PLCδ-PH-GFP Is a Specific Probe for 4,5-PIP2 in Macrophages
The PH domain of PLCδ was shown to bind to 4,5-PIP2
in vitro and in vivo with high affinity and specificity (Lemmon et al. 1995
). Therefore, we used a fusion of this domain with GFP (PLCδ-PH-GFP) to analyze phospholipid metabolism in RAW 264.7 cells. As reported for other cell types (Stauffer et al. 1998
; Varnai and Balla 1998
), PLCδ-PH-GFP associated predominantly with the plasmalemma of RAW cells, with little binding to organellar membranes ( A). Activation of endogenous PLC by elevation of cytosolic Ca2
+ using ionomycin caused extensive translocation of the chimera to the cytoplasm ( B), suggesting that PLCδ-PH-GFP is predominantly anchored to the plasma membrane through 4,5-PIP2
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 (more ...)
In vitro, the PH domain of PLCδ can also bind to phosphatidylinositol-3,4-bisphosphate (3,4-PIP2
) and phosphatidylinositol-3,4,5-trisphosphate (3,4,5-PIP3
), though with comparatively low affinity (Kavran et al. 1998
). To assess the relative abundance of these lipids, RAW macrophages were transfected with the GFP-tagged PH domains of either Akt ( and ) or Btk (not illustrated), which recognize specifically 3,4-PIP2
(Kavran et al. 1998
; Varnai et al. 1999
). As depicted for Akt-PH-GFP, these fusion proteins were almost entirely in the cytosol before and after treatment with ionomycin ( and ), implying that 3′-phosphorylated inositides are not abundant in unstimulated macrophages and that their contribution to the distribution of PLCδ-PH-GFP is likely negligible.
We also transfected RAW macrophages with PM-GFP, a myristoylated/palmitoylated GFP (Teruel et al. 1999
). PM-GFP was preferentially targeted to the plasma membrane, although internal structures were also decorated ( E). Importantly, the distribution of PM-GFP was unaltered upon addition of ionomycin ( F), indicating that activation of PLC did not induce wholesale redistribution of the plasmalemmal lipids.
Redistribution of PLCδ-PH-GFP during Phagocytosis
To monitor the distribution of 4,5-PIP2 during phagocytosis, RAW cells transfected with PLCδ-PH-GFP were allowed to bind IgG-opsonized RBCs and fluorescence was measured in real time by confocal microscopy. Soon after addition of the opsonized particles, pseudopods labeled with PLCδ-PH-GFP extended from the macrophage surface and made contact with the RBCs ( A, open arrows). A biphasic change in fluorescence intensity was observed in the area of the membrane involved in phagocytosis. During pseudopod extension PLCδ-PH-GFP appeared to accumulate in phagocytic cups compared with the rest of the plasmalemma ( A, see also ). This is best illustrated in panel A2 of , where the downward-pointing open arrow denotes an area that is clearly more intense than the rest of the plasma membrane and where particle engulfment is about to occur (see 3–6-min sequence). In sharp contrast, once the phagocytic vacuole sealed, PLCδ-PH-GFP was no longer associated with the phagosomal membrane ( A, solid arrows). This is most clearly appreciated in the region of the phagosomal membrane most distant from the plasmalemma (i.e., the “base” of the phagosome), where differentiation of the two membranes is unambiguous. The distinction between surface and phagosomal membrane is clearly illustrated in and , which are enlargements of the inset areas in the 4- and 6-min acquisitions of the top panel.
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 (more ...)
Figure 4 Quantification of phagosomal fluorescence. Confocal images of RAW cells undertaking phagocytosis were acquired as in the legends to and . The digitized fluorescence intensity was quantified along line scans traversing both a phagosomal cup (more ...)
The precise onset of the disappearance of 4,5-PIP2 from the phagosomal membrane was difficult to assess using RBCs, since the time required for completion of phagocytosis and the distance between the PLCδ-PH-GFP–rich pseudopods and the base of the phagosome were short (e.g., open circle in B). Therefore, we used larger particles (8-μm latex beads, IgG-opsonized) to define whether the second phase of 4,5-PIP2 metabolism commences before or only after phagosomal sealing is complete. As in the case of opsonized RBCs, the larger beads were effectively internalized by RAW cells and, as before, fully formed phagosomes were devoid of PLCδ-PH-GFP ( and , filled circle). In addition, the larger size of the beads facilitated the detection of intermediate stages of phagosome formation and it was often possible to find phagosomal cups where the base of the phagosome was devoid of PLCδ-PH-GFP, whereas the fluorescent chimera was clearly detectable in the pseudopods ( and , open circle). This suggests that the onset of 4,5-PIP2 conversion preceded phagosomal sealing.
