PI(4,5)P(2) diffuses freely in the plasma membrane even within high-density effector protein complexes

The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P(2)] is a master regulator of plasma membrane (PM) function. Its effector proteins regulate transport, signaling, and cytoskeletal processes that define PM structure and function. How a single type of lipid regulates so many parallel processes is unclear. We tested the hypothesis that spatially separate PI(4,5)P(2) pools associate with different PM complexes. The mobility of PI(4,5)P(2) was measured using biosensors by single-particle tracking. We found that PM lipids including PI(4,5)P(2) diffuse rapidly (∼0.3 µm(2)/s) with Brownian motion, although they spend one third of their time diffusing more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P(2)-dependent complexes did not slow PI(4,5)P(2) lateral mobility. Only the spectrin and septin cytoskeletons showed reduced PI(4,5)P(2) diffusion. We conclude that even structures with high densities of PI(4,5)P(2) effector proteins, such as clathrin-coated pits and focal adhesions, do not corral unbound PI(4,5)P(2), questioning a role for spatially segregated PI(4,5)P(2) pools in organizing and regulating PM functions.