献。我将发表讲话 这有两个目的，使用几个强大的生物物理工具。在目标1中，我将研究其力学性能 通过使用飞秒激光纳米外科手术切割单个SFS来进行功能丧失研究。 我将使用荧光显微镜来检查由 切断了SF与周围细胞骨架网络的连接。此外，我还将检查所施加的牵引力 使用基于模型的牵引力通过每个应力纤维亚群到细胞外基质上 显微镜。在目标2中，我将研究应力纤维亚群在培养的细胞中的作用。 聚丙烯酰胺微通道，这已经被证明捕捉受限的重要特征 体内侵袭性迁移。我对研究这些复杂的环境如何影响应力纤维很感兴趣 亚群形成。我还将重复AIM中概述的激光纳米外科和牵引力实验 1在这些微通道中培养的细胞中。最后，我将比较应力纤维亚群在 目标1中的理想化2D衬底和目标2中的微通道。通过这些研究，我希望 增强该领域对肌动蛋白细胞骨架如何调节张力和细胞形状的理解 迁移。

Abstract Directed cell migration is important in many normal and pathological processes including embryogenesis, wound healing, and tumor progression. The tensed actomyosin stress fiber (SF) network is largely responsible for generating contractile forces to establish the cell structures and shape needed for polarized migration. Over the past ten years, the field has classified SFs into three subpopulations, each differing in their connections to focal adhesions, molecular composition, and localization in a migrating cell. However, it is unclear how the mechanical properties of individual SFs in each of the subpopulations contribute to maintaining cell shape, tension, and migration. Furthermore, much of the existing knowledge on SFs has been indirectly inferred from observational studies of cells cultured on idealized two-dimensional substrates, which are not representative of the in vivo tissue microenvironment. In this fellowship proposal, I will study the mechanical properties of single SFs in each subpopulation and their contribution to generating tension to maintain cell shape and migration. I will address this in two aims, using several powerful biophysical tools. In Aim 1, I will investigate the mechanical properties of individual SFs by using femtosecond laser nanosurgery to sever single SFs to conduct loss-of-function studies. I will use fluorescence microscopy to examine changes in the redistribution of the tension released by the severed SF to the surrounding cytoskeletal network. Furthermore, I will also examine the traction forces exerted onto the extracellular matrix by each of the stress fiber subpopulations using model-based traction force microscopy. In Aim 2, I will investigate the role of the stress fiber subpopulations in cells cultured in polyacrylamide microchannels, which have been shown previously to capture important features of confined invasive migration in vivo. I am interested in studying how these complex environments affect stress fiber subpopulation formation. I will also repeat the laser nanosurgery and traction force experiments outlined in Aim 1 in cells cultured in these microchannels. Finally, I will compare the roles of the stress fiber subpopulations in both the idealized 2D substrates in Aim 1 and the microchannels in Aim 2. Through these studies, I hope to enhance the field’s understanding of how the actin cytoskeleton regulates tension and cell shape for directed migration.