The biphasic behavior of PLCδ-PH-GFP during phagocytosis closely resembled that of F-actin, which was revealed by staining with rhodamine-phalloidin. As reported previously, F-actin accumulated transiently underneath the phagosomal cup, where it is thought to propel the extension of pseudopods (Greenberg et al. 1991
). As shown in and (open arrows), sites of F-actin accumulation coincided with those regions where PLCδ-PH-GFP fluorescence was most intense. Upon particle internalization, F-actin was rapidly lost from the sealed phagosomes (indicated by filled circles in G). The dissociation of actin paralleled the virtual loss of PLCδ-PH-GFP from the phagosomes ( F), signaling depletion of 4,5-PIP2
. These observations are consistent with a role of 4,5-PIP2
in controlling actin remodeling during phagocytosis.
Distribution of PM-GFP during Phagocytosis
The phagosomal membrane is known to undergo an active maturation process that involves progressive budding and concomitant fusion with endosomes and lysosomes (Beron et al. 1995
). It is therefore conceivable that the observed loss of fluorescence from the early phagosomes could result from rapid replacement with endomembranes, which are ostensibly devoid of PLCδ-PH-GFP. This possibility was examined using PM-GFP, an acylated form of GFP that partitions preferentially to the inner monolayer of the plasma membrane (Teruel et al. 1999
). Typical results are illustrated in A. As expected, pseudopods labeled with PM-GFP were observed extending over RBCs (open arrows). However, in contrast to PLCδ-PH-GFP, the association of PM-GFP with the phagosomal membrane was persistent ( A, solid arrows). These observations are clearly illustrated in and , which are magnified images of the enclosed areas in , A0
. In fact, PM-GFP remained bound with phagosomes for at least 20 min after formation (not illustrated). Because RAW cells internalize multiple opsonized particles, this resulted in the progressive intracellular accumulation of brightly labeled phagosomes ( A6
Figure 3 Phagocytosis in macrophages expressing PM-GFP. (A) DIC image and time course of phagocytosis, as described in the legend to A. (B and C) Enlargements of the areas identified by boxes in A0 and A6. (D) Phagocytosis of 8-μm latex beads. (E) (more ...)
The persistence of PM-GFP on the phagosome was also evident when using 8-μm latex beads. In this system, fluorescence was seen to surround the phagosome throughout the sealing process and to remain therein long after ( and , open and filled circles, respectively). These data indicate that rapid membrane turnover is not a likely explanation for the loss of PLCδ-PH-GFP from nascent phagosomes. Rather, the loss of PLCδ-PH-GFP is occurring due to local changes in the availability of 4,5-PIP2. This may result from sequestration, removal, or enzymatic conversion of the phosphoinositide during FcγR-mediated phagocytosis.
Accumulation of PLCδ-PH-GFP in Nascent Phagosomes
To support our visual impression that PLCδ-PH-GFP, but not PM-GFP, was preferentially accumulated in phagosomal cups we quantified multiple digital images of early stages of phagocytosis. Line scanning of the base of the phagosome and of a contralateral area of the plasma membrane devoid of ruffles was performed as illustrated in , and the results of multiple determinations are summarized in E. In the phagosomal membrane PLCδ-PH-GFP had peak intensities ranging from 1.3 to 2.3 times that of the plasma membrane ( and ). The average ratio was 1.61 ± 0.08 (mean ± SE; n
= 64, E). In contrast, phagocytic cups labeled with PM-GFP had peak intensities ranging from 0.9 to 1.6 times that of the plasma membrane ( and ). The average ratio in this case was 1.25 ± 0.08 (n
= 35, E), which is significantly different from unity (t
≤ 0.005). This moderate, yet significant increase may reflect partition of PM-GFP into lipid rafts (Cheng et al. 1999
) that may in turn accumulate in the phagosome. Regardless of the mechanism of accumulation of PM-GFP, a different process must underlie at least part of the accumulation of PLCδ-PH-GFP, which was significantly greater (P
< 0.005). Increased net biosynthesis or preferential unmasking of 4,5-PIP2
are likely possibilities.
We also quantified the amount of PM-GFP and PLCδ-PH-GFP in the formed (intracellular) phagosome. In this case, the density of phagosomal and plasmalemmal PM-GFP was virtually identical, whereas PLCδ-PH-GFP was essentially undetectable in the phagosome ( E). This quantitation confirms that bulk exchange of membrane during maturation is unlikely to account for the disappearance of PLCδ-PH-GFP from the sealed phagosome.
