Cell mechanics and mechanical transduction by ion channels

描述(申请人提供)：动物细胞的行为与环境产生的机械压力和机械化学密切相关，肌肉收缩和听力最能说明这一点。有大量的生理学和疾病与力量和感觉有关，包括血流动力学、协调运动、触摸、心律失常、肌肉营养不良、水肿、青光眼、耳聋、高血压等。细胞皮质形成了环境和细胞之间的接口，这个项目解决了应力在细胞皮质中的分布以及机械敏感离子通道(MSCs)如何感知它。我们将分析特定的细胞骨架蛋白如何分担压力，以及机械敏感离子通道是如何感受压力的。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外推到细胞行为奠定了基础。贴片实验将定位贴片内不同的通道和细胞骨架蛋白；在贴片受到压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；根据膜张力测量和量化内源性和Trek-1通道动力学；使用通道动力学、贴片电容、标记蛋白质的荧光成像和特定标记细胞骨架蛋白中的应力来表征贴片内微域的特性。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相结合的方法来检测细胞骨架蛋白和通道的分布，以及当细胞受到SICM压力时细胞骨架蛋白中的应力。我们将使用贴片中校准的MSCs作为额外的双层应力探针。与公共健康相关：生物学中的机械力可以解释听力、肌肉收缩、血压调节等更多因素。正如预期的那样，它们也参与了许多病理过程，如心律失常、高血压和肌肉营养不良。这项建议分析了应力如何在分子、膜和细胞中分布，以及如何通过离子通道进行信号转导。

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.