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 测定、极性、运动性和组织形态发生。在活动细胞内的SFS已经被广泛地 分为三种不同的特殊“亚型”(背侧纤维、横弧和腹侧纤维)。 前后位置和网络连接。除了推动正常的组织发育和 动态平衡、SFS和类似的收缩结构有助于组织内肿瘤的侵袭， 其中一个显著的例子是致命性脑肿瘤多形性胶质母细胞瘤的血管周围浸润。 (GBM)。已经假设背侧纤维、横弧和腹侧纤维彼此紧张，并且 细胞外基质以非常特殊的方式控制细胞的形状、极性和运动性。然而，这种模式存在以下问题 有几个关键的限制。例如，还没有直接证明每个SF子类型生成 通常假设的张力，而这反过来又源于缺乏对SF机械的直接测量 活细胞中的属性。此外，虽然这些亚型被广泛理解为在分子上有所不同 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是如何 不同的SF亚型会产生不同的收缩能力差异。最后，也许也是最重要的，它是 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下。在本提案中，我们将解决所有这三个问题 结合单细胞生物光子学技术、传统细胞和分子生物学的关键开放问题 方法、工程培养系统和体外组织模型。这些研究的关键支持工具 (我们的团队在过去十年中开创的)是飞秒激光纳米外科(FLN)，它使 US选择性地切割活细胞中的单个SFs，从而使我们能够推断出所承担的机械载荷 通过SF及其对细胞其余部分的结构贡献。在目标1中，我们将应用FLN选择性地切割 从每个典型子类型中提取SFS，以映射这些力学特性和结构贡献。我们会 还将Fln与单细胞微图案化和基于荧光的分子张力读数相结合，以 确定单个SFs如何将张力分配到整个细胞，并有助于EGF依赖的极化 和能动性。在目标2中，我们将研究特定的化学计量学和机械力化学性质 肌球蛋白II亚型相互协作以确定整个SF的机械特性。在《目标3》中，我们将 将这些方法与我们用脑片范式开发的微流体模型结合起来，以确定 特定的SF亚型及其肌球蛋白亚型如何影响GBM的血管周围侵袭。致我们的 知识，AIM 3研究将代表对哺乳动物SF机制和功能的第一次测量 组织。总而言之，该项目将结合创新的单细胞和培养技术，以解决主要 围绕细胞形状、极性和运动性的微尺度生物物理机制的悬而未决的问题。

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.