PIPKIα Concentrates in Phagocytic Cups
The preferential accumulation of PLCδ-PH-GFP near the forming phagosome may reflect net formation of 4,5-PIP2
, as postulated for other processes where active actin remodeling occurs (Shibasaki et al. 1997
; Martin 1998
; Tolias et al. 2000
). To examine whether increased biosynthesis of the phosphoinositide occurs at the site of phagocytosis, we studied the distribution of PIPKIα isoforms by immunofluorescence. Although our antibodies were unable to detect PIPKIβ in RAW cells, positive immunoreactivity for PIPKIα was readily detected in these macrophages. Strikingly, upon addition of opsonized and fluorescently labeled particles, PIPKIα migrated to the nascent phagosome ( and , open arrow). The kinase dissociated from the phagosomal membrane shortly after completion of phagocytosis ( and , solid arrow). The transient association of PIPKIα with the nascent phagosome is consistent with the notion that 4,5-PIP2
is locally accumulated during FcγR-mediated phagocytosis.
PLCγ Localizes to Phagocytic Cups
Based on prior knowledge that PLCγ undergoes tyrosine phosphorylation during FcγR cross-linking (Azzoni et al. 1992
; Liao et al. 1992
), we hypothesized that the phase of dissociation of PLCδ-PH-GFP from phagosomes could be explained at least partly by PLC-mediated hydrolysis of 4,5-PIP2
. To validate this notion we studied the distribution of PLCγ2, the isoform expressed exclusively in hemopoietic cells, during the course of phagocytosis, using immunofluorescence. In unstimulated RAW macrophages, PLCγ2 was mostly cytosolic with slight accumulation in ruffling membranes (not illustrated). Significantly, we found that PLCγ2 accumulated intensely at sites of phagocytosis ( and ). PLCγ is known to be recruited to the vicinity of activated receptors through interaction of its dual SH2 domains with phosphotyrosine residues on receptor–adaptor complexes (Rhee and Bae 1997
). A similar mechanism is likely involved during phagocytosis since, when transfected into RAW cells, the dual SH2 domains of PLCγ1 fused to GFP (PLCγ-SH2-GFP) were often seen to concentrate around phagocytic cups ( and ).
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 (more ...)
Localized Generation of Diacylglycerol during Phagocytosis
Activation of the PLCγ recruited to the phagocytic cup could account for the observed decrease in 4,5-PIP2
. To test this possibility, we examined the appearance of DAG using fusion proteins of the C1 domain of PKCδ with either GFP or YFP, hereafter named C1δ-GFP and C1δ-YFP, respectively. The C1 domain of PKCs was shown earlier to be an effective probe of DAG distribution in vivo (Oancea and Meyer 1998
; Oancea et al. 1998
). Its use in RAW cells is illustrated in . In otherwise untreated cells, C1δ-GFP was exclusively associated with endomembranes, concentrating in a juxtanuclear complex ( A). Upon activation of endogenous PLC by calcium-ionomycin, a sizable fraction of C1δ-GFP translocated to the plasma membrane ( B). Likewise, treatment of the cells with TPA ( C) or DiC8
(not shown) induced displacement of C1δ-GFP and C1δ-YFP to the plasmalemma. This implies that translocation of the C1δ fusion proteins during PLC activation occurs in response to the release of endogenous DAG and not to the concomitant increases in IP3
and calcium concentration. Translocation of the C1δ chimeras to the plasma membrane persisted at 15°C ( C). This is consistent with the notion that the C1δ domain reaches the plasmalemma by diffusion across the cytosol, after net dissociation from endomembrane binding sites. An alternative model, namely fusion of internal vesicles bearing C1δ domains with the plasma membrane, is unlikely due to the persistence of translocation at 15°C, since at this temperature vesicular traffic essentially ceases in mammalian cells. Moreover, we also observed modest yet reproducible accumulation of the C1 domain of PKCγ in forming phagosomes (not shown). In resting cells this domain is almost exclusively in the cytosol, ruling out translocation by vesicular fusion.
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 (more ...)
