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、极性、运动性和组织形态发生。运动细胞内的SF已经广泛地 分为三个专门的“亚型”（背侧纤维、横弧和腹侧纤维），它们的功能不同 前后位置和网络连接。除了驱动正常组织发育和 稳态，SF和类似的收缩结构有助于肿瘤在组织内的侵袭， 其中一个显著的例子是致命的脑肿瘤多形性胶质母细胞瘤的血管周围浸润 （GBM）。据推测，背侧纤维、横弧和腹侧纤维相互拉紧， ECM以非常特定的方式控制细胞的形状、极性和运动性。然而，这种模式受到 几个关键的限制。例如，尚未直接证明每个SF亚型产生 张力，这反过来又源于缺乏直接测量SF机械 活细胞的特性。此外，虽然这些亚型被广泛理解为在分子水平上不同， 它们包含的马达（即肌球蛋白II亚型），我们几乎不知道这些分子尺度的 这些差异导致SF亚型之间的收缩性差异。最后，也许也是最重要的， 尚不清楚这种亚型分类是否与组织内细胞的持续迁移有关， 特别是在由异常细胞迁移驱动的疾病状态中。在本提案中，我们将解决所有这三个问题 通过结合单细胞生物光子技术，传统的细胞和分子生物学， 方法、工程化培养系统和离体组织模型。这些研究的一个关键工具 （我们的团队在过去十年中开创的）是飞秒激光纳米手术（FLN），它使 我们选择性地切割活细胞中的单个SF，从而使我们能够推断出所承受的机械载荷 通过SF和它对细胞其他部分的结构贡献。在目标1中，我们将应用FLN选择性地切割 SF从每个典型的亚型映射这些机械性能和结构的贡献。我们将 还将联合收割机FLN与单细胞微图案化和基于荧光的分子张力读数相结合， 确定单个SF如何在整个细胞中分配张力并促进EGF依赖性极化 和运动性。在目标2中，我们将研究如何化学计量和机械化学性质的具体 肌球蛋白II同种型协作以确定整个SF的机械性质。在目标3中，我们 联合收割机将这些方法与我们用脑切片范例开发的微流体模型相结合，以确定 特定的SF亚型和肌球蛋白亚型如何促进GBM血管周围浸润。对我们 目标3研究将代表哺乳动物SF力学和功能的首次测量， 组织.总之，该项目将结合创新的单细胞和培养技术，以解决主要的 围绕细胞形状、极性和运动性的微观生物物理机制的开放性问题。

PROJECT SUMMARY/ABSTRACT Actomyosin stress fibers (SFs) enable cells to tense the extracellular matrix (ECM), a process key to cell shape determination, polarity, motility, and tissue morphogenesis. SFs within motile cells have been broadly classified into three specialized “subtypes” (dorsal fibers, transverse arcs, and ventral fibers) that differ in their antero-posterior location and network connectivity. In addition to driving normal tissue development and homeostasis, SFs and analogous contractile structures contribute to the invasion of tumors within tissue, a notable example of which is the perivascular infiltration of the deadly brain tumor glioblastoma multiforme (GBM). It has been hypothesized that dorsal fibers, transverse arcs, and ventral fibers tense each other and the ECM in very specific ways to govern cell shape, polarity, and motility. However, this paradigm suffers from several critical limitations. For example, it has not been directly demonstrated that each SF subtype generates tension as commonly assumed, which in turn derives from a lack of direct measurement of SF mechanical properties in living cells. Additionally, while these subtypes are broadly understood to vary in the molecular motors they contain (i.e. myosin II isoforms), we know virtually nothing about how these molecular-scale differences create the contractility differences across SF subtypes. Finally, and perhaps most importantly, it is unclear whether this subtype classification is relevant to the persistent migration of cells within tissue, particularly in disease states driven by aberrant cell migration. In this proposal we address all three of these critical open questions by combining single-cell biophotonic technologies, traditional cell and molecular biology approaches, engineered culture systems, and ex vivo tissue models. A key enabling tool for these studies (which our team has pioneered over the past decade) is femtosecond laser nanosurgery (FLN), which enables us to selectively cut single SFs in living cells, thereby allowing us to deduce both the mechanical loads borne by that SF and its structural contributions to the rest of the cell. In Aim 1, we will apply FLN to selectively incise SFs from each canonical subtype to map these mechanical properties and structural contributions. We will also combine FLN with single-cell micropatterning and fluorescence-based readouts of molecular tension to determine how single SFs distribute tension throughout the cell and contribute to EGF-dependent polarization and motility. In Aim 2, we will investigate how the stoichiometry and mechanochemical properties of specific myosin II isoforms collaborate to determine the mechanical properties of the entire SF. In Aim 3, we will combine these approaches with a microfluidic model we developed with a brain-slice paradigm to determine how specific SF subtypes and the myosin isoforms therein contribute to perivascular invasion in GBM. To our knowledge, Aim 3 studies will represent the first measurements of SF mechanics and function in mammalian tissue. In summary, this project will marry innovative single-cell and culture technologies to address major open questions surrounding the microscale, biophysical mechanisms of cell shape shape, polarity, and motility.