In vivo analysis of mechanotransduction

描述（由申请人提供）：机械转导，即对机械力的感觉和响应，在人类生理学中具有根本重要性。例如，对压力、伸展和流动的适当感觉和反应对于心血管健康至关重要。尽管生物物理学和细胞生物学对工程基质有深入的了解，但人们对细胞如何将机械信息（例如拉伸）转化为控制体内组织功能的生化信号知之甚少。在这里，我们介绍了一种新颖且简便的体内系统，用于研究机械转导，即秀丽隐杆线虫生殖系统的拉伸敏感和响应细胞。我们发现，卵母细胞进入称为精子卵泡膜的管状器官时，会触发扫过精子卵泡膜的 Ca 2 波，最终形成平滑挤压，排出受精胚胎。我们建议检验以下假设：卵母细胞进入会拉伸分子应变计 FLN-1/细丝蛋白，从而导致小 GTP 酶 RHO-1/Rho、PLC-1/磷脂酶 C-ε 的激活、IP3 触发的钙释放以及受精囊组织的协调收缩。在目标 1 中，将研究机械输入在触发钙信号传导中的重要性。基于 FRET 的应力和应变传感器将用于量化 FLN-1 分子所经历的力，响应拉伸所需的 FLN-1 结构域将用于通过质谱法分离关键的相互作用蛋白质。在目标 2 中，将通过对表达 IP3 和 Ca 2 生物传感器的动物进行基因操作来确定 FLN-1 和 RHO-1 在 PLC-1 激活、IP3 产生和 Ca 2 释放中的作用。将研究肌动球蛋白收缩性的下游调节和 Ca 2 信号传导的反馈。我们发现新型调节剂 TAG-341 是防止钙信号过早激活所必需的。 TAG-341 包含 Rho 家族 GTPases 的 GAP 结构域、可以结合和弯曲膜的 BAR 结构域以及可以允许分子响应 DAG 的 C1 结构域。在目标 3 中，将确定这些结构域在 IP3 和 Ca 2 信号传导的激活和响应中的功能以及 TAG-341 拉伸敏感支架中对 FLN-1 和 PLC-1 的需求。这项研究将有助于更好地理解细胞将机械信息转化为生化信号的基本机制，以及如何整合这种信号来调节组织功能。

DESCRIPTION (provided by applicant): Mechanotransduction, the sensation of and response to mechanical forces, is of fundamental importance in human physiology. For example, proper sensation and response to pressure stretch, and flow is essential for cardiovascular health. Despite insights from biophysics and from cell biology on engineered substrates, very little is known about how cells convert mechanical information, such as stretch, into the biochemical signals that control tissue function in vivo. Here, we introduce a novel and facile in vivo system for the study of mechanotransduction, the stretch-sensitive and responsive cells of the C. elegans reproductive system. We have discovered that oocyte entry into the tube-shaped organ known as the sperm theca triggers waves of Ca+2 that sweep across the sperm theca, culminating in a smooth squeeze that expels the fertilized embryo. We propose to test the hypothesis that oocyte entry stretches the molecular strain gauge FLN-1/filamin, leading to activation of the small GTPase RHO-1/Rho, PLC-1/phospholipase C-epsilon, IP3- triggered calcium release, and coordinated contraction of the spermathecal tissue. In Aim 1, the importance of mechanical input in triggering calcium signaling will be investigated. FRET-based stress and strain sensors will be used to quantify the forces experienced by the FLN-1 molecule, and FLN-1 domains needed for response to stretch will be used to isolate key interacting proteins by mass spectrometry. In Aim 2, the role of FLN-1 and RHO-1 in PLC-1 activation, IP3 production and Ca+2 releases will be determined using genetic manipulation of animals expressing IP3 and Ca+2 biosensors. Downstream regulation of actomyosin contractility and feedback on Ca+2 signaling will be investigated. We have discovered that the novel regulator TAG-341 is required to prevent premature activation of calcium signaling. TAG-341 contains a GAP domain for Rho family GTPases, a BAR domain that may bind and bend membranes, and a C1 domain that may allow the molecule to respond to DAG. In Aim 3, the function of these domains in activation of and response to IP3 and Ca+2 signaling and the requirement for FLN-1 and PLC-1 in the stretch-sensitive scaffolding of TAG-341 will be determined. This research will lead to an improved understanding of the fundamental mechanism by which cells convert mechanical information into biochemical signals, and how this signaling is integrated to regulate tissue function.