Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as fences around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D similar to 1 mu m(2)/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (> 100 s) after photobleaching the entire forming phagosome but recovers rapidly (similar to 10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane-anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.