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

项目总结/摘要 肌动球蛋白应力纤维（SF）使细胞能够拉紧细胞外基质（ECM），这是细胞形状的关键过程 决定力、运动力和形态发生。在过去的15年以上，包括R 01的过去时期 支持，我们已经作出了重大贡献，该领域的理解SF力学和贡献 细胞结构。我们的工作特别值得注意的是飞秒激光纳米手术（FLN）的使用， 已经使我们能够表明，三个典型的SF亚型-背侧纤维，横弧，腹侧， 纤维-共同加强了前后张力梯度，这是二维（2D）间质 迁移我们还表明，SF网络架构可以机械地增强各个SF，这 在定向迁移和力通过细胞的传播过程中， 单层。有了这个知识基础，我们的更新应用程序转向两个重要的 问题：SF网络中的张力极化是如何经典地由分子信号编码的 建立前后极性我们对2D SF网络的了解如何转化为 像在组织中发现的那些受限的迁移几何形状？我们将通过两个方面来解决这些问题。 具体目标，这两个都建立在这个奖项的出版物。在具体目标1中，我们将研究 cofilin-1对SF前后张力极化建立和维持的机制作用 在迁移过程中。我们假设cofilin-1通过促进SF张力的前后极化， 横弧的组装和收缩成熟。通过结合生物物理工程和细胞 生物学工具，我们将确定关键的分子和基于力的信号，调节cofilin-1的招募， 形成横向弧形。在与布鲁斯古德博士（布兰迪斯）的创新合作中，我们将 在微流控装置中重建肌动蛋白束，并量化张力和cofilin之间的关系， 1订婚。在具体目标2中，我们将剖析SF网络对受限环境中迁移的贡献。 几何形状，其中ECM施加轴向线索并且在空间上排除2D SF网络的精细化。我们 假设增加限制将SF组装从2D背侧纤维-横弓-腹侧 纤维组装路径朝向从头平行SF组装。我们将联合收割机 平台，单细胞机械工具和超分辨率成像，以探测限制引起的变化， SF装配、建筑和机械。目标2将利用两个已建立的富有成效的合作： 与乌尔里希施瓦茨博士（美国）。海德堡），我们将开发多尺度计算模型， 网络结构和机制，细胞迁移在有限的空间。神经外科医生Manish Aghi （UCSF），我们将通过询问胶质母细胞瘤干细胞的局限性迁移来测试我们观察的临床价值。 细胞是体内侵袭模式的回顾性预测。我们的研究将创造前所未有的新见解 以创新的方法和与人类疾病的密切联系来研究SF如何促进移民。

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.