Cell mechanics and mechanical transduction by ion channels

描述（由申请人提供）：动物细胞行为与环境产生的机械应力和机械化学密切相关，肌肉收缩和听觉最生动地说明了这一点。有大量的生理和疾病连接到力和传感，包括血液动力学，协调运动，触摸，心律失常，肌肉萎缩症，水肿，青光眼，耳聋，高血压等细胞皮层形成环境和细胞之间的界面，该项目解决了细胞皮层中的应力分布以及如何通过机械敏感离子通道（MSC）进行感知。我们将分析压力是如何共享特定的细胞骨架蛋白，脂质双层，并通过机械敏感离子通道。该项目有两个具体目标，分别针对斑块和整个细胞，并为从高分辨率斑块数据推断细胞行为奠定基础。贴片实验将定位贴片内的不同通道和细胞骨架蛋白;当贴片受到应力时测量细胞骨架蛋白中的应力;创建具有最小细胞骨架的贴片以简化应力分布;测量和量化膜张力方面的内源性和TREK-1通道动力学;使用通道动力学，贴片电容，标记蛋白的荧光成像和特异性标记细胞骨架蛋白的应力。全细胞实验将使用扫描电导显微镜（SICM）、全细胞膜片钳和荧光探针的组合来检查细胞骨架蛋白和通道的分布以及细胞受到SICM应激时细胞骨架蛋白中的应激。我们将使用在贴片中校准的MSC作为双层应力的额外探针。公共卫生相关性：生物学中的机械力解释了听力、肌肉收缩、血压调节等诸多问题。正如对这种普遍存在的过程所预期的那样，它们也参与许多病理学，如心律失常、高血压和肌肉萎缩症。该提案分析了应力如何分布在分子、膜和细胞中，以及如何通过离子通道导致信号转导。

DESCRIPTION (provided by applicant): Animal cell behavior is tightly linked to mechanical stresses produced by the environment and mechanochemistry illustrated most vividly by muscle contraction and hearing. There is an enormous amount of physiology and disease connected to forces and sensing including hemodynamics, coordinated movement, touch, cardiac arrhythmias, muscular dystrophy, edema, glaucoma, deafness, high blood pressure, etc. The cell cortex forms the interface between the environment and the cell and this project addresses the distribution of stress in the cell cortex and how it is sensed by mechanosensitive ion channels (MSCs). We will analyze how stress is shared by specific cytoskeletal proteins, the lipid bilayer, and sensed by mechanosensitive ion channels. The project has two specific aims directed at patches and whole cells and creating a basis for extrapolating from high resolution patch data to cell behavior. The patch experiments will localize different channels and cytoskeletal proteins within the patch; measure stress in cytoskeletal proteins as the patch is stressed; create patches with minimal cytoskeleton to simplify the stress distribution; measure and quantify endogenous and TREK-1 channel kinetics in terms of membrane tension; characterize the properties of microdomains within the patch using channel kinetics, patch capacitance, fluorescence imaging of labeled proteins and the stress in specific labeled cytoskeletal proteins. The whole cell experiments will use a combination of scanning conductance microscopy (SICM), whole cell patch clamp and fluorescent probes to examine the distribution of cytoskeletal proteins and channels and the stress in cytoskeletal proteins as the cell are stressed by the SICM. We will use MSCs calibrated in the patch as additional probes of bilayer stress. PUBLIC HEALTH RELEVANCE: Mechanical forces in biology account for hearing, muscle contraction, blood pressure regulation and a great deal more. As expected for such ubiquitous processes, they are also involved in much pathology such as cardiac arrhythmias, high blood pressure and muscular dystrophy. This proposal analyses how stresses are distributed in molecules, membranes and cells and how that leads to signal transduction by ion channels.