Correspondence to: Sergio Grinstein, Hospital for Sick Children, Division of Cell Biology, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel:(416) 813-5727 Fax:(416) 813-5028 E-mail:sga{at}sickkids.on.ca.
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Abstract |
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Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis. The presence of 3'PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3'PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3'PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3'PI at the cup. 3'PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3'PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.
Key Words:
Fc receptors, PH domain, phosphoinositide, lipid mobility, FRAP
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Introduction |
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Phagocytosis, the engulfment of foreign bodies by specialized myeloid cells, is an essential component of the innate immune response. Invading microorganisms and other particles are ingested by phagocytic cells through a receptor-mediated mechanism that involves extensive cytoskeletal rearrangement and membrane remodelling (
Activation of type I phosphatidylinositol 3-kinase (PI3K), the enzyme responsible for generation of PI(3,4,5)P3, appears to be essential for the extension and/or fusion of pseudopods around the ingested particle. This view is supported by two lines of evidence: first, inhibition of the kinase with wortmannin or LY294002 virtually eliminates phagocytosis of large particles ( receptors, recruitment of PI3K to the membrane is initiated by activation of Syk, a tyrosine kinase. Syk itself is brought to the phagosomal cup by the interaction of its tandem Src homology 2 domains with a pair of appropriately spaced phosphotyrosine residues on the immunoreceptor or on its ancillary
chain (
Very recently, advances in our understanding of proteinlipid interactions have provided a novel approach to analyze the distribution of certain phospholipids in their native environment. Specifically, pleckstrin homology (PH) domains of several proteins were found to interact with varying degrees of affinity and specificity with either PI(3,4,5)P3 or PI(4,5)P2 (
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Materials and Methods |
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Reagents
Sheep red blood cells (SRBCs) and anti-IgG were obtained from ICN Biomedicals. Polystyrene beads (8-µm diameter) were obtained from Bangs Beads. FuGene 6 was obtained from Boehringer. Wortmannin was from Calbiochem. Earl's -MEM was obtained from Cellgrow. Antibodies to PTEN were raised in rabbits and affinity purified as described (
cDNA Constructs
The Btk-PHGFP and (R28C)Btk-PHGFP constructs were prepared as previously described (RIIa-GFP, Syk-GFP, and p85-GFP constructs were constructed by subcloning the respective cDNA into pEGFP obtained from CLONTECH Laboratories, Inc. The construct encoding SHIP1 tagged with the HA epitope YPYDVPDYAS was a gift from Dr. J. Penninger (Amgen Institute). The Gab1GFP construct was provided by Dr. E. Skolnik (Skirball Institute, New York, NY).
Cell Culture and Transfection
The macrophage RAW 264.7 cell line was obtained from the American Type Culture Collection. RAW 264.7 macrophages were cultured in Earl's -MEM with 10% FCS. Cells were scraped and seeded onto 2.5-cm glass coverslips at
30% confluence. After 24 h, the cells were transfected using FuGene 6 according to the manufacturer's directions. For p85-GFP, the cells were transfected 810 h before experiments. All other constructs were expressed for 1224 h.
Phagocytosis Assay
SRBCs and 8-µm polystyrene beads were opsonized for 1 h at 37°C with rabbit anti-SRBC or human IgG, respectively. For phagocytosis, RAW 264.7 cells on 2.5-cm glass coverslips were exposed to opsonized particles in HPMI medium at 37°C. To score phagocytic efficiency, macrophages were incubated with SRBCs for 15 min. External SRBCs were then lysed by brief hypotonic shock, and the cells were fixed with 4% paraformaldehyde overnight at 4°C. The number of phagosomes per cell was counted by differential interference contrast (DIC) microscopy.
Frustrated Phagocytosis Model
RAW macrophages were transfected 24 h before resuspension in 1 ml of HPMI with 2 mM EDTA. The cells were gently rotated for 12 h before the experiments. 100 µl of the suspension was then added to 1 ml of HPMI containing an additional 2 mM of MgCl2 and allowed to sediment on uncoated glass coverslips or coverslips coated with poly-L-lysine or human IgG (10 mg/ml). At the indicated time, the distribution of the fluorescent chimeras was analyzed by confocal microscopy.
