Divalent Cation-Induced PIP2 Clustering in Cholesterol-Containing Membranes: How PIP2 Lateral Distribution affects PIP2-Gelsolin Interaction

Abstract

Phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) is involved in many important cellular events, but the mechanism by which this relatively rare lipid selectively regulates specific proteins is unclear. While many proteins bind to PIP2 with similar affinities in vitro, only a small subset interact with PIP2 at specific sites during specific cellular processes. Previously, we showed that lateral distribution of PIP2 in a background of neutral lipids is affected by divalent cations through counterion-mediated attraction. We further hypothesize that spatial control of PIP2 concentration can alter the binding of this lipid to its protein targets. To test this hypothesis, we determine if Ca2+-induced PIP2 clustering affects the interaction between PIP2 and PIP2-binding proteins with polybasic domains under liquid order/liquid disorder (Lo/Ld) phase demixing conditions.We first characterize the effect of divalent cations on Lo/Ld phase-demixing. The addition of Ca2+ induces a surface pressure drop and lowers the transition surface pressure. In contrast, Mg2+ increases the transition surface pressure and has a minimum condensing effect. Topography measurements through AFM show that Ca2+-induced PIP2-rich clusters co-localize with the Lo phase. The effect of PIP2 microdomain/cluster formation on the regulation of gelsolin was studied using an actin filament severing assay and Ca2+-insensitive gelsolin fragments (NtGSN). This functional assay suggests that membrane partitioning of NtGSN is sensitive to PIP2 local concentration upon phase separation, which also depends on the temperature. Cholesterol-induced phase demixing strongly inhibits the severing function of gelsolin, and the presence of PIP2 clusters formed by micromolar Ca2+ also improves the inhibition efficiency. These observations suggest that changes in local PIP2 distribution might be a major mechanism to determine how proteins interact with PIP2 in the membrane. This research may shed a light in studying the interplay between PIP2, cholesterol and Ca2+-signaling.