EAGER: Membrane Allostery: How membrane mechanics regulates activity of membrane receptors

EAGER：膜变构：膜力学如何调节膜受体的活性

• 批准号: 2022385
• 负责人: Atul Parikh
• 金额: $ 30万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2022385&HistoricalAwards=false
• 关键词: EAGER; Membrane; Allostery; How; membrane

Many proteins function by relaying information of chemical activity at one site to a distant site on the same molecule. This simple concept of long-distance communication – collectively referred to as allostery – reverberates across much of biology by providing the most rapid, direct, and efficient means to switch proteins between functional and non-functional forms. But can such action-at-distance also arise when the two sites reside on different, but closely interacting, biological assemblies? This project seeks to address this question by experimentally investigating long-distance communication between cellular membranes and a ubiquitous protein called E-cadherin, which is embedded within these membranes. This project uses single-molecule measurements with model membranes and living cells to tease-out relations between the mechanical properties of the membrane and the molecular-level organization and function of E-cadherin. These studies will address how cell membranes control the reorganization of E-cadherins on the cell surface and consequently regulate cell interactions and cell migration during tissue formation and wound healing. The effort also provides benefit to the broader scientific community by establishing a new paradigm that links the global material properties of cellular membranes with biochemical behaviors of individual proteins. The work will (1) train graduate and undergraduate students in interdisciplinary collaborative research combining tools, techniques, and perspectives from membrane biophysics, single molecule science, soft matter, mechanobiology, and biochemistry and (2) implement proactive mechanisms to engage underrepresented minorities and contribute directly to enhancing diversity, inclusion, and equity with the STEM fields. It thus integrates outreach, education, and research in basic interdisciplinary research at the interface between the physical and biological sciences. The researchers will address a central hypothesis that changes in membrane mechanical properties – such as those elicited by an upstream mechanical stimuli – can induce allosteric activation of embedded membrane proteins. They will combine approaches, methodologies, and tools developed in single-molecule biophysics, functional protein dynamics, and membrane mechanics. Specifically, they will use (1) quantitative characterization methods including single molecule force - fluorescence microscopy, wide-field and high-content spinning disk confocal fluorescence microscopy, and phase contrast optical microscopy; (2) molecularly-tailored and mechanically activated membrane models (e.g., giant lipid vesicles subjected to well-defined osmotic stresses); and (3) living cells expressing fluorescently tagged, embedded membrane proteins. As a test-bed, the proposal investigates the force-induced clustering of E-cadherin, a key cell-surface adhesion receptor, which is essential in orchestrating complex movement of cells during tissue formation and in maintaining tissue integrity. The PI and co-PI will focus their efforts to resolve key determinants of the force-induced protein clustering in a minimal model and establish biophysical determinants for allosteric protein clustering on live cell surfaces. Successful completion of this EAGER application will establish the experimental rules underlying membrane allostery, in the specific context of cell-cell adhesion. These principals would be broadly applicable to membrane allostery across multiple aspects of cellular organization, dynamics, and function including signaling, homeostasis and adaptation, and mechanobiology. Even more generally, the insights obtained from this research should provide experimental data for determining whether global mechanical properties can influence properties of single molecules.This award is supported by the Molecular Biophysics and Cellular Dynamics and Function clusters of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

许多蛋白质通过将一个位点的化学活性信息传递到同一分子上的遥远位点来发挥作用。这个长距离交流的简单概念（共同称为变构）通过提供最快，直接和有效的手段来在功能和非功能形式之间切换蛋白质，从而在生物学中恢复生物学。但是，当两个站点居住在不同但密切相互作用的生物组件上时，这种动作距离也会出现吗？该项目旨在通过实验研究细胞膜与普遍存在的蛋白质E-钙粘着蛋白之间的长距离通信来解决这个问题，该蛋白嵌入了这些膜中。该项目使用与模型膜和活细胞的单分子测量结果来逗留膜的机械性能与E-钙粘着蛋白的分子级组织和功能之间的关系。这些研究将解决细胞膜如何控制细胞表面电子钙蛋白的重组，从而调节组织形成和伤口愈合期间细胞相互作用和细胞迁移。这项工作还通过建立一个将细胞膜的全球物质特性与单个蛋白质的生化行为联系起来的新范式，从而为更广泛的科学界提供了好处。这项工作将（1）在跨学科合作研究中训练毕业生和本科生，结合了膜生物物理学，单分子科学，软物质，机制和生物化学的工具，技术和观点，并（2）实施积极主动的机制，以直接占据了特定的少数群体，并促进多样性的领域，并促进多样性的领域，并促进各种各样的派对，并促进各种词性，等分，等于steg，等于steg，等于该词性，平等和平等，等于较少的领域。因此，在物理科学和生物科学之间的界面基本跨学科研究中，它将外展，教育和研究整合在一起。研究人员将解决一个中心假设，即膜机械性能的变化（例如上游机械刺激引起的）可以诱导嵌入式膜蛋白的变构激活。它们将结合单分子生物物理学，功能蛋白动力学和膜力学中开发的方法，方法和工具。具体而言，他们将使用（1）定量表征方法，包括单分子力 - 荧光显微镜，宽场和高内感旋转磁盘共聚焦荧光显微镜以及相位对比度光学显微镜； （2）分子定制和机械活化的膜模型（例如，经受明确定义的渗透应激的巨型脂质蔬菜）； （3）表达荧光标记的嵌入式膜蛋白的活细胞。作为测试床，该提案研究了钥匙细胞表面粘附受体的力引起的E-钙粘着蛋白的聚类，这对于在组织形成过程中和维持组织完整性过程中策划细胞的复杂运动至关重要。 PI和CO-PI将集中精力解决最小模型中力诱导的蛋白聚类的关键决定剂，并为在活细胞表面上的变构蛋白聚类建立生物物理确定剂。在细胞 - 细胞粘合剂的具体情况下，成功完成此急切的应用将建立膜变构基础的实验规则。这些原理将广泛适用于跨细胞组织，动力学和功能的多个方面的膜变构，包括信号传导，稳态和适应性以及机制。甚至更一般地，从这项研究中获得的见解应提供实验数据，以确定全球机械性能是否可以影响单分子的特性。这项奖项得到了分子生物物理学以及细胞动力学以及分子和细胞生物学家的细胞动力学以及功能群集的支持。这一奖项通过NSF的法规及其构成的稳定性影响，这是NSF的范围的范围。 标准。