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Profilin is a small (12-15 kDa) actin binding protein. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with polyphosphoinositides (PPI). To date, profilin's interaction with PPI has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of phosphatidylinositol (4,5) bis-phosphate [PI(4,5)P₂] molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P₂ and PI(3,4,5)P₃. Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. Despite the low affinity for submicellar concentration of PI(4,5)P₂, profilin bind to Giant unilamellar vesicles (GUV) in presence of 1% mole fraction of PI(4,5)P₂. We also found that profilin destabilize the membrane of the GUVs, resulting eventually in the complete destructions of the GUVs. Finally, we investigated the effect of varying the mole fraction of PI(4,5)P₂ on the GUV morphology in absence of profilin. These results are discussed and compared with the effect of profilin on the GUV membrane. |
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