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

Abstract

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 ∼ 1 μm²/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 (∼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.