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

单个肌动球蛋白应力纤维对细胞形态和功能的生物物理控制

• 批准号: 9977697
• 负责人: Sanjay Kumar
• 金额: $ 29.84万
• 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: 2017-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977697
• 关键词: Actins; Actomyosin; Address; Apoptosis; Automobile Driving; Biological; Biophotonics; Biophysical Process; Biophysics; Brain; Brain Neoplasms; Cell Culture Techniques; Cell Shape; Cells; Cellular biology; Classification; Custom; Cytoskeleton; Development; Disease; Dorsal; EGF gene; Elements; Engineering; Extracellular Matrix; Family; Fiber; Fluorescence; Fluorescence Resonance Energy Transfer; Genetic; Glioblastoma; Homeostasis; Image; Individual; Infiltration; Interphase Cell; Lasers; Location; Malignant neoplasm of brain; Maps; Measurement; Measures; Mechanics; Methods; Microfilaments; Microfluidics; Modeling; Molecular; Molecular Biology; Molecular Motors; Morphogenesis; Motor; Myosin ATPase; Myosin Type II; Neoplasm Metastasis; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Normal tissue morphology; Oncogenic; Phenotype; Process; Property; Protein Isoforms; Regulation; Shapes; Signal Transduction; Slice; Stress Fibers; Structure; System; Technology; Thinking; Tissue Model; Tissues; Traction; Traction Force Microscopy; Tumor Cell Invasion; Ursidae Family; Work; base; cell motility; in vivo; innovation; interest; loss of function; mechanical load; mechanical properties; migration; molecular scale; nanosurgery; sensor; stoichiometry; tool; transmission process; two-dimensional; virtual; viscoelasticity

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.

项目概要/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 已被广泛 分为三种专门的“亚型”（背侧纤维、横弓和腹侧纤维），它们的作用不同 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侵入组织内， 著名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）。据推测，背侧纤维、横弓和腹侧纤维彼此紧张， ECM 以非常特定的方式控制细胞形状、极性和运动性。然而，这种范式存在以下问题： 几个关键的限制。例如，尚未直接证明每种 SF 亚型都会产生 通常假设的张力，这又源于缺乏对 SF 机械的直接测量 活细胞中的特性。此外，虽然人们广泛认为这些亚型在分子结构上有所不同 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如何 差异导致 SF 亚型之间的收缩性差异。最后，也许也是最重要的一点是 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，我们解决了所有这三个问题 通过结合单细胞生物光子技术、传统细胞和分子生物学来解决关键的开放问题 方法、工程培养系统和离体组织模型。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）是飞秒激光纳米手术 (FLN)，它使 我们有选择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 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 选择性切割 来自每个规范子类型的 SF 用于映射这些机械特性和结构贡献。我们将 还将 FLN 与单细胞微图案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有助于 EGF 依赖性极化 和动力。在目标 2 中，我们将研究特定物质的化学计量和机械化学性质。 肌球蛋白 II 亚型共同决定整个 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 亚型和其中的肌球蛋白亚型如何导致 GBM 血管周围侵袭。致我们的 知识，目标 3 研究将代表哺乳动物中 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来解决主要问题 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。