Abstract
Phospjatidylinositol 4,5-bisphosphated(PIP2) is known to interact with many cytoskeletal proteins electrostatically. When calcium ion channels open, local calcium levels are estimated up to 500uM. Experiments in model lipid bilayer have shown that such calcium level is sufficient to affect PIP2 in the inner leaflet of plasma membrane (PM).PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between lipid acyl-chains, stabilized by cholesterol, lead to transient submicroscopic nano-domains, and head-group interactions of charged lipids with divalent ions may cluster lipids. In addition, membrane cytoskeleton may impose further structure on membrane. Here we study influence of calcium on formation of PIP2 clusters in intact cells. We study changes of 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 formation of PIP2 clusters in the PM, we use GFP-PHPLCdelta to directly mark PIP2. We observe that opening TRPV1 channels leads to a transient rise in calcium as imaged using GCaMP5G, increases interaction strength between GFP-PHPLCdelta and PIP2 domains, which decreases to base lines within 1-2 minutes. Using ionophores, we determine that the increasing interaction strength is due to calcium-PIP2 interaction. By Vinculin depletion or actin filaments (de-)polymerization, we find that both anchor proteins and actin filaments play important roles in PIP2 clusters, and the formation of larger PIP2 clusters can potentially further activate Plasma membrane Ca2+ ATPase. These results suggest that in intact cells, calcium ions are able to induce changes of PIP2 clusters that are also regulated by cytoskeleton structure, changes of PIP2 clusters can further affect other signaling events.
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