The plasma membrane of eukaryotic cells is organized into lipid and protein microdomains, which have been observed in many cellular processes including cell growth, neuronal signaling, and cell adhesion. Such biochemically distinct compartments are believed to play an important role in the transmission of signals between the extracellular environment and the cytoplasm. However, the mechanisms by which receptors assemble into micron-scale structures are incompletely understood.
Our lab previously demonstrated that the podocyte transmembrane protein Nephrin and its downstream signaling molecules Nck and N-WASP can produce phase separated polymers via multivalent interactions, both in solution and on model membranes. My project follows from this in vitro work to study the micron-scale assembly of Nephrin/Nck/N-WASP at the basal plasma membrane of cells upon triggered phosphorylation of Nephrin. I have found that the formation of protein clusters in cells is dependent on the concentration of Nephrin and Nck in the cytoplasm, as well as on the number of Nck SH3 domains. These clusters can readily exchange components with their surroundings, suggesting they are polymers analogous to those previously observed in vitro. The clusters form independently of the actin cytoskeleton, but their movement on the membrane depends on the acto-myosin contractility. Nephrin phosphorylation also causes formation of larger clusters at the cell periphery, which are enriched in the actin cytoskeleton and cause changes in cell morphology. These observations suggest that even in the crowded cellular environment with many competing molecules, multivalent interactions between proteins at the plasma membrane are sufficient to promote micron-scale organization of signaling molecules, which can both respond to and regulate the actomyosin cytoskeleton.