Transient Effect of Calcium Influx on PIP2 Clusters and Cholesterol-Stabilized Nano-Domains in the Inner Plasma Membrane Leaflet of Intact Cells

Abstract

When calcium ion channels open, local calcium levels are estimated to reach up to 500uM, which may be sufficient to affect negatively charged lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2), in the inner leaflet of the plasma membrane (PM). The PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between the acyl-chains of the lipids, stabilized by Cholesterol, lead to transient submicroscopic nano-domains, and lipid head-group interactions of charged lipids with divalent ions may cluster lipids. Here, we study the influence of calcium on the formation of PIP2 clusters and cholesterol-stabilized nano-domains in intact cells. We study changes of these domains over time and upon calcium channel activation by analyzing diffusion of GFP-tagged inner-leaflet membrane proteins. Using bimFCS, developed by us, we measure diffusion on multiple length scales simultaneously to derive information about domains. To study the formation of PIP2 clusters we use GFP-PHPLCdelta to directly mark PIP2, and as marker for cholesterol-stabilized nano-domains, we use Lck-mGFP. We observe that opening TRPV1 channels increases the interaction both between GFP-PHPLCdelta and PIP2 domains, Lck-mGFP and cholesterol domains. The interaction between GFP-PHPLCdelta and PIP2 domains decreases to base lines within 1-2 minutes, while the interaction of Lck-mGFP and cholesterol domains takes another 2-3 minutes to decrease as the cell down-regulates the intracellular calcium level after stimulation. To control for large scale signaling, we image the membrane cytoskeleton using mCherry-alpha-actinin, and use GT46-GFP to mark transmembrane domains. These results suggest a concentration dependence of calcium-induced PIP2 clusters and cholesterol-stabilized nano-domains in the PM at calcium levels, which may be reached in intact cells locally by opening of ion channels.