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

肌动蛋白应力纤维亚群对细胞形状和迁移的机械调节

• 批准号: 9257062
• 负责人: Stacey Lee
• 金额: $ 3.9万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9257062
• 关键词: Actins; Actomyosin; Address; Affect; Area; Atomic Force Microscopy; Binding Proteins; Biology; Biomechanics; Biosensor; Cell Shape; Cells; Cellular Structures; Classification Scheme; Complex; Cultured Cells; Cytoskeleton; Dorsal; Embryonic Development; Energy Transfer; Engineering; Environment; Extracellular Matrix; Fellowship; Fluorescence Microscopy; Focal Adhesions; Goals; Homeostasis; Individual; Knowledge; Lasers; Length; Measurement; Measures; Mechanics; Microfilaments; Modeling; Molecular; Morphology; Motor; Observational Study; Pathologic Processes; Play; Process; Proteins; Regulation of Cell Shape; Research; Research Personnel; Role; Shapes; Spatial Distribution; Stress; Stress Fibers; Structure; Subcellular structure; Testing; Tissue Engineering; Tissues; Traction; Work; Wound Healing; anticancer research; base; biophysical techniques; biophysical tools; cell motility; directional cell; experimental study; in vivo; insight; interest; live cell imaging; loss of function; mechanical load; mechanical properties; migration; nanosurgery; network architecture; polyacrylamide; predictive modeling; tool; tumor progression; two-dimensional; virtual

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.

摘要 细胞定向迁移在许多正常和病理过程中起重要作用，包括胚胎发生、创伤、 愈合和肿瘤进展。紧张的肌动球蛋白应力纤维（SF）网络主要负责 产生收缩力以建立极化迁移所需的细胞结构和形状。来 在过去的十年里，该领域将SF分为三个亚群，每个亚群与焦点的联系都不同。 粘附、分子组成和在迁移细胞中的定位。然而，目前还不清楚机械 每个亚群中的单个SF的性质有助于维持细胞形状、张力， 迁移此外，有关SF的大部分现有知识都是从观察中间接推断出来的 在理想化的二维基质上培养的细胞的研究，其不代表体内 组织微环境在这个奖学金计划中，我将研究每种材料中单个SF的力学性能， 亚群及其对产生张力以维持细胞形状和迁移的贡献。我将向 这有两个目的，使用几个强大的生物物理工具。在目标1中，我将研究机械性能 通过使用飞秒激光纳米外科手术切断单个SF进行功能丧失研究。 我将使用荧光显微镜检查由细胞释放的张力重新分布的变化。 切断SF与周围细胞骨架网络的联系。此外，我还将研究施加的牵引力 通过每个应力纤维亚群使用基于模型的牵引力 显微镜在目标2中，我将研究应力纤维亚群在细胞培养中的作用。 聚丙烯酰胺微通道，其先前已被证明能够捕获受限微通道的重要特征。 体内侵入性迁移。我感兴趣的是研究这些复杂的环境如何影响应力纤维 亚群形成我还将重复Aim中概述的激光纳米手术和牵引力实验 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的作用， 目标1中的理想化2D基底和目标2中的微通道。通过这些研究，我希望 加强该领域的理解如何肌动蛋白细胞骨架调节张力和细胞形状，为定向 迁移