In vivo analysis of mechanotransduction

描述（由申请人提供）：机械力传导，即对机械力的感觉和响应，在人体生理学中具有根本的重要性。例如，对压力、拉伸和流动的适当感觉和反应对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了深入的了解，但人们对细胞如何将力学信息（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，我们介绍了一种新的和简单的在体内系统的力学转导的研究，拉伸敏感和响应细胞的C。线虫生殖系统我们已经发现，卵母细胞进入被称为精子膜的管状器官会触发Ca+2波，这些波扫过精子膜，最终在平滑的挤压中排出受精胚胎。我们建议测试的假设，卵母细胞进入拉伸的分子应变计FLN-1/细丝蛋白，导致激活的小GTdR RHO-1/Rho，PLC-1/磷脂酶C-β，IP 3触发的钙释放，并协调收缩的受精囊组织。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，而响应拉伸所需的FLN-1结构域将用于通过质谱分离关键的相互作用蛋白。在目标2中，FLN-1和RHO-1在PLC-1激活、IP 3产生和Ca+2释放中的作用将使用表达IP 3和Ca+2生物传感器的动物的遗传操作来确定。将研究肌动球蛋白收缩性的下游调节和对Ca+2信号传导的反馈。我们已经发现，需要新型调节剂TAG-341来防止钙信号的过早激活。TAG-341含有Rho家族GTP酶的GAP结构域、可结合和弯曲膜的BAR结构域和可允许分子对DAG作出反应的C1结构域。在目标3中，将确定这些结构域在激活和响应IP 3和Ca+2信号传导中的功能以及TAG-341的拉伸敏感性支架中对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以调节组织功能。

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.