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 (PIP(2)), several investigators have proposed that there are separate pools of PIP(2) in the plasma membrane. Recent experiments show the surface concentration of PIP(2) is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP(2) are also concentrated in these regions. However, how is the PIP(2) 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 PIP(2) 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 PIP(2). FCS measurements show that PIP(2) diffuses rapidly (D ~ 1 μm(2)/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP(2) 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 PIP(2) into and out of forming phagosomes.

FIGURE 1:

Methods used to study the diffusion of fluorescent PIP₂ in the forming phagosomes of macrophages. (A) Cartoon showing a J774a.1 macrophage and adjacent microinjector needle loaded with micelles containing Bodipy-TMR-PIP₂. (B) After microinjection, monomers of fluorescent PIP₂ incorporate rapidly into the inner leaflet of the plasma membrane, which is now colored red. The cell is then exposed to 8-μm-diameter latex beads coated with human IgG. One bead, colored gray, is shown in the process of landing on top of the cell. (C) The cell begins to ingest the bead by the process of Fcγ receptor–mediated phagocytosis. The laser focus (green hourglass) is positioned on the top membrane in the middle of the forming phagosome to obtain the FCS data from fluorescent PIP₂ molecules diffusing into and out of this area.

FIGURE 2:

FCS measurements of PIP₂ diffusion in the phagosomal cups of macrophages. (A) Fluorescence intensity scan in the z-direction through the center of the phagosomal cup region of a J774a.1 macrophage. The cell was injected with arachidoyl–Lyso-PC/Bodipy-TMR-PIP₂ micelles prior to addition of beads. The peaks correspond to the positions of the plasma membrane. We focused on the top membrane to perform FCS measurements. T = 25°C. (B) Autocorrelation function, G(τ), of Bodipy-TMR-PIP₂ diffusing in the forming phagosomal cup of the J774a.1 macrophage shown in A. The red curve represents the fit of Eq. 1, the equation for free Brownian diffusion in two dimensions, to the data. The average residency or correlation time (approximate midpoint of curve) is 24 ms for this example. (C) Average diffusion coefficients of Bodipy-TMR-PIP₂ in the plasma membrane of macrophages (cyan), in the forming phagosomal cups of macrophages (red), and in the plasma membrane of a different cell, the Rat1 fibroblast (green). The dots represent the 5th and 95th percentiles, the vertical bars represent the standard deviations, and the heights of the boxes represent the standard errors. The diffusion coefficient of PIP₂ in the phagosomal cup (red; 0.9 μm²/s) is not significantly lower than in the area outside the cup (cyan; 1.1 μm²/s).

FIGURE 3:

FRAP measurements on fluorescent PIP₂ are consistent with a fence around the forming phagosome. FRAP measurements of TopFluor-PIP₂ in the phagosomal cup of a J774 cell. (A) Cell injected with TopFluor-PIP₂ prior to the bleach. The rectangle of dashed white lines indicates the region of the phagosomal cup we bleached. (B) The same cell immediately after a 1.5-s bleach. (C) The same cell ∼100 s after the bleach. (D) Time trace of average fluorescence intensity per pixel in the region of the forming phagosome indicated by the dashed lines. T = 25°C. Result representative of experiments on nine different cells. (E) Control experiment that shows that the fluorescence due to TopFluor-PIP₂ recovers rapidly (time constant ∼10 s) and fully when we bleach a similar area in an unstimulated J774 macrophage (or in a region outside of the cup).

FIGURE 4:

Fluorescence due to PM-GFP within a phagocytic cup does not recover after photobleaching. RAW 264.7 macrophages transfected with a GFP construct that is targeted to the plasma membrane (PM-GFP) were allowed to engage 8-μm-diameter opsonized beads at 37°C (A, top; bead location indicated by white arrow). (A) Top, bleach of cup. PM-GFP within the cup was bleached, and selected images are shown from the time course of recovery. The frame “bleach” indicates the first frame after execution of the bleach cycle (∼2-s duration). Note that the fluorescence recovers only slightly in 100 s. Bottom, bleach of plasma membrane (PM) outside of the cup. Note that the fluorescence does recover after bleach of a similar area in the same cell. The white dashed circles indicate the bleached areas in both cases. (B) Fluorescence recovery curves for the two experiments shown in A. Inside the cup (filled black squares, red line) the fluorescence recovers only 20%; outside of the cup (open circles, green line) the fluorescence recovers 70%. The figure is representative of five similar results where the entire cup was bleached.

FIGURE 5:

Diffusion of proteins in the actin-rich region at the perimeter of the forming phagosome. (A) RAW macrophages with stable expression of mCherry-actin were allowed to phagocytose 8-μm-diameter latex beads opsonized with human IgG. A time course of actin dynamics during the phagocytosis is shown. Following initial contact and nascent phagosome formation (0´´), F-actin continues to surround the bead (30´´). When the F-actin reaches around ∼1/2 the bead, clearance of the F-actin occurs at the base of the cup (60´´). The cleared area continues to grow as the bead is engulfed by the advancing F-actin (90´´). Scale bar, 10 μm. T = 37°C. (B) Time course of GFP-actin dynamics during frustrated phagocytosis. Stable GFP/actin–expressing RAW macrophages were “parachuted” onto a coated coverslip. The coverslip was coated with BSA, followed by opsonization with mouse anti-BSA monoclonal antibody. Initial contact with coverslip is shown (0´´), followed by a time course of the macrophage forming a phagocytic “cup” with a thick F-actin ring. The ring advances along the coverslip, with clearing of F-actin from the center of the “cup” (30–90´´). The cell eventually becomes “frustrated” and the cup disassembles (not shown). Dynamics of F-actin ring formation and clearance are similar to those during phagocytosis of a particle as shown in A. Scale bar, 10 μm. (C) Example of photobleaching performed for FRAP experiments with macrophage expressing both mCherry-actin (red) and PM-GFP (green) following parachuting of cells onto opsonized coverslips and formation of the frustrated cup. Spinning disk confocal image of cell after photobleaching of a 3-μm-diameter circle (white dashed lines) inside an area cleared of F-actin, as well as 3-μm-diameter area in the F-actin ring of the frustrated cup. Scale bar, 5 μm. (D) Diffusion coefficients determined by FRAP for tested constructs. FRAP areas were either within the F-actin–cleared frustrated cup (Center) or within the strong F-actin ring of the frustrated cup (Edge). FRAP was also done on control cells grown on coverslips without opsonin present (Control). PM-GFP is the N-tail 10 residues of Lyn (myristoylated and palmitoylated) fused to GFP. tH-Ras-GFP is the minimal membrane targeting sequence from H-Ras (palmitoylated and farnesylated) fused to GFP. GT46-GFP is a nonraft transmembrane chimera fused to GFP, and GPI-GFP is an outer leaflet glycoslyphosphatidylinositol-anchored GFP fusion protein. Intracellular lipid-anchored probes are significantly slowed in the F-actin ring of the frustrated cup, but the effect is small (less than twofold). The recovery rate of the GPI probe on outer membrane leaflet is not affected. Asterisk indicates statistical difference with p < 0.05. Error bars indicate SEM (of n = 4–16 measurements per condition).