Having validated the sensitivity of these C1δ chimeras as indicators of DAG in RAW cells, we proceeded to study its distribution during phagocytosis. RAW macrophages were cotransfected with C1δ-YFP and PLCδ-PH-CFP, which enabled us to concomitantly examine the distributions of 4,5-PIP2 and DAG in individual live cells by confocal microscopy. D illustrates the course of redistribution of the two fluorophores after the addition of opsonized RBCs. C1δ-YFP was only weakly associated with the early phagocytic cup ( D1, inset Y). At this stage, PLCδ-PH-CFP was enriched in the extending pseudopods ( D1, inset C). As phagocytosis progressed, it was often possible to resolve the two membranes that constitute the phagocytic cup: the outside bilayer of the pseudopod remained labeled with PLCδ-PH-CFP throughout and eventually sealed to restore plasmalemmal continuity. In comparison, PLCδ-PH-CFP fluorescence diminished progressively in the inner bilayer of the pseudopods, i.e., the one more closely apposed to the particle and later becomes the phagosomal membrane ( D3.5, inset C). The dissociation of PLCδ-PH-CFP from the inner bilayer was accompanied by recruitment of C1δ-YFP ( D3.5, inset Y). Initially, this accumulation was most prominent at the base of the phagocytic cup, but it then extended toward the tips of the pseudopods before phagosome sealing. Importantly, C1δ-YFP labeling was usually strongest immediately after phagosome closure, at which time PLCδ-PH-CFP was completely absent from the phagosome ( D6). The association of C1δ-YFP with the phagosome declined gradually thereafter ( D7.5), becoming undetectable after several minutes. The full dissociation of C1δ-YFP from formed phagosomes can be clearly appreciated by inspecting the three sealed phagosomes indicated by filled circles in D1. Similar results were obtained with C1δ-GFP alone (not shown). These observations suggest that DAG is locally synthesized by PLC-mediated hydrolysis of 4,5-PIP2 during phagosome formation and is subsequently converted in the sealed phagosome to other metabolites.
Reduced Availability of 4,5-PIP2 Attenuates FcγR-mediated Phagocytosis
The localized activation of 4,5-PIP2
metabolism in the forming phagosome suggests a functional role for this phosphoinositide in phagocytosis. To verify this hypothesis, we tested the effects of several methods to reduce the availability of 4,5-PIP2
on the ability of RAW cells to internalize opsonized particles. Neomycin sulfate is a somewhat permeant divalent cation that can compete with endogenous ligands for binding to 4,5-PIP2
. Overnight treatment with 5 and 10 mM neomycin reduced the phagocytic index by 30 ± 8 and 45 ± 5%, respectively (n
= 15, P
< 0.001; A). Because neomycin may have nonspecific effects, we also tested other, more specific means of interfering with 4,5-PIP2
. PLCδ-PH-GFP can be used not only to monitor the distribution of 4,5-PIP2
, but when expressed at sufficiently high levels, also to competitively inhibit other 4,5-PIP2
–dependent processes. Accordingly, overexpression of this construct was shown recently to interfere with anchoring of the actin cytoskeleton to the plasma membrane (Varnai and Balla 1998
; Raucher et al. 2000
). Effective scavenging of 4,5-PIP2
by PLCδ-PH-GFP would only be expected to occur if the concentration of the latter can approximate or exceed that of its phosphoinositide ligand. Therefore, we estimated the intracellular concentration of PLCδ-PH-GFP in transfected RAW cells by comparison with microscopic droplets of standard solutions containing known concentrations of recombinant EGFP. As shown in B, interpolation of the cell-associated fluorescence in such calibration curves yielded intracellular concentrations of ≈25 μM. This concentration should suffice to exert a significant scavenging effect on 4,5-PIP2
, which can be estimated to exist in the micromolar range, based on existing determinations of phospholipid content in several cell types.
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 (more ...)
Therefore, we proceeded to test the effects of PLCδ-PH-GFP expression on phagocytosis. In cells expressing PLCδ-PH-GFP the phagocytic index was reduced moderately (30 ± 2%; n = 12), yet significantly (P < 0.0001; A). In contrast, the soluble, unmodified form of GFP had no significant effect on phagocytosis ( A).
We also attempted to decrease the availability of 4,5-PIP2
by enzymatic means, expressing PM-5′-phosphatase-GFP in RAW macrophages. This chimeric protein consists of the yeast INP54p
, a 4,5-PIP2
–specific 5′-phosphatase, attached to the acylation sequence of Lyn to promote its association with the membrane, and to GFP to facilitate detection. This yeast phosphatase has been shown to reduce the content of 4,5-PIP2
in COS cells (Raucher et al. 2000
). The subcellular distribution of PM-5′-phosphatase-GFP was indistinguishable from PM-GFP when expressed in RAW macrophages but the cells had a tendency to round up (not illustrated). We found that expression of PM-5′-phosphatase-GFP reduced the phagocytic index of RAW macrophages by 42 ± 2% (n
= 12, P
< 0.0001; A). This effect may not be entirely attributable to the phosphatase activity of the construct, since transfection with PM-GFP alone produced a modest, yet reproducible inhibition of phagocytosis ( A). It is conceivable that the acylated GFP displaces endogenous Src family kinases, which are also acylated and required for efficient phagocytosis. Jointly, these observations suggest that unhindered availability of 4,5-PIP2
is required for optimal phagocytosis.
