Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers

Fluorescent derivatives of phosphatidyl inositol (PtdIns)-(4,5)-P-2 were sy nthesized and used to test the effects of the PtdIns-(4,5)-P-2-regulated pr oteins gelsolin, tau, cofilin, and profilin on labeled PtdIns-(4,5)-P-2 tha t was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7-nitrobenz-2-oxa -1,3-diazole (NBD)- or pyrene-labeled PtdIns-(4,5)-P-2 and NBD-PtdIns-(3,4, 5)-P-3. Cofilin and profilin produced no detectable change at equimolar rat ios to PtdIns-(4,5)-P-2, while tau decreased NBD-PtdIns-(4,5)-P-2 fluoresce nce. Fluorescence enhancement by gelsolin of NBD-PtdIns-(4,5)-P-2 in mixed lipid vesicles depended on the mole fraction of PtdIns-(4,5)-P-2 in the bil ayer. Specific enhancement of 3% NBD-PtdIns-(4,5)-P-2 : 97% PtdCho was much lower than that of 10% PtdIns-(4,5)-P-2: 90% PtdCho, but the enhancement o f 3% NBD-PtdIns-(4,5)-P-2 could be increased by addition of 7% unlabeled Pt dIns-(4,5)-P-2. The gelsolin-dependent increase in NBD-PtdIns-(4,5)-P-2 flu orescence was reversed by addition of Ca2+ or G-actin. Significant, but wea ker, fluorescence enhancement was observed with the gelsolin N-terminal dom ain (residues 1-160) and a peptide comprised of gelsolin residues 150-169. Fluorescence energy transfer from gelsolin to pyrene-PtdIns-(4,5)-P-2 was m uch stronger with intact gelsolin than the N-terminal region of gelsolin co ntaining the PtdIns-(4,5)-P-2 binding sites, suggesting that PtdIns-(4,5)-P -2 may bind near a site formed by the juxtaposition of the N- and C-termina l domains of gelsolin.