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Regulation of the dynamics and stability of supramolecular clusters on artificial membranes

Nephrin is a transmembrane receptor that is essential for the formation and maintenance of the glomerular filtration barrier in kidneys. The glomerular filtration barrier is composed of cells, called podocytes, that form a physical barrier at cell-cell junctions to prevent large molecules, such as proteins, from leaving the blood stream while allowing small molecules to freely diffuse out of the blood to produce urine. This barrier is formed and maintained through the apparent clustering of Nephrin in the membranes of podocytes, which promotes Nephrin-Nephrin interactions between neighboring podocytes. Upon injury, this barrier fails, resulting in proteinuria, a disease in which protein is lost from the blood stream and excreted from the organism. Studies have shown that phosphorylation of three tyrosines in the cytoplasmic tail of Nephrin are necessary for the formation and maintenance of the glomerular filtration barrier.

Phosphorylation of the three tyrosines in the Nephrin tail creates docking sites for the Src homology (SH) 2/SH3 adaptor protein Nck. Upon binding to Nephrin, Nck recruits and activates N-WASp, which recruits and activates the Arp2/3 complex to promote actin polymerization. Our lab has shown that the multivalent interactions of phosphorylated Nephrin, Nck and N-WASp on artificial membranes promote the formation of large supramolecular protein clusters and that cluster formation is dependent on the degree of nephrin phosphorylation, suggesting that cluster formation can be regulated by kinase and phosphatase activity. However, the effect of kinase/phosphatase activity on Nephrin/Nck/N-WASp cluster formation has not been demonstrated. I am currently studying how kinase and phosphatase activity regulate the kinetics of formation and dissolution, respectively, of these clusters.

Like Nephrin, the transmembrane adaptor protein LAT contains four cytoplasmic tyrosine residues that, upon phosphorylation, function as docking sites for the SH2 domain-containing proteins PLCγ1, Grb2 and Gads. Membrane-bound microclusters of LAT have been observed following antigen-stimulated activation of T cells. Experiments have suggested that the formation of LAT microclusters is essential for T cell signaling downstream of LAT. Two potential LAT signaling modules exist that may promote LAT clustering through multivalent interactions: Grb2 and Sos1 or PLCγ1, Gads and SLP-76. How these two modules contribute to the stability and dynamics of LAT microclusters is unknown.

I am currently collaborating with Xiaolei Su, a postdoc in Ron Vale’s group at the University of California San Francisco, to determine the biochemical mechanism of LAT clustering on artificial membranes. These studies will reveal how the multivalent interactions of phosphorylated LAT, Grb2, Sos1, PLCγ1, Gads and SLP-76 regulate microcluster formation and function.