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.