FRAP
Cells transfected with Akt-PHGFP or PMGFP were allowed to sediment onto poly-lysine or human IgGcoated or uncoated coverslips for 1020 min. An initial image of an optical slice in the plane of the adherent (basal) membrane was obtained using the LSM 510 confocal microscope. Next, an 3-µm diameter spot was photobleached using the 488-nm laser line at full power. Subsequent images were obtained every 2 s in the case of Akt-PHGFP or every 10 s in the case of PMGFP with the laser at
1% of full power for excitation. A similar protocol was used to measure FRAP in the phagosomal cup in cells allowed to interact with IgG-opsonized latex beads of 8-µm diameter. Diffusion coefficients were calculated from FRAP as described (
Immunolocalization of SHIP1 and PTEN
RAW cells were fixed with ice-cold 4% paraformaldehyde in PBS between 3 and 8 min after addition of opsonized SRBCs. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min before blocking for 1 h with 5% BSA in PBS, followed by staining with the primary antibody for 1 h. Affinity purified rabbit anti-PTEN antibody was used to measure endogenous PTEN (
Image Analysis
Both live and fixed samples were analyzed by confocal microscopy using a ZEISS LSM 510 laser scanning confocal microscope with a 100x oil immersion objective. GFP-FITC and Cy3 were examined using the conventional standard laser excitation and filter sets. 16-bit digital images were analyzed with Java Image software. The accumulation of soluble proteins such as Btk-PH, Syk, or p85 at the phagosomal cup (p) was normalized by comparing it to the value of the surrounding cytoplasm (c) using the ratio ([p-c]/c). The enrichment of membrane (m)-bound proteins such as PMGFP or FcRIIa at the cup was quantified by the ratio (p/m). Digital images were prepared using Adobe Photoshop® 4.0 (Adobe Systems, Inc.) and CorelDraw 8.0 (Corel Corp.).
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Results |
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Accumulation of PI(3,4,5)P3 in the Phagosomal Cup
The distribution of 3-phosphoinositides in resting and stimulated phagocytes was studied by transient transfection of a fusion protein consisting of the PH domain of Akt linked to GFP (Akt-PHGFP). In otherwise untreated cells, this chimera localized to the cytoplasm and nucleus with little or no association with the plasma membrane (Fig 1 A, inset). Similar results were obtained using Btk-PHGFP, Gab1-PHGFP, or unconjugated GFP, though the cytosol-to-nucleus ratio varied somewhat between constructs (not shown). Initiation of phagocytosis by presentation of IgG-opsonized SRBCs induced a marked and rapid redistribution of Akt-PHGFP, which accumulated at the phagosomal cup (Fig 1, AB). A similar pattern was observed using Gab1-PHGFP (Fig 1, CD) and a weaker but reproducible accumulation of Btk-PHGFP was also observed (Fig 1 F, inset), whereas unconjugated GFP remained in the cytosol throughout (not illustrated).
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The accumulation of several different PH domains with high affinity for PI(3,4,5)P3 suggests that this lipid concentrates in the phagosomal cup. Two lines of evidence support this notion: first, the redistribution of Akt-PHGFP was completely eliminated by pretreatment of the macrophages with 100 nM wortmannin, a dose that is predicted to inhibit fully type I PI3K ( receptors are cross-linked by ligation of opsonized particles. For simplicity, the concentration of Akt-PHGFP at the membrane will be equated hereafter to accumulation of PI(3,4,5)P3.
Time Course of PI(3,4,5)P3 Redistribution during Phagocytosis
A more detailed analysis of the course of accumulation of PI(3,4,5)P3 at the phagosomal membrane is presented in Fig 2 and Fig 3. PI(3,4,5)P3 accumulation was detectable shortly after the plasmalemma established contact with the opsonized particles (Fig 2, AB) and became more pronounced as the cup developed (Fig 2 C). The accumulation was still evident at the time of phagosomal sealing, which we designated arbitrarily as time 0 (Fig 2 D, 0 s) and declined sharply thereafter (Fig 2 E).
