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

许多蛋白质的功能是将一个位置的化学活动信息传递给同一分子上的另一个位置。这种简单的远距离交流的概念--统称为变构--通过提供最快速、直接和有效的手段在功能形式和非功能形式之间切换蛋白质，在生物学的大部分领域产生反响。但是，当这两个位置位于不同但密切相互作用的生物组装上时，这种远距离作用也会发生吗？这个项目试图通过实验研究细胞膜和嵌入在细胞膜中的一种名为E-钙粘蛋白的普遍存在的蛋白质之间的远程通信来解决这个问题。该项目使用模型膜和活细胞的单分子测量，梳理出膜的机械性能与E-钙粘蛋白的分子水平组织和功能之间的关系。这些研究将阐述细胞膜如何控制细胞表面E-钙粘附素的重组，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，将细胞膜的全球材料属性与单个蛋白质的生化行为联系起来，从而为更广泛的科学界提供了好处。这项工作将(1)培训研究生和本科生进行跨学科协作研究，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，让未被充分代表的少数群体参与进来，直接促进STEM领域的多样性、包容性和公平性。因此，它将在物理科学和生物科学之间的基础跨学科研究中的外展、教育和研究结合在一起。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那些--可以诱导嵌入的膜蛋白的变构激活。他们将结合在单分子生物物理学、功能蛋白质动力学和膜力学中开发的方法、方法和工具。具体地说，他们将使用(1)定量表征方法，包括单分子力荧光显微镜、广场和高含量旋转圆盘共聚焦荧光显微镜和相差光学显微镜；(2)分子定制和机械激活的膜模型(例如，受到明确渗透压力的巨大脂泡)；以及(3)表达荧光标记的嵌入膜蛋白的活细胞。作为一个试验台，该提案研究了E-钙粘附素的力诱导聚集，E-钙粘附素是一种关键的细胞-表面黏附受体，在组织形成期间协调细胞的复杂运动和维持组织完整性方面是必不可少的。PI和co-PI将致力于在最小模型中解析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。这一急切应用的成功完成将建立在细胞-细胞黏附的特定背景下的膜变构的实验规则。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、动态平衡和适应以及机械生物学。更广泛地说，从这项研究中获得的见解应该为确定全球机械性能是否可以影响单分子的性质提供实验数据。该奖项得到了分子和细胞生物科学的分子生物学和细胞动力学与功能簇的支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.