Inhibition of PLC Blocks Diacylglycerol Synthesis and Ablates Phagocytosis
To test the role of PLC in phagocytosis, we used U73122 and ET-18-OCH3, two structurally different PLC-specific antagonists (Chen et al. 1996b
). Inhibition of the phosphoinositide-specific PLC by U73122 was confirmed using the DAG-binding C1δ-GFP construct. Treatment with 6 μM U73122 for 15 min precluded accumulation of C1δ-GFP at the sites of RBC attachment ( and ). In contrast, C1δ-GFP was recruited normally to the phagocytic cups in the presence of U73343, an inactive analogue of U73122, mirroring the effects of these agents on phagocytosis ( C). Importantly, U73122 did not block TPA-dependent recruitment of C1δ-GFP to the plasma membrane (not illustrated), indicating that U73122 does not directly bind to C1δ-GFP.
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 (more ...)
The dependence of FcγR-mediated phagocytosis on PLC was examined next. We found that U73122 inhibited phagocytosis in a dose-dependent fashion, with half-maximal effects at ≈3 μM ( D). In contrast, U73343 had no significant effect at up to 6 μM and had only a minor antagonistic effect at 12 μM ( D). Importantly, ET-18-OCH3, a structurally and mechanistically unrelated antagonist of PLC, also reduced phagocytosis in a dose-dependent fashion, with half-maximal inhibition at ≈25 μM ( E).
A more specific approach to inhibit PLCγ was also undertaken. Cells were transfected with two catalytically inactive fragments of PLCγ, namely PLCγ-SH2-GFP and PLCz, which were expected to exert a dominant-negative effect. PLCz contains the dual SH2 and SH3 domains of PLCγ1 and previously described PLC-inhibitory motif (Chen et al. 1996b
). As illustrated in F, macrophages transfected with PLCγ-SH2-GFP or PLCz had a markedly impaired ability to internalize opsonized RBCs. In both instances the phagocytic index dropped by >50% (n
= 12; P
< 0.0001). As before, paired experiments in which GFP alone was transfected showed no effect on phagocytosis. While we cannot rule out that PLCγ-SH2-GFP or PLCz bound to phosphotyrosine groups unrelated to those that anchor PLCγ, the unique sequence and spacing of these dual SH2 domains favor preferential association with the physiological target of the phospholipase. Therefore, these findings suggest that PLCγ is an important regulator of FcγR-mediated phagocytosis.
Inactivation of PLC Abolishes Pseudopod and Actin Cup Formation
To better define the stage of the phagocytic sequence requiring PLC, we examined whether pseudopod extension and actin polymerization occurred in cells treated with U73122 and ET-18-OCH3. Pseudopod morphology was evaluated by SEM and TEM. In control cells (either untreated or treated with the inactive analogue U73343), the formation of pedestals below the RBCs and the extension of pseudopods around them were readily observable ( A). In contrast, cells treated with U73122 were unable to extend pseudopods or form pedestals, even though RBCs appeared tightly attached to the surface of the macrophages ( B). This impression was verified using TEM. As before, in control cells pseudopods were observed elongating along the surface of RBCs (not illustrated). By comparison, the extension of pseudopods in U73122-treated cells was minimal.
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 (more ...)
Not only was pseudopod extension inhibited by U73122, but actin remodeling under the cup was similarly obliterated. Although in normal cells marked F-actin accumulation delineates the phagocytic cup ( and ), as reported by others (Sheterline et al. 1984
; Greenberg et al. 1991
; Kwiatkowska and Sobota 1999
), virtually no accumulation was noted in cells treated with the PLC blocker, despite normal adherence of RBCs to their surface ( and ). Similar results were obtained by treating cells with 80 μM ET-18-OCH3 in the presence of 10% serum (not depicted). These observations differ from those described in cells incubated with the PI3K inhibitor wortmannin. While wortmannin-treated cells fail to internalize particles, they nevertheless display marked actin reassembly under the adherent RBCs (Araki et al. 1996
; Cox et al. 1999
; and our own observations, not shown).
The inability of cells to form pseudopods and actin cups upon incubation with U73122 and ET-18-OCH3 is not the result of a detrimental effect of the drug on cellular viability. These effects were fully reversible. Moreover, in the presence of the drug, pinocytosis of FITC-dextran appeared to be unimpaired (data not shown). We conclude that inhibition of PLC arrests phagocytosis at an early stage, and is required for actin remodeling during pseudopod formation.