ROLE OF INTRACELLULAR STRAIN IN MECHANOTRANSDUCTION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 血管内皮细胞（EC）由于脉动血压而不断受到周期性拉伸。在直的、无分支的动脉中，EC是细长的，其主轴与动脉血管的主轴定向，即垂直于周向拉伸的方向。 垂直于拉伸方向排列的纤维比平行于拉伸方向排列的纤维承受更小的张力。因此，应力纤维的垂直取向用于降低拉伸诱导的张力，从而使单轴拉伸诱导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与拉伸的垂直取向，而Rho活性的抑制导致应力纤维平行于拉伸方向取向[1]。这些结果使我们提出，Rho GTdR通过控制应力纤维重组在调节细胞内机械应变的拉伸调制中起着核心作用。该模型的实验测试需要阐明细胞粘弹性的作用，部分是由于细胞骨架，在表面应力的传输和转导。应激的转导将导致Src激酶信号传导[2，3]，我们可以通过遗传编码的Src激酶FRET传感器真实的监测这一事件[3]。我们将使用拟议的LAMMP空间调制微束（SMM）系统，通过激光镊子微珠相互作用施加应力，以研究细胞力学（表面应力，应变和应变率）和应力转导之间的关系，通过监测机械诱导的Src信号。实验将在用GFP融合的线粒体蛋白转染或用荧光微珠显微注射的人脐静脉内皮细胞（HUVEC或EC）上进行，并且还用Src传感器转染，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.