Repurposing Bacterial Mechanosensitive Channel as a Membrane Tension Biosensor

机械转导中的缺陷--将机械刺激转化为生化的细胞过程 信号--与多种疾病的发展有关，包括心肌病， 肌肉营养不良和癌症进展。质膜的张力越来越大。 被公认为积极调节许多细胞过程，包括细胞迁移和膜运输。 传统的认为质膜是嵌入了膜的二维流体脂双层 蛋白质导致了一种想法，即膜张力可以远距离传递力量来调节细胞 极性和迁徙。然而，最近的研究表明，膜张力的局部变化可以 调节不同的亚细胞过程。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。这严重限制了关键技术的研究进展 细胞机械转导中的问题。为了解决这一未得到满足的需求，拟议工作的目标是 目的：将细菌机械敏感通道MSCL作为膜张力生物传感器。大的 从结构和生物物理研究预测的构象变化与现象 近年来在基于蛋白质的生物传感器方面的成功使MSCL成为工程应用的理想候选者 薄膜张力传感器。我们实验室最近的工作证明了骨髓间充质干细胞的功能重建 哺乳动物细胞。此外，我们已积累了一系列创新的方法，以重组MSCL在 用于体外操作和测量细胞的力学性能。在目标1中，我们将插入圆形 骨髓间充质干细胞周质环中的置换绿色荧光蛋白(CpGFP)及其系统工程 响应性和敏感度。我们将鉴定重组成脂质双层囊泡的cpGFP-MSCL 并选择在活细胞中进行最理想的传感器实验。在目标2中，我们将建立连接 膜张力和细胞收缩能力之间的关系，因为两者之间的这种重要关系从来没有 已经确定了，但却是假设的。我们将测量具有不同扩散区域的细胞的膜张力 并评价低渗休克时细胞膜张力的动态变化。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。由荧光提供的高空间和时间分辨率- 基于膜张力的生物传感器有望在膜生物物理学中产生革命性的影响， 发育生物学(发生戏剧性形态动力学的领域预计将提高 膜张力)，以及机械生物学。

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.