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反应，在人体生理学中具有重要意义。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深入的了解，但对于细胞如何将机械信息（如拉伸）转化为体内控制组织功能的生化信号，我们知之甚少。在这里，我们介绍了一种新的和简单的体内系统，用于研究秀丽隐杆线虫生殖系统的机械转导，拉伸敏感和反应细胞。我们发现，卵母细胞进入管状器官——精子膜，会触发钙离子波，横扫精子膜，最终平滑挤压，排出受精胚胎。我们提出验证假设，卵母细胞进入拉伸分子应变计FLN-1/丝蛋白，导致小GTPase Rho -1/Rho， PLC-1/磷脂酶C-epsilon的激活，IP3-触发钙释放，并协调精子组织收缩。在Aim 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，FLN-1结构域需要响应拉伸，将用于通过质谱分离关键的相互作用蛋白。在Aim 2中，FLN-1和RHO-1在PLC-1激活、IP3产生和Ca+2释放中的作用将通过对表达IP3和Ca+2生物传感器的动物进行遗传操作来确定。肌动球蛋白收缩性的下游调控和Ca+2信号的反馈将被研究。我们已经发现新的调节剂TAG-341是防止钙信号过早激活所必需的。TAG-341含有一个针对Rho家族GTPases的GAP结构域，一个可能结合和弯曲膜的BAR结构域，以及一个可能允许分子对DAG作出反应的C1结构域。在Aim 3中，将确定这些结构域在IP3和Ca+2信号的激活和响应中的功能，以及在TAG-341的拉伸敏感支架中对FLN-1和PLC-1的需求。这项研究将有助于我们更好地理解细胞将机械信息转化为生化信号的基本机制，以及这种信号是如何整合来调节组织功能的。

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.