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. 2000 Dec 25;151(7):1353-68.
doi: 10.1083/jcb.151.7.1353.

Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis

R J Botelho  1 , M Teruel, R Dierckman, R Anderson, A Wells, J D York, T Meyer, S Grinstein
Affiliations

Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis

R J Botelho et al. J Cell Biol. .

Abstract

Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

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Figures

Figure 1
Figure 1
Distribution of PLCδ-PH-GFP, Akt-PH-GFP, and PM-GFP in RAW macrophages. RAW cells were transfected with PLCδ-PH-GFP (A and B), Akt-PH-GFP (C and D), and PM-GFP (E and F). Shown are representative cells before (A, C, and E) and after 10 min incubation in the presence of 10 μM ionomycin in medium containing 1.2 mM calcium (B, D, and F). Bar, 10 μm.
Figure 2
Figure 2
Distribution of PLCδ-PH-GFP during phagocytosis. Macrophages expressing PLCδ-PH-GFP were exposed to IgG-opsonized particles to induce phagocytosis. (A–C) Phagocytosis of RBCs. First panel is DIC image corresponding to the fluorescence image at t = 0 min. Subscripts (e.g., A0) refer to the time in minutes at which the fluorescence images were captured. The times refer to the onset of recording, not to the time of addition of RBCs. Open arrows point to nascent phagosomes and solid white arrows show formed phagosomes. B and C are enlargements of the areas identified by boxes in A4 and A6. (D) Phagocytosis of 8-μm latex beads. (E) Corresponding DIC image. Nascent and formed phagosomes are indicated by open and filled circles, respectively, in B–E. (F and G) Macrophages expressing PLCδ-PH-GFP (F) were allowed to initiate phagocytosis of RBCs, followed by fixation and staining for F-actin with rhodamine-phalloidin (G). Open arrows point to phagocytic cups while the small filled white circles indicate formed phagosomes. Bars, 10 μm.
Figure 3
Figure 3
Phagocytosis in macrophages expressing PM-GFP. (A) DIC image and time course of phagocytosis, as described in the legend to Fig. 2 A. (B and C) Enlargements of the areas identified by boxes in A0 and A6. (D) Phagocytosis of 8-μm latex beads. (E) Corresponding DIC image. Bars, 10 μm.
Figure 4
Figure 4
Quantification of phagosomal fluorescence. Confocal images of RAW cells undertaking phagocytosis were acquired as in the legends to Fig. 2 and Fig. 3. The digitized fluorescence intensity was quantified along line scans traversing both a phagosomal cup (x) and the contralateral plasma membrane (y). Representative images and corresponding quantitations are illustrated. (A and B) Cell transfected with PLCδ-PH-GFP. (C and D) Cell transfected with PM-GFP. (E) Ratio of the fluorescence at the phagosomal cup to the plasmalemma (two leftmost bars) or of the membrane of sealed phagosomes to the plasmalemma (two rightmost bars). The PLCδ-PH-GFP fluorescence of sealed phagosomes was negligible, yielding a ratio of ≈0. Means ± SE of 35, 64, 63, and 14 determinations, from left to right. (F) Distribution of PIPKIα during phagocytosis of Texas red–labeled zymosan particles (shown in G). Open arrow, nascent phagosome. Solid arrow, sealed phagosome. Bars, 10 μm.
Figure 7
Figure 7
Sequestration and degradation of 4,5-PIP2 attenuates phagocytosis. (A) Phagocytosis was quantified as described in Materials and Methods in RAW cells treated as follows (from left to right): control (untreated/untransfected); preincubated overnight with 5 or 10 mM neomycin sulfate; transfected with PLCδ-PH-GFP; transfected with GFP; transfected with PM-5′-phosphatase-GFP; transfected with PM-GFP. The phagocytic index was normalized to that of controls performed the same day. Data are means ± SE of 1,200 cells per condition. (B) Estimation of the concentration of PLCδ-PH-GFP expressed by RAW macrophages. To construct a calibration curve, droplets of solutions of recombinant EGFP at varying concentrations were placed under mineral oil and analyzed by confocal microscopy. The corresponding fluorescence is plotted versus EGFP concentration (squares). Cells transfected with PLCδ-PH-GFP were treated with ionomycin as in the legend to Fig. 1 and confocal images were acquired. Mean fluorescence of three experiments, each with ≥25 cells, is shown (circles with error bars representing SEM).
