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Regulation of transmembrane signaling by phase separation

The plasma membrane not only provides a barrier between the intracellular and extracellular space but also serves as a platform for processing and transducing information.  I am interested in understanding how protein phase separation organizes transmembrane receptors and their cytosolic binding partners into discrete signaling clusters on membranes.  My approach is to use in vitro reconstitution of signaling clusters on model membranes to dissect the molecular mechanisms that promote cluster formation and regulate downstream signaling.  I study the cell-cell adhesion receptor Neprhin as well as the cell-matrix adhesion receptor Integrin.

Nephrin is a transmembrane receptor that regulates cell-cell adhesion in podocyte cells of the kidney to form the glomerular filtration barrier.  The intracellular domain of Nephrin contains multiple Tyrosine residues that, when phosphorylated, bind to the SH2 domain of Nck.  Nck also contains three SH3 domains that bind to proline rich motifs in the actin regulatory protein N-WASP.  Multivalency in these molecules enables their assembly and concomitant phase separation, forming micron-scale signaling clusters on membranes that polymerize actin.  To understand how the phase separation of N-WASP regulates its actin assembly activity, I have developed a quantitative Total Internal Reflection Fluorescence (TIRF) microscopy assay to measure actin polymerization in the reconstituted Nephrin/Nck/N-WASP clusters.  I aim to understand how N-WASP activity is enhanced and regulated by phase separation.

            Focal adhesions are plasma membrane-associated compartments that serve as signaling hubs where cells sense biochemical and physical cues in the environment and control processes including cell migration, differentiation and death. Local clustering of integrin receptors, coupled with rapid recruitment and concentration of cytosolic binding partners, is critical for formation of signaling-competent focal adhesions in cells.  I am developing an in vitro experimental system to study the formation of integrin signaling complexes on model membranes.  I aim to understand how specific multivalent interactions between focal adhesion proteins regulate integrin clustering, focal adhesion formation and downstream signaling.