In vivo analysis of mechanotransduction

描述(申请人提供)：机械转导，即对机械力的感觉和反应，在人类生理学中具有基本的重要性。例如，适当的感觉和对压力拉伸和流动的反应对心血管健康是必不可少的。尽管来自生物物理学和工程基质上的细胞生物学的见解，但人们对细胞如何将拉伸等机械信息转化为体内控制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而简便的体内系统，用于研究机械转导--线虫生殖系统的拉伸敏感和反应细胞。我们已经发现，卵母细胞进入被称为精子膜的管状器官时，会触发钙波横扫精子膜，最终以平滑的挤压排出受精胚胎。我们建议检验这一假设，即卵母细胞进入拉伸分子应变计Fln-1/细丝蛋白，导致小GTP酶Rho-1/Rho，PLC-1/磷脂酶C-epsilon的激活，IP3触发的钙释放，以及精鞘组织的协调收缩。在目标1中，将研究机械输入在触发钙信号转导中的重要性。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学分离关键相互作用的蛋白质。在目标2中，将通过对表达IP3和Ca2生物传感器的动物的遗传操作来确定Fln-1和Rho-1在PLC-1激活、IP3产生和钙释放中的作用。肌动球蛋白收缩的下游调节和对钙信号的反馈将被研究。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包含一个Rho家族GTP酶的GAP结构域，一个可以结合和弯曲细胞膜的BAR结构域，以及一个可能允许分子对DAG做出反应的C1域。在目标3中，将确定这些结构域在IP3和钙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。这项研究将使人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以调节组织功能的。

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.