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Densitometry of line scans was used to quantify more precisely the course and extent of accumulation of Akt-PHGFP at the phagosomal membrane. A representative experiment is illustrated in Fig 3A and Fig B. In Fig 3 C, the density of the chimera at the phagosome was corrected by subtracting the cytosolic fluorescence and was normalized with respect to the expression level to allow comparison among cells and between experiments. As shown in Fig 3 C (), which represents the average of six experiments, maximal accumulation of Akt-PHGFP was attained nearly 30 s before phagosomal sealing and decayed rapidly after the phagosome pinched off from the plasmalemma, reaching near baseline levels 12 min later. Similar results were obtained using Gab1-PHGFP (Fig 3 D), whereas no significant concentration of unconjugated GFP was observed at the cup at any time (Fig 3 C,
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Fig 3 D compares the course of accumulation of PI(3,4,5)P3 with the changes in PI(4,5)P2 detected using the PH domain of PLC. As reported previously (
-PHGFP indicates that the kinetics of Akt-PHGFP or Gab1-PHGFP binding to the phagosomal membrane is not limited by diffusion through the actin meshwork that lines the phagosomal cup.
In Fig 4, the temporal course of PI(3,4,5)P3 accumulation is compared with that of the Fc receptor clustering (Fig 4 A) and with the recruitment of Syk (Fig 4 B) and PI3K (Fig 4 C). In all cases, GFPchimeric constructs were transfected into RAW 264.7 cells, which were challenged with SRBCs, as described for the PH domain chimeras (as described in Materials and Methods). The receptors clustered rapidly under the adherent particle, and although their density decreased somewhat by the time of phagosomal closure, receptor concentration in the sealed phagosomal membrane clearly exceeded that of the surface membrane. Accumulation of receptors under the phagocytic particle resulted from their lateral displacement along the plane of the bilayer and not from localized membrane ruffling, since an acylated form of GFP (PMGFP) used as a nonspecific membrane marker (
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The tyrosine kinase Syk and the p85 regulatory subunit of the type I PI3K also accumulated at the phagosomal cup before membrane sealing (Fig 4B and Fig C). By contrast, soluble GFP remained in the cytosol throughout the phagocytic sequence (Fig 3 C, ), confirming the specificity of the measurements. Binding of Syk and recruitment of PI3K are thought to be downstream of receptor clustering and to precede the de novo biosynthesis of PI(3,4,5)P3. The site and time of recruitment of these proteins described in Fig 4 are compatible with the notion that the accumulation of the PH domainGFP chimeras is indeed an accurate reflection of the localized biosynthesis of PI(3,4,5)P3.
A Model of Frustrated Phagocytosis to Analyze Lipid Mobility
The experiments in Fig 1 and Fig 2 illustrate the fact that the PI(3,4,5)P3 generated at the nascent phagosome is precisely confined to the small region of the membrane apposed to the particle and fails to diffuse noticeably to the contiguous regions of the plasma membrane, despite their physical continuity before phagosomal closure. This observation suggests that the PI(3,4,5)P3 generated at the phagosomal cup has a limited ability to diffuse laterally. This idea could in principle be tested by estimating the lateral mobility of phagosomal lipids by FRAP.
The size (4 µm), shape, and dynamic nature of phagosomes formed around SRBCs constrained our ability to accurately measure FRAP. To circumvent these limitations, we implemented a system where the clustering of Fc
receptors by IgG occurred on a planar surface, extending the area and the time available for measurement of fluorescence, since under these conditions internalization of the opsonized surface is precluded. Human IgG was adhered to a glass coverslip and suspended RAW 264.7 cells transfected with Akt-PHGFP were allowed to land and spread on this surface while the process was monitored continuously by DIC microscopy. As controls for nonspecific interactions with the surface, uncoated, polylysine- or BSA-coated coverslips were also used. The typical morphology of a cell allowed to spread on IgG for 20 min is illustrated in Fig 5 A. Such cells adhered firmly and spread wide and thin lamellipodia on the coated surface. Importantly, Akt-PHGFP concentrated greatly on the membrane apposed to the IgG with no obvious accumulation elsewhere. As in the case of bona fide phagosomes, the accumulation of Akt-PHGFP (that is, PI[3,4,5]P3) was confined to the area of the membrane in direct contact with IgG with little evidence for lateral spreading for at least 10 min. By contrast, cells that adhered to either glass or poly-lysine for comparable periods remained more rounded and extended shorter lamellipodia (Fig 5 C). Only a slight accumulation of Akt-PHGFP at sites of contact was detected (Fig 5 D). In five determinations, the concentration at the membrane was 70% higher than the cytosol compared with 230% higher in the case of IgG. These findings suggest that the recruitment of Akt-PHGFP to the IgG-coated surface reflects the specific receptor-induced accumulation of PI(3,4,5)P3 and that this system is an adequate surrogate for the phagosomal cup.
