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和机械化学产生的机械应力密切相关，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与力和传感有关，包括血液动力学、协调运动、触觉、心律失常、肌肉萎缩、水肿、青光眼、耳聋、高血压等。细胞皮层形成了环境和细胞之间的界面，该项目解决了压力在细胞皮层中的分布以及它是如何被机械敏感离子通道（MSCs）感知的。我们将分析压力是如何由特定的细胞骨架蛋白、脂质双分子层和机械敏感离子通道所感知的。该项目有两个特定的目标，分别针对贴片和整个细胞，并为从高分辨率贴片数据推断细胞行为奠定基础。贴片实验将在贴片内定位不同的通道和细胞骨架蛋白；测量贴片受压时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；根据膜张力测量和量化内源性和TREK-1通道动力学；利用通道动力学、贴片电容、标记蛋白的荧光成像和特定标记细胞骨架蛋白的应力表征贴片内微结构域的特性。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在SICM胁迫下细胞骨架蛋白中的应力。我们将使用贴片中校准的间充质干细胞作为双层应力的附加探针。公共卫生相关性：生物学中的机械力与听力、肌肉收缩、血压调节等有关。正如人们所期望的那样，它们也参与了许多病理，如心律失常、高血压和肌肉萎缩症。本文分析了应力如何在分子、膜和细胞中分布，以及这如何导致离子通道的信号转导。

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.