Biophysical Control of Cell Form and Function by Single Actomyosin Stress Fibers

项目摘要/摘要 肌动球蛋白应激纤维(SFS)使细胞能够紧张细胞外基质(ECM)，这是细胞形状的关键过程 决定、能动性和形态发生。过去15年以上，包括过去的R01时期 支持，我们为外地对SF机制和贡献的理解做出了重大贡献 细胞结构。我们的工作特别值得注意的是飞秒激光纳米外科的使用，它 使我们能够展示三种典型的SF亚型-背侧纤维、横弧和腹侧 纤维-共同加强二维(2D)间充质的前后张力梯度 迁移。我们还表明，SF网络体系结构可以机械地增强单个SFS，这 对定向迁移过程中的对称性破坏和通过细胞的力传播具有重要意义 单层。有了这个知识基础，我们的续签申请就变成了两个重要的 问题：由分子信号编码的SF网络中的张力极化是如何经典的 明白建立前后两极吗？我们对2D SF网络的知识如何转化为 像在组织中发现的那样的受限迁移几何形状？我们将通过两个方面来解决这些问题 具体目标，这两个目标都建立在该奖项的出版物基础上。在具体目标1中，我们将调查 Cofilin-1在SF前-后张力极化建立和维持中的作用 在迁移过程中。我们假设，cofilin-1通过促进SF张力建立前后极化 横弧的组装和收缩成熟。通过将生物物理、工程和细胞相结合 生物工具，我们将确定关键的分子和基于力量的信号，这些信号调节Cofilin-1的招募 形成横向弧线。在与布鲁斯·古德(Brandeis)博士的创新合作中，我们将 重建微流控装置中的肌动蛋白束，并量化张力和粘连蛋白之间的关系- 1个订婚。在具体目标2中，我们将剖析SF网络对受限地区移民的贡献 ECM施加轴向线索并从空间上排除2D SF网络精化的几何图形。我们 假设增加的限制使SF集合体从2D背侧纤维-横弧-腹侧重定向 纤维组装路径朝向从头开始的平行SF组装。我们将结合微工程培养 平台，单细胞机械工具和超分辨率成像，以探测限制诱导的变化 SF装配、建筑和机械。AIM 2将利用两个已建立的富有成效的协作： 与乌尔里希·施瓦茨博士(U·海德堡)一起，我们将开发与SF相关的多尺度计算模型 从网络架构和机制到受限空间中的蜂窝迁移。与神经外科医生Manish Aghi博士 (加州大学旧金山分校)，我们将通过询问胶质母细胞瘤干细胞的受限迁移来测试我们观察的临床价值。 细胞可追溯地预测体内的侵袭模式。我们的研究将创造前所未有的新见解 通过创新的方法和与人类疾病的密切联系，探讨SFS如何为移徙作出贡献。

PROJECT SUMMARY/ABSTRACT Actomyosin stress fibers (SFs) enable cells to tense the extracellular matrix (ECM), a process key to cell shape determination, motility, and morphogenesis. Over the past 15+ years, including the past period of R01 support, we have made significant contributions to the field’s understanding of SF mechanics and contributions to cell structure. Our work is particularly notable for the use of femtosecond laser nanosurgery (FLN), which has enabled us to show that the three canonical SF subtypes – dorsal fibers, transverse arcs, and ventral fibers – collectively enforce a front-back tension gradient that underlies two-dimensional (2D) mesenchymal migration. We also showed that the SF network architecture can mechanically reinforce individual SFs, which has significant implications for symmetry breakage during directed migration and force propagation through cell monolayers. With this intellectual foundation in place, our renewal application turns to two important questions: How is polarization of tension in the SF network encoded by molecular signals classically understood to establish front-back polarity? And how does our knowledge of 2D SF networks translate to confined migration geometries like those found in tissue? We will address these questions through two specific aims, both of which build upon publications from this award. In Specific Aim 1, we will investigate mechanistic contributions of cofilin-1 to establishment and maintenance of SF front-back tension polarization during migration. We hypothesize that cofilin-1 establishes front-back polarization of SF tension by promoting the assembly and contractile maturation of transverse arcs. By combining biophysical, engineering, and cell biological tools, we will identify key molecular and force-based signals that modulate recruitment of cofilin-1 to developing transverse arcs. In an innovative new collaboration with Dr. Bruce Goode (Brandeis) we will reconstitute actin bundles in microfluidic devices and quantify the relationship between tensile force and cofilin- 1 engagement. In Specific Aim 2, we will dissect contributions of SF networks to migration in confined geometries where the ECM imposes axial cues and sterically precludes elaboration of 2D SF networks. We hypothesize that increasing confinement redirects SF assembly from the 2D dorsal fiber-transverse arc-ventral fiber assembly pathway towards de novo parallelized SF assembly. We will combine microengineered culture platforms, single-cell mechanical tools, and superresolution imaging to probe confinement-induced changes in SF assembly, architecture, and mechanics. Aim 2 will leverage two established, productive collaborations: With Dr. Ulrich Schwarz (U. Heidelberg), we will develop multiscale computational models that relate SF network architecture and mechanics to cell migration in confined spaces. With neurosurgeon Dr. Manish Aghi (UCSF), we will test the clinical value of our observations by asking if confined migration of glioblastoma stem cells is retrospectively predictive of in vivo invasion patterns. Our studies will create unprecedented new insight into how SFs contribute to migration, with innovative methodology and close connection to human disease.