In vivo analysis of mechanotransduction

力转导的体内分析

• 批准号: 9321991
• 负责人: Erin Jean Cram
• 金额: $ 38.79万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9321991
• 关键词: Actins; Actomyosin; Animals; Behavior; Binding; Binding Sites; Biochemical; Biophysics; Biosensor; Caenorhabditis elegans; Calcium; Calcium Oscillations; Calcium Signaling; Cells; Cellular biology; DAG/PE-Binding Domain; Development; Embryo; Engineering; Esthesia; Family; Feedback; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate Phosphohydrolases; Health; Human; Imaging Device; Knowledge; Mass Spectrum Analysis; Mechanics; Membrane; Molecular; Monomeric GTP-Binding Proteins; Morphogenesis; Oocytes; Organ; Phospholipase; Phospholipase C; Physiology; Play; Process; Production; Proteins; Regulation; Reproductive system; Research; Role; Scaffolding Protein; Signal Transduction; Stimulus; Stress; Stretching; Structure; System; Testing; Tissues; Tube; Work; base; cancer cell; cardiovascular health; experience; filamin; genetic manipulation; improved; in vivo; in vivo imaging; insight; mechanical force; mechanical properties; mechanotransduction; novel; phospholipase C epsilon; premature; pressure; prevent; public health relevance; response; rho; rho GTP-Binding Proteins; rho GTPase-activating protein; scaffold; sensor; sperm cell

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.

描述（由申请人提供）：机械转导的（对机械力的感觉和反应）在人类生理学中至关重要。例如，适当的感觉和对压力拉伸的反应，流动对于心血管健康至关重要。尽管生物物理学和工程底物的细胞生物学有所了解，但对于细胞如何将机械信息（例如拉伸）转化为控制组织在体内功能功能的生化信号的知识知之甚少。在这里，我们介绍了一种新颖且容易的体内系统，用于研究机械转移的研究，这是秀丽隐杆线虫生殖系统的拉伸敏感细胞。我们已经发现，卵母细胞进入管状器官，称为精子theca触发Ca+2的波浪，它们横穿精子theca，以平滑的挤压为顶，以排出受精的胚胎。我们建议测试卵母细胞进入的假设会拉伸分子应变测量表FLN-1/丝蛋白，从而导致小GTPase Rho-1/Rho，PLC-1/PLC-1/磷脂酶C-EPSILON，IP3-触发的钙释放，并协调了上层状组织组织的相互构造。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，而拉伸响应所需的FLN-1域将用于通过质谱分离键相互作用的蛋白质。在AIM 2中，将使用表达IP3和Ca+2生物传感器的动物的遗传操纵来确定FLN-1和RHO-1在PLC-1激活中的作用，IP3产生和Ca+2释放。将研究肌动蛋白收缩性的下游调节和对CA+2信号的反馈。我们发现，需要新型调节剂TAG-341来防止钙信号的过早激活。 TAG-341包含一个用于RHO家族GTPases的间隙结构域，一个可能结合和弯曲膜的条形域，以及一个可能允许该分子响应DAG的C1结构域。在AIM 3中，将确定这些域在对IP3和Ca+2信号的激活和响应中的功能，以及在TAG-341的拉伸敏感脚手架中对FLN-1和PLC-1的要求。这项研究将导致对细胞将机械信息转换为生化信号的基本机制的基本机制，以及如何整合该信号以调节组织功能。