The cellular plasma membrane is a complex, crowded mixture of lipids and proteins. The lipid environment in which proteins find themselves can influence protein function, but the interplay between lipids and proteins that regulates function remains elusive. The working hypothesis of this project is that transient compartmentalization of proteins in membrane regions enriched in particular lipids and the resulting intermolecular interactions in biomembranes must control biological function. However, current conventional approaches lack the resolution and the ability to manipulate single molecules or groups of molecules that are required to probe the dynamics of these interactions on living cells. The PI and co-PI will develop and use cutting edge imaging approaches to investigate quantitatively the molecular interactions and their dynamics that occur between cholesterol-rich membrane regions and immune receptor signaling in mast cells. Key to the cross-disciplinary experimental and analytical approach is a combination of non-invasive and quantitative fluorescence microscopy, spectroscopy and manipulation that will be applied to living mast cells and artificial (biomimetic) membranes. The project includes experiments to investigate the dynamics of lipids and proteins in living mast cells during immune receptor signaling, using fluorescence microscopy and spectroscopy with high spatial and temporal resolution, and testing whether it is possible to nucleate membrane microdomains (regions enriched in particular lipids, including cholesterol) in artificial membranes using dynamic holographic optical trapping and fluorescence microscopy and spectroscopy. More broadly, these results and tools will be directly applicable to other signaling pathways (e.g., other immunoreceptors, growth factor receptors and other receptors) that may be influenced by membrane microdomains. Broader Impacts: The project will result in development of cutting edge discovery-based tools that should find broad applications in biophysics and cell biology. This cross-disciplinary research program serves as an ideal platform to train the next generations of scientists at the interface of cell biology, biophysics, chemistry, bioengineering and optics. The PI and co-PI also are active in public outreach, for example, by exhibiting their dramatic imaging technologies at a booth at the annual County Fair.
