ROLE OF INTRACELLULAR STRAIN IN MECHANOTRANSDUCTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 由于血压波动，血管内皮细胞(ECs)不断受到周期性拉伸。在笔直的无分支动脉中，内皮细胞被延长，其主轴与动脉血管的主轴取向，即垂直于周向拉伸的方向。垂直于拉伸方向排列的纤维所承受的张力小于与拉伸方向平行排列时的张力。因此，应力纤维的垂直取向有助于减小拉伸诱导的张力，从而使单轴拉伸引起的细胞内机械应变最小化。初步实验表明，内皮细胞中Rho活性的增加增强了应力纤维的垂直拉伸方向，而抑制Rho活性则使应力纤维平行于拉伸方向[1]。这些结果使我们认为Rho GTP酶通过控制应力纤维的重组，在调节细胞内机械应变的拉伸调制中起着核心作用。模型的实验测试需要阐明细胞粘弹性特性在表面应力的传递和传递中的作用，部分原因是细胞骨架。压力的转导将导致Src激酶信号[2，3]，我们可以通过基因编码的Src激酶FRET传感器实时监测这一事件[3]。我们将使用所提出的LAMP空间调制微束(SMM)系统通过激光镊子-微珠相互作用来施加应力，以通过监测机械诱导的Src信号来研究细胞力学(表面应力、应变和应变率)与应力传递之间的关系。这些实验将在人脐静脉内皮细胞(HUVEC或EC)上进行，其中人脐静脉内皮细胞(HUVEC或EC)被导入GFP融合的线粒体蛋白或显微注射荧光微珠，同时也被导入Src传感器，以确定细胞内力学的分子和细胞相关性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Vascular endothelial cells (ECs) are constantly subjected to cyclic stretch due to pulsatile blood pressure. In straight, unbranched arteries, ECs are elongated with their major axes oriented with that of the arterial vessel, i.e. perpendicular to the direction of circumferential stretch. Fibers aligned perpendicular to stretch bear less tension than when they are aligned parallel to stretch. Thus, perpendicular orientation of the stress fibers serves to reduce the stretch-induced tension and thus minimize the intracellular mechanical strain induced by uniaxial stretch. Preliminary experiments have shown that an increase of Rho activity in ECs enhances the perpendicular orientation of stress fibers to stretch and that the inhibition of Rho activity causes the stress fibers to be oriented parallel to the direction of stretch [1]. These results led us to propose that Rho GTPase plays a central role in regulating the stretch-modulation of intracellular mechanical strain through the control of stress fiber re-organization. Experimental testing of the model requires an elucidation of the role of cell viscoelastic properties, due in part to the cytoskeleton, in the transmission and transduction of surface stresses. Transduction of the stresses will lead to Src kinase signaling [2, 3], an event that we can monitor in real time through a genetically encoded Src kinase FRET sensor [3]. We will use the proposed LAMMP Spatially Modulated Microbeams (SMM) system to apply stresses through laser tweezer-microbeads interactions in order to study the relationship between cell mechanics (surface stresses, strain and strain rates) and stress transduction by monitoring the mechanically induced Src signaling. The experiments will be conducted on Human Umbilical Vein Endothelia Cells (HUVEC or EC) transfected with a GFP-fused mitochondrial protein or microinjected with fluorescent microbeads, and also transfected with the Src sensor in order to determine the molecular and cellular correlates of the intracellular mechanics.