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.

许多蛋白质的功能是将同一分子上一个位点的化学活性信息传递到另一个位点。这种简单的远距离通信概念-统称为变构-通过提供最快速，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，在生物学的大部分领域都产生了反响。但是，当这两个位点位于不同但密切相互作用的生物集合体上时，这种远距离作用也会出现吗？该项目旨在通过实验研究细胞膜和一种名为E-cadherin的普遍存在的蛋白质之间的长距离通信来解决这个问题，这种蛋白质嵌入在这些膜中。该项目使用模型膜和活细胞的单分子测量来梳理膜的机械特性与E-钙粘蛋白的分子水平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的E-钙粘蛋白的重组，从而调节组织形成和伤口愈合过程中的细胞相互作用和细胞迁移。这项工作还通过建立一种新的范式，将细胞膜的整体材料特性与单个蛋白质的生化行为联系起来，为更广泛的科学界带来了好处。这项工作将（1）培训研究生和本科生进行跨学科合作研究，结合膜生物物理学，单分子科学，软物质，机械生物学和生物化学的工具，技术和观点，（2）实施积极主动的机制，以吸引代表性不足的少数民族，并直接促进STEM领域的多样性，包容性和公平性。 因此，它在物理科学和生物科学之间的界面上整合了基础跨学科研究的推广，教育和研究。 研究人员将解决一个中心假设，即膜机械特性的变化-例如由上游机械刺激引起的变化-可以诱导嵌入的膜蛋白的变构激活。 他们将结合联合收割机的方法，方法和单分子生物物理学，功能蛋白质动力学和膜力学开发的工具。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、宽视场和高含量旋转盘共聚焦荧光显微镜和相差光学显微镜;（2）分子定制和机械活化的膜模型（例如，巨大的脂质囊泡经受明确的渗透应力）;和（3）表达荧光标记的包埋膜蛋白的活细胞。作为一个试验平台，该提案研究了力诱导的E-钙粘蛋白的聚集，E-钙粘蛋白是一种关键的细胞表面粘附受体，在组织形成过程中协调细胞的复杂运动和维持组织完整性方面至关重要。PI和co-PI将集中精力在最小模型中解决力诱导蛋白质聚集的关键决定因素，并建立活细胞表面变构蛋白质聚集的生物物理决定因素。EAGER应用的成功完成将在细胞-细胞粘附的特定背景下建立膜变构的实验规则。 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、稳态和适应以及机械生物学。甚至更一般地，从这项研究中获得的见解应该为确定整体力学性质是否会影响单个分子的性质提供实验数据。该奖项由分子和细胞生物科学的分子生物物理学和细胞动力学和功能集群支持。该奖项反映了NSF的法定使命，并被认为值得通过利用基金会的智力价值和更广泛的影响审查标准。