Single-component optogenetic tools to bidirectionally control RhoA in mechanotransduction

摘要 RhoA是Rho家族中的一员，主要调节肌动蛋白的组织和肌球蛋白的收缩能力。RhoA动力学协调肌动蛋白应激纤维的形成，最终决定细胞如何产生细胞骨架张力，以将机械力传递到相邻的细胞-细胞连接以及与细胞外基质(ECM)的局部粘连。因此，控制RhoA信号动力学的新工具可能会加强对细胞如何对机械刺激做出决定的理解。我们建议创建光遗传工具来双向(激活和失活)控制RhoA信号，通过一系列在收缩和机械转导方面的生理测试来系统地表征它们的功能，并将它们的性能与其他已报道的光遗传技术进行比较。我们将使用我们最近发现的BcLOV4光感受器，它通过光诱导的蛋白质-脂质与质膜的直接相互作用动态移位，使II能够强大地对外周膜蛋白进行单组分光遗传控制，这在细胞类型和原代细胞之间都是强大的。为了展示该工具箱的独特功能，我们将通过同步光遗传扰动和多报告成像，定量地映射已知的RhoA/Y AP机械传导反馈环在细胞骨架重塑和持续细胞运动中的细胞骨架信号和拉伸动力学。这个工具箱将通过促进对普遍存在的RhoA信号的控制来探索其在细胞收缩、运动性、机械转导和再生中的不同调节作用，从而广泛影响细胞和细胞骨架生物学。

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.