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是小型GTPases的Rho-Family的成员，中央调节肌动蛋白组织和肌动蛋白收缩力。 RhoA动力学坐标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，以将机械力传输到/从相邻的细胞细胞连接和与细胞外基质（ECM）的局灶性粘附。因此，控制RhoA信号动力学的新工具可以增强对细胞如何对机械刺激做出决策的理解。我们建议创建对RhoA信号传导的双向（激活和灭活）控制的光遗传学工具，并通过一系列在收缩率和机械传输方面的生理测定法对其功能进行系统的特征，并根据其他报道的Optogenotic Technologies对其性能进行基准测试。我们将使用我们最近发现的BCLOV4光感受器，该光感受器通过直接光诱导的蛋白 - 脂质与质膜进行动态易位，这使II使II强大，可用于对外围膜蛋白的单晶上遗传学控制，这是跨细胞类型和主要细胞的可靠的外围膜蛋白。为了证明该工具箱的独特功能，我们将通过同时选择通过同时选择的，通过同时选择的，持续的细胞重塑和持续的细胞运动率来定量地绘制已知RhoA/Y AP机械转移反馈回路的细胞骨架信号和张力动力学，并通过同时选择的遗传细胞运动性。该工具箱将通过控制无处不在的RhoA信号传导的控制来探讨其在细胞收缩性，运动，机械转导和再生中的多种调节作用，从而广泛影响细胞和细胞骨架生物学。