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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

基本信息

批准号：
9399083
负责人：
Sanjay Kumar
金额：
$ 29.97万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9399083
关键词：
Actins Actomyosin Address Alpha Cell Apoptosis Atomic Force Microscopy 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 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

项目摘要

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.

项目总结/文摘