Single-component optogenetic tools to bidirectionally control RhoA in mechanotransduction

在力转导中双向控制 RhoA 的单组分光遗传学工具

基本信息

批准号：
10521872
负责人：
Joel D Boerckel
金额：
$ 34.51万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10521872
关键词：
3T3 Cells Actins Actomyosin Animals Behavior Benchmarking Biological Assay Biology Biophysics Cell Line Cell membrane Cells Cellular Morphology Collaborations Cytoskeleton Decision Making Dependence Development Dimensions Dissection Dominant-Negative Mutation Engineering Equilibrium Extracellular Matrix F-Actin Family Feedback Focal Adhesions Future GTPase-Activating Proteins Gene Expression Generations Genetic Genetic Transcription Goals Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Heterodimerization Heterogeneity Homeostasis Human Image Intercellular Junctions Light Link Lipids Maps Measures Membrane Membrane Proteins Metabolic Metabolic stress Methods Microscopy Molecular Monomeric GTP-Binding Proteins Natural regeneration Nodal Optics Outcome Output Pattern Performance Peripheral Pharmacology Photoreceptors Physiological Population Proteins Regulation Reporter Reporting Resolution Resources Role Signal Transduction Spatial Distribution Spectrum Analysis Stress Fibers Techniques Technology Testing Tissues Traction Zebrafish base cell motility cell type density experimental study flexibility genetic payload innovation insight loss of function mechanical force mechanical stimulus mechanotransduction member mesenchymal stromal cell migration novel operation optogenetics polymerization recruit research and development response rho spatial integration spatiotemporal tool

项目摘要

ABSTRACT RhoA, a member of the Rho-family of small GTPases, centrally regulates actin organization and actomyosin contractility. RhoA dynamics coordinate actin stress fiber formation, which ultimately determines how cells generate cytoskeletal tension to transmit mechanical forces to/from neighboring cell-cell junctions and focal adhesions with the extracellular matrix (ECM). Thus, new tools to control the dynamics of RhoA signaling may enhance understanding of how cells make decisions in response to mechanical stimuli. We propose to create optogenetic tools for bi-directional (activation and inactivation) control over RhoA signaling, systematically characterize their function through a set of physiological assays in contractility and mechanotransduction, and benchmark their performance against other reported optogenetic technologies. We will use our recently discovered BcLOV4 photoreceptor that dynamically translocates via a direct light-induced protein-lipid interaction with the plasma membrane, making ii powerful for single-component optogenetic control over peripheral membrane proteins that is robust across cell types and primary cells. To demonstrate the unique capabilities of the toolbox, we will quantitatively map the cytoskeletal signaling and tensional dynamics of a known RhoA/Y AP mechanotransductive feedback loop in cytoskeletal remodeling and persistent cell motility, through simultaneous optogenetic perturbation and multi-reporter imaging. This toolbox will broadly impact cell and cytoskeletal biology by advancing control over ubiquitous RhoA signaling to probe its diverse regulatory roles in cell contractility, motility, mechanotransduction, and regeneration.

抽象的 RhoA 是小 GTP 酶 Rho 家族的成员，集中调节肌动蛋白组织和肌动球蛋白收缩性。 RhoA 动力学协调肌动蛋白应力纤维的形成，最终决定细胞如何产生细胞骨架张力，以将机械力传递到/来自邻近的细胞-细胞连接以及与细胞外基质 (ECM) 的粘着斑。因此，控制 RhoA 信号动力学的新工具可能会增强对细胞如何响应机械刺激做出决策的理解。我们建议创建用于双向（激活和失活）控制 RhoA 信号传导的光遗传学工具，通过一系列收缩性和机械转导的生理测定系统地表征其功能，并将其性能与其他报道的光遗传学技术进行比较。我们将使用我们最近发现的 BcLOV4 光感受器，它通过直接光诱导的蛋白质-脂质与质膜的相互作用进行动态易位，使 ii 能够强大地对外周膜蛋白进行单组分光遗传学控制，该控制在细胞类型和原代细胞中都具有鲁棒性。为了展示该工具箱的独特功能，我们将通过同步光遗传学扰动和多报告者成像，定量绘制已知 RhoA/Y AP 机械传导反馈环路在细胞骨架重塑和持久细胞运动中的细胞骨架信号传导和张力动力学。该工具箱将通过推进对普遍存在的 RhoA 信号传导的控制来广泛影响细胞和细胞骨架生物学，以探究其在细胞收缩性、运动性、机械转导和再生中的多种调节作用。