Repurposing Bacterial Mechanosensitive Channel as a Membrane Tension Biosensor

机械转导缺陷-将机械刺激转化为生物化学物质的细胞过程 信号-与多种疾病的发展有关，包括心肌病， 肌肉萎缩症和癌症进展。质膜的张力越来越大， 被认为积极调节许多细胞过程，包括细胞迁移和膜运输。 传统观点认为质膜是一个二维的流体脂质双层， 蛋白质导致了这样的想法，即膜张力可以远距离传递力来调节细胞 极性和迁移。然而，最近的工作指出，局部变化的膜张力， 介导不同的亚细胞过程。虽然存在测量膜张力的方法，但 技术需要专门的设置和专业知识。这严重限制了关键领域的研究进展。 细胞机械传导中的问题。为了满足这一未满足的需求，拟议工作的目标是 将细菌机械敏感通道MscL改造为膜张力生物传感器。大 从结构和生物物理研究预测的构象变化， 近年来在基于蛋白质的生物传感器上的成功使MscL成为工程化生物传感器的理想候选者。 薄膜张力传感器我们实验室最近的工作已经证明了MscL的功能重建， 哺乳动物细胞此外，我们已经积累了一系列创新的方法来重建MscL， 体外和用于操纵和测量细胞力学性质。在目标1中，我们将插入圆形 在MscL的周质环中置换GFP（cpGFP），并系统地工程化它以增加 反应能力和灵敏度。我们将表征重组到脂质双层囊泡中的cpGFP-MscL 并在活细胞中选择最佳的传感器实验。在目标2中，我们将建立连接 膜张力和细胞收缩性之间的关系，因为这两者之间的重要关系从来没有 被认定，但假设。我们将测量不同铺展面积的细胞膜张力 并且还评估了经受低渗休克的细胞中膜张力的动态变化。这些 实验将解决已知的细胞过程中膜张力的时空动力学， 有膜张力的变化。高空间和时间分辨率提供的荧光- 基于膜张力的生物传感器有望在膜生物物理学中产生变革性的影响， 发育生物学（其中发生的戏剧性形态动态预计将提高 膜张力）和机械生物学。

Defects in mechanotransduction – the cellular processes that convert mechanical stimuli into biochemical signals – are implicated in the development of a wide range of diseases, including cardiomyopathies, muscular dystrophies, and cancer progression. Tension of the plasma membrane has been increasingly recognized to actively regulate many cellular processes, including cell migration and membrane trafficking. The conventional view that the plasma membrane is a two-dimensional fluid lipid bilayer with embedded proteins has led to the idea that membrane tension could transmit forces over long-range to regulate cell polarity and migration. However, recent work has pointed to local variation of membrane tension can mediate distinct sub-cellular processes. While there exist approaches to measure membrane tension, the techniques require specialized setup and expertise. This has severely limited research progress in key questions in cell mechanotransduction. To address this unmet need, the objective of the proposed work is to repurpose bacterial mechanosensitive channel MscL as a membrane tension biosensor. The large conformational changes predicted from structural and biophysical studies coupled with phenomenal successes in recent years on protein-based biosensors make MscL an ideal candidate for engineering a membrane tension sensor. Recent work in our lab has demonstrated functional reconstitution of MscL in mammalian cells. Further, we have accrued a range of innovative methodologies for reconstituting MscL in vitro and for manipulating and measuring cell mechanics properties. In Aim 1, we will insert circular permutated GFP (cpGFP) in the periplasmic loop of MscL and systematically engineer it for increased responsiveness and sensitivity. We will characterize cpGFP-MscL reconstituted into lipid bilayer vesicles and select the most optimal sensor experiments in living cells. In Aim 2, we will establish the connection between membrane tension and cell contractility, as this important relationship between the two has never been determined but assumed. We will measure membrane tension in cells with different spreading areas and also evaluate dynamic changes of membrane tension in cells subjected to hypo-osmotic shock. These experiments will resolve the spatiotemporal dynamics of membrane tension in cellular process known to have membrane tension changes. The high spatial and temporal resolution afforded by the fluorescence- based membrane tension biosensor is expected to have transformative impact in membrane biophysics, developmental biology (where dramatic morphological dynamics that takes place is expected to elevate membrane tension), and mechanobiology.