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Lipid Mobility in Control and IgG-stimulated Cells
The complex formed between PI(3,4,5)P3 and Akt-PHGFP is reversible, which confounds measurements of lateral mobility by FRAP. Following bleaching, the recovery of fluorescence will be effected at least in part by dissociation and replacement of a bleached Akt-PHGFP by fluorescent cytosolic Akt-PHGFP rather than by lateral displacement of the lipid. Because reversibly bound PH domains are inappropriate for the measurement of PI(3,4,5)P3 mobility and covalently labeled fluorescent derivatives of this lipid are not suitable, we chose instead to measure the mobility of a model lipid. Since the lipid distribution and properties of the two monolayers of the plasma membrane differ greatly, we felt it was important to measure lipid mobility in the inner monolayer. This was accomplished by transfecting the cells with a vector encoding the 10 amino acid myristoylation/palmitylation sequence from Lyn fused to GFP (PMGFP). This acylated form of GFP partitions preferentially to the inner monolayer of the plasma membrane (
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In adherent yet unstimulated cells, PMGFP diffused rapidly on the plane of the membrane: the mobile fraction of the probe approached 65%, and when the bleached area averaged 4 µm in diameter the t1/2 for recovery was 10 s (Fig 6 C), corresponding to a diffusion coefficient of
3.5 x 10-9 cm2/s, comparable to that reported for lipids in the inner monolayer of the plasma membrane (
receptors were engaged: only
20% of the fluorescence was restored after 30 s (Fig 6 C). This finding indicates that lipid mobility is restricted in the stimulated cells, possibly accounting for the confinement of PI(3,4,5)P3 within the phagosomal cup.
To ensure that the observed changes in lipid mobility were not unique to the frustrated phagocytosis model in which IgG-coated coverslips are used, experiments were performed using large (8-µm) IgG-coated poly styrene beads. Such beads are effectively internalized albeit more slowly than SRBCs, facilitating determination of FRAP. As in the case of frustrated phagocytosis, the mobility of PMGFP at the phagosomal cup was greatly reduced compared with that estimated before presentation of the beads (not illustrated).
Rapid Dissociation/Reassociation of Akt-PHGFP
The decreased lipid mobility observed in Fig 6 is consistent with the restricted diffusion of PI(3,4,5)P3 observed using the PH domains (for example, Fig 2). However, it was conceivable that the latter was not an intrinsic property of the lipid but was instead a consequence of its interaction with Akt-PHGFP. This concern was also addressed using FRAP. A typical determination is illustrated in Fig 7AD. The rapid (t1/2 5 s) and virtually complete (80%) recovery of the fluorescence in the bleached region is noteworthy. This rapid recovery contrasts markedly with the persistence of differential fluorescence intensity between the adherent (basal) membrane and the nonadherent regions of the plasma membrane, even after much longer periods (Fig 5 B). These observations may be interpreted to mean that PI(3,4,5)P3 is corralled within the area of contact with the IgG-coated surface, being capable of movement within but not across this boundary. However, it is more likely that the data reflect the rapid dissociation and reassociation of Akt-PHGFP with the headgroup of PI(3,4,5)P3. This was concluded after analyzing the shape of the photobleaching pattern as a function of time (Fig 7 E). The optical features of the system used for illumination rendered a Gaussian photobleaching pattern. As shown in Fig 7 E, the peak of the Gaussian curve diminishes as the bleaching recovers; yet, the width of the curve remains unaltered. A widening of the Gaussian curve would be anticipated if lateral motion of the lipidprobe complex contributes significantly to the recovery process (for details see
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Contribution of Phosphatases to the Restricted Accumulation of PI(3,4,5)P3
Although reduced lipid mobility appears to influence the distribution of PI(3,4,5)P3, other mechanisms may also contribute to its restricted localization. One conceivable mechanism involves the combination of focal synthesis with rapid degradation of the lipid. If the catabolic enzymes are present outside the area of synthesis, the rate of degradation would outstrip the (modest) diffusion of the lipid out of the restricted area of biosynthesis, limiting its distribution. Such a "focal source-peripheral degradation" mechanism could contribute to PI(3,4,5)P3 localization at the phagosomal cup only if the turnover of the lipid is high, that is, if its rate of hydrolysis during the course of phagocytosis is significant. This was