Influence of Calcium Concentration on Lipid Domains in the Inner and Outer Leaflets of the Plasma Membrane

Many cellular functions are modulated by the spatial organization of membrane lipids. The effects of head-group-mediated lipid interactions and cytosolic factors play an important role for the lateral organization of lipids, but is less well understood than the role of the lipid fatty acid tails. The interaction of the lipid headgroups can be modulated by divalent ions. It has been reported that divalent calcium ions stabilize domains in monolayers containing phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how the local concentration of calcium affects the formation of lipid rafts in the much more complex, PIP2 containing plasma membrane (PM). Here, we study the influence of calcium concentration on domain formation in PM sheets created from intact cells by sonication. We analyze the diffusion of GFP-tagged membrane proteins, which interact with the domains, using bimFCS, which measures diffusion on multiple length scales simultaneously. PIP2 is a cytosolic leaflet lipid with negatively charged head groups. To study the aggregation of PIP2 in the PM, we use GPI-GFP to study the outer leaflet of the PM, and Lyn-GFP and fluorescent PIP2 for the inner leaflet of the PM. Our results that these marker proteins reside longer in the PIP2 lipid domains at 2mM of divalent calcium ions, while this residence time is reduced rapidly as we decrease the calcium concentration. Theses results suggest a concentration dependence of calcium-induced aggregation of PIP2 in the PM at calcium levels, which may be reached in intact cells locally by opening of ion channels.