Gelsolin-Polyphosphoinositide Interaction: Full Expression of Gelsolin-inhibiting Function by Polyphosphoinositides in Vesicular form and Inactivation by Dilution, Aggregation, or Masking of the Inositol Head Group

Abstract

Abstract Calcium activates, and the polyphosphoinositides phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibit the mechanical severing of actin filaments by gelsolin. Previous work indicated that the physical state of the two phospholipids is important for their effects in this system. This study correlates tests of gelsolin's severing function with quasielastic light scattering measurements of the size of mixed lipid particles and shows that the previously demonstrated diminution of the maximal effect of PIP2 in micellar form by aggregation of the micelles or mixing with other phospholipids is not the result of an absolute requirement for small lipid particles, but rather the masking of critical sites by aggregation, by sequestration in multilamellar vesicles, or by dilution of the polyphosphoinositides below a critical concentration. Large unilamellar vesicles of PIP and, importantly, PIP2 at low molar ratios (less than 3%) in mixed lipid vesicles of composition similar to plasma membranes are as active as PIP2 micelles. Aggregation or masking of polyphosphoinositide head groups by neomycin or profilin, respectively, blocked inhibition of gelsolin. Experiments with bilayer-forming phospholipids or with Triton X-100 indicate that a critical number of PIP2 molecules may be required for incipient effects on a gelsolin molecule. The actin and polyphosphoinositide binding protein profilin competed with gelsolin for binding PIP2 with a stoichiometry also suggesting binding to multiple PIP2 molecules. The membrane constituents sphingosine and cholesterol blocked the effect of PIP2 on gelsolin when added alone, but did not affect PIP2 when incorporated into mixed lipid bilayers containing phosphatidylinositol. The results suggest that profilin, small changes in membrane lipid composition, and, especially, membrane PIP2 concentration could have large effects on the modulation of gelsolin function in vivo.