evaluated by measuring the rate of disappearance of PI(3,4,5)P3 from the phagosomal cup when biosynthesis was suddenly interrupted by addition of wortmannin. Large polystyrene beads were used in these experiments to slow the internalization process thereby extending the period of accumulation of PI(3,4,5)P3 at the cup. Fig 8 shows that PI(3,4,5)P3 accumulated at the base of the cup decreases modestly over 80 s when the vehicle, dimethylsulfoxide, is added. In contrast, the inositide disappeared rapidly (t1/2 50 s) following the addition of the PI3K inhibitor. This is a low estimate of the rate of ongoing PI(3,4,5)P3 degradation because inhibition of PI3K is unlikely to occur instantaneously upon addition of wortmannin, which must enter the cell and react covalently with the enzyme. Moreover, hydrolysis of PI(3,4,5)P3 by a 5-phosphatase would yield phosphatidylinositol 3,4-bisphosphate (PI[3,4]P2), which would still retain Akt-PHGFP. Fig 8 also illustrates the distribution of actin in cells treated with or without wortmannin during the course of phagocytosis. Note that the actin cup formed under nascent phagosomes persists for
1 min, despite inhibition of PI3K. The rapid dissociation of the chimera under these conditions indicates that the accumulation and restricted mobility of Akt-PHGFP are not attributable to its nonspecific binding or trapping by the actin cytoskeleton.
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In the immunological synapse of lymphocytes, receptors and the associated molecules are arranged concentrically, generating microdomains where signaling is restricted (
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The distribution of the 5'-phosphatase SHIP1 was studied by transfection of an epitope-tagged form of the enzyme. As described recently by
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Discussion |
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Probe Specificity
Two lines of evidence indicate that the PH domainGFP chimeras used in our studies are suitable indicators of the distribution of 3-phosphoinositides in intact macrophages. First, their accumulation at the phagosome was eliminated by treatment with wortmannin (Fig 1) or LY294002 (not shown), which are potent inhibitors of PI3Ks (
The main probe used in our studies, namely the PH domain of Akt, binds with comparable affinity to PI(3,4,5)P3 and PI(3,4)P2 (
It is also noteworthy that the probes did not alter the function of the transfected cells. Phagocytic efficiency in cells expressing Akt-PHGFP was indistinguishable from untransfected controls. This observation is compatible with the notion that the probe is in a fast dynamic equilibrium with the phosphoinositides, as suggested by the pattern of recovery after photobleaching (Fig 7) and by the ability of phosphatases to access and rapidly degrade PI(3,4,5)P3 following addition of wortmannin (Fig 8).
Time Course of PI(3,4,5)P3 Accumulation
Accumulation of PI(3,4,5)P3 at sites of phagocytosis was an early event detectable during the course of pseudopod extension before closure of the phagosomal vacuole (Fig 2). The appearance of PI(3,4,5)P3 coincides with the equally localized disappearance of PI(4,5)P2 from the base of the cup (Fig 3;
The early stages of the phagocytic process involve active actin remodelling and focal exocytosis of endomembranes. It is likely that the generation of PI(3,4,5)P3 plays a role in these processes, although the requirement for PI3K in actin polymerization remains controversial. On one hand, clustering of membrane-bound type I PI3K suffices to induce actin assembly and phagocytosis (
The accumulation of PI(3,4,5)P3 at the phagosome is transient, becoming undetectable within 23 min of phagosomal sealing. The disappearance of the phosphoinositide follows closely the detachment of type I PI3K, suggesting the presence of active catabolic enzymes at the phagosome. Accordingly, SHIP1 was found to accumulate on the phagosomal membrane, where it persisted throughout the period of PI(3,4,5)P3 degradation. Interestingly, the time of disappearance of PI(3,4,5)P3 coincides approximately with the disassembly of phagosomal F-actin and with the onset of maturation. The earliest identified stages of phagosomal maturation are thought to be controlled by Rab5 (
Mechanism of Focal Accumulation of PI(3,4,5)P3
