Unrestricted Diffusion of Exogenous and Endogenous PIP2 in Baby Hamster Kidney and Chinese Hamster Ovary Cell Plasmalemma

Abstract

We used two approaches to characterize the lateral mobility of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the plasmalemma of baby hamster kidney and Chinese hamster ovary fibroblasts. First, nitrobenzoxadiazole-labeled C6-phosphatidylcholine and C16-PIP(2) were incorporated into plasma membrane "lawns" ( approximately 20 x 30 microm) from these cells and into the outer monolayer of intact cells. Diffusion coefficients determined by fluorescence recovery after photobleaching were similar for the two lipids and were higher in lawns, approximately 0.3 microm(2)/s, than on the cell surface, approximately 0.1 microm(2)/s. For membrane lawns, the fractional recoveries (75-90%) were close to those expected from the fraction of total membrane bleached, and labeling by the probes was several times greater than for intact cells. Second, we analyzed cells expressing M1 muscarinic receptors and green fluorescent protein fused with PIP(2)-binding pleckstrin-homology domains, Tubby domains or diacylglycerol (DAG)-binding C1 domains. On-cell gigaseal patches were formed with pipette tips >5 microm in diameter. When the agonist carbachol (0.3 mM: ) was applied either within or outside of the pipette, lipid signals crossed the pipette barrier rapidly in both directions and membrane blebbing occurred on both membrane sides. Accurate simulations of lipid gradients required diffusion coefficients >1 microm(2)/s. Exogenous DAG also crossed the pipette barrier rapidly. In summary, we found no evidence for restricted diffusion of signaling lipids in these cells. The lower mobility and incorporation of phospholipid at the extracellular leaflet may reflect a more ordered and condensed extracellular monolayer, as expected from previous studies.