Figure 5
Figure 5
Distribution of PLCγ during phagocytosis. (A) Distribution of PLCγ2 during phagocytosis of Texas red–labeled zymosan particles (shown in B). (C) Distribution of PLCγ1-SH2-GFP during phagocytosis of RBCs (shown in D). Arrows in A–D point to nascent phagosomes. Bars, 10 μm.
Figure 6
Figure 6
Distribution of C1δ-YFP and PLCδ-PH-CFP during phagocytosis. (A–C) Representative cells expressing C1δ-GFP or C1δ-YFP immediately before (A) and 10 min after addition of 10 μM ionomycin (B), or after treatment with 200 nM TPA for 10 min at 15°C (C). (D) Confocal fluorescence images of RAW cells coexpressing C1δ-YFP (yellow) and PLCδ-PH-CFP (blue) undergoing phagocytosis of RBCs. Subscripts (e.g., D1) refer to the time in minutes at which the fluorescence images were captured. D1, D3.5, D6, and D7.5 show overlays of the PLCδ-PH-CFP and C1δ-YFP signals. The insets show separately the fluorescence of PLCδ-PH-CFP (labeled C) and C1δ-YFP (labeled Y) of the areas boxed by the dotted line. In D1 and D3.5, three formed phagosomes are indicated with filled white circles. Bars, 10 μm.
Figure 8
Figure 8
PLC is required for phagocytosis. (A–C) Macrophages expressing C1δ-GFP were pretreated with 6 μM U73122 (A) or U73343 (C) and subsequently allowed to interact with opsonized RBCs. B is a DIC image corresponding to A, showing adherent RBCs (arrowheads). In C, open arrowhead points to nascent phagosome, solid arrowhead to sealed phagosome. (D and E) RAW cells were pretreated for 15 min with the indicated concentrations of either U73343 (D, filled symbols), U73122 (D, open symbols), or ET-18-OCH3 (E) and phagocytosis was measured in the presence of the drugs. Data are means ± SE of three experiments (200 cells counted in each). (F) Phagocytosis was quantified in RAW cells treated as follows (from left to right): control (untransfected); transfected with the SH2 domains of PLCγ1; transfected with PLCz and GFP; transfected with GFP alone. The phagocytic index was normalized to that of controls performed the same day. Data are means ± SE of at least 1,200 cells per condition.
Figure 9
Figure 9
Effect of U73122 on pseudopod extension and actin cup formation. (A and B) RAW cells were treated without (A, C, and D) or with 5 μM U73122 (B, E, and F), allowed to bind RBCs on ice, and then warmed up for 4 min to initiate phagocytosis. The cells were then fixed and analyzed by SEM (A and B) or stained with rhodamine-phalloidin and analyzed by confocal microscopy (C–F). Arrows point to adherent RBCs. Bars, 10 μm.
Figure 10
Figure 10
Lipid signaling and actin redistribution during phagocytosis. (A) Putative events mediating 4,5-PIP2 metabolism at the nascent phagosome. (B) Diagrammatic representation of the sequence of events during phagosome formation and internalization. The font size is indicative of the relative abundance of the components (e.g., more 4,5-PIP2 at the phagosomal cup than in the plasmalemma during formation). Note that: (a) actin and 4,5-PIP2 are enriched in the phagosomal cup during formation; (b) DAG appears during formation, but is most abundant shortly after completion of phagocytosis (Early stage); (c) neither 4,5-PIP2 nor DAG are detectable in “late” phagosomes (Late stage); (d) phagosomal actin decreases in parallel with or shortly after 4,5-PIP2; (e) 4,5-PIP2 is present at the nonphagosomal plasma membrane throughout the phagocytic process; and (f) we postulate that PLC activity is required to induce actin disassembly and remodeling during cup formation and phagosomal closure.
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