The diffusion coefficients estimated for a variety of lipids in biological membranes, including 3'PI, range from 230 x 10-9 cm2/s (25 µm from the site of phagocytosis before sealing of the vacuole. This distance is much greater than the circumference of the phagosomal cup, and noticeable diffusion of PI(3,4,5)P3 out of the phagosomal boundary would be predicted on this basis. This prediction was clearly not fulfilled, and two mechanisms that could account for the unexpected behavior were contemplated. On one hand, we considered a model of "focal sourceperipheral degradation," whereby diffusion of PI(3,4,5)P3 synthesized focally at the cup would be limited by degradative enzymes (likely phosphatases) localized peripherally. In an extreme scenario, the phosphatases would have a circumferential distribution delimiting the phagosomal cup. This model requires that PI3K be accumulated at the site of particle attachment and that PI(3,4,5)P3 turn over rapidly. As shown in Fig 4 and Fig 8, both of these conditions were met. However, we failed to detect a phosphatase located in the periphery of the phagosome: PTEN was not found at the plasma membrane, and SHIP1 appeared to concentrate at the phagosome but with comparable density throughout. SHIP1 is known to associate with the immunoreceptor tyrosine inhibition motif of Fc
IIB receptors (
RIIB by the opsonized particles, which is expected to occur homogeneously throughout the area of contact with the particle, is likely to account for the accumulation of SHIP1 at the phagocytic cup. Despite our failure to detect peripheral phosphatases, the "focal sourceperipheral degradation" model cannot be disregarded and remains an attractive possibility. Microdomains of SHIP1 or PTEN may have escaped our detection and other phosphatases not tested (for example, SHIP2 or other heretofore unidentified enzymes) may distribute in the periphery of the phagosome.
A second mechanism that could contribute to the restricted distribution of PI(3,4,5)P3 is the reduction in lipid mobility observed when Fc receptors are engaged. Though 3'PI mobility could not be measured directly, we found that a di-acylated GFP construct that, like PI(3,4,5)P3, inserts into the inner leaflet of the plasmalemma could be used reliably for FRAP measurements. Although the majority of this probe (95%) diffused freely before stimulation of the cells with a coefficient in the range reported for other lipids in biological membranes (3.5 x 10-9 cm2/s), we found that its mobility was severely curtailed upon cross-linking of Fc receptors. Aggregation of lipid rafts could account for this phenomenon, since proteins acylated with saturated chains (such as our PMGFP probe) and polyphosphoinositides (
Regardless of the underlying mechanism, the occurrence of focal accumulation of lipids that serve messenger roles has important implications. PI(3,4,5)P3 and/or other 3'PIs are expected to recruit to the phagosomal cup a variety of proteins containing PH domains, the same way they direct the accumulation of the GFP probes. Proteins such as Vav and Arf nucleotide exchange factors, which possess such PH domains, are likely to play a critical role in actin and membrane remodelling. In addition, kinases like Akt and Btk will influence the activity of phospholipases and other downstream effectors. Many if not all of these processes likely require precise spatial coordinates that are provided by the restricted distribution of 3'PI. The subsequent degradation of the inositides may signal the termination of some of these early events and in addition may provide secondary messengers, such as PI(3)P or other derivatives, to initiate phagosomal maturation.
In summary, formation of phagosomes appears to depend on the accurate spatial and temporal control of the distribution of inositides. The mechanisms responsible for such fine control cannot be easily studied by conventional biochemical means and will require the application and further development of noninvasive approaches to study lipid metabolism.
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Footnotes |
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1 Abbreviations used in this paper: DIC, differential interference contrast; GFP, green fluorescent protein; HA, influenza virus hemagglutinin; PH, pleckstrin homology; PI(3)P, phosphatidylinositol 3-phosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphatidylinositol 3-kinase; SRBC, sheep red blood cell; 3'PI, 3' phosphoinositide.
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Acknowledgements |
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This work was supported by the Medical Research Council of Canada, the Arthritis Society of Canada, the National Sanatorium Association, and by National Institutes of Health grant HL28207. J.G. Marshall is supported by a fellowship from the Research Institute of the Hospital for Sick Children. J.W. Booth is the recipient of a fellowship from Canadian Cystic Fibrosis Foundation. S. Grinstein is an International Scholar of the Howard Hughes Medical Institute and the current holder of the Pitblado Chair in Cell Biology.
Submitted: 7 February 2001
Revised: 11 May 2001
Accepted: 18 May 2001
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References |
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