Differential Role of Actin, Clathrin, and Dynamin in Fcgamma Receptor-mediated Endocytosis and Phagocytosis*

Shirley M. L. TseDagger §||, Wendy Furuya§, Elizabeth Gold**, Alan D. SchreiberDagger Dagger , Kirsten Sandvig§§, Robert D. Inman¶¶, and Sergio Grinstein§||||

From the Departments of Dagger  Pediatrics, § Cell Biology, and ¶¶ Medicine,  Division of Rheumatology, Hospital for Sick Children, Institute of Medical Science, and University Health Network, University of Toronto, Toronto, Ontario M5G 1X8, Canada, ** Department of Immunology, University of Washington, Seattle, Washington 98195, Dagger Dagger  Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283, and §§ Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

Received for publication, August 5, 2002, and in revised form, November 4, 2002

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Clustering of macrophage Fcgamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fcgamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (>= 3 µm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (<= 1 µm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Receptors on the surface of phagocytes are essential in the recognition and elimination of microorganisms. Some phagocytic receptors interact directly with the pathogens, by recognizing motifs that are conserved on the surface of various types of microorganisms (1, 2). A second group of receptors interact instead with opsonins, host molecules such as antibodies, or complement components that coat the surface of the microorganisms (3). The latter class includes Fcgamma receptors, which bind to the constant region of IgG. Fcgamma receptors are involved in the recognition and elimination of IgG-coated microbes, erythrocytes, and platelets and in the clearance of IgG-containing immune complexes, thereby playing a role in antigen presentation (4, 5). Abnormal function or regulation of Fcgamma receptors may lead to the ineffective clearance of infectious organisms or can cause immunohematologic and autoimmune disorders (4).

In phagocytes the mode of internalization of Fcgamma receptors depends on the size of the opsonized complex. Small immune complexes and soluble aggregated IgG are internalized through receptor-mediated endocytosis (6-8). In contrast, the internalization of IgG-opsonized particles occurs via phagocytosis (9). Although cross-linking of Fcgamma receptors triggers both internalization processes, the molecular mechanisms underlying phagocytosis and endocytosis differ markedly. Endocytosis requires assembly of clathrin at the site of receptor clustering, yet is not strictly dependent on the cytoskeleton and does not require class I phosphatidylinositol 3-kinase activity (10). In contrast, phagocytosis involves acute assembly of F-actin and is blocked by inhibitors of class I phosphatidylinositol 3-kinase, such as wortmannin (11, 12). A recent study further highlighted the mechanistic differences between the two modes of Fcgamma receptor internalization (13); endocytosis, but not phagocytosis, was found to require receptor-induced ubiquitination.

Even though phagocytosis is thought to be driven by remodeling of actin, assembly of clathrin and of the associated proteins dynamin and amphiphysin has been reported to occur at sites of phagocytosis (14-16). The purpose of this clathrin recruitment may be to drive the budding and fission of vesicles from the nascent phagosomes, contributing to their maturation process. In this regard, it is noteworthy that phagosome maturation is akin to the progression of the endocytic pathway and that clathrin assembles on endocytic vesicles and is thought to contribute to the maturation of endosomes (17, 18). To date, however, the role of clathrin-coated vesicles in phagosome maturation has not been studied. The paucity of information likely stems from the fact that, unexpectedly, interference with the formation and/or detachment of the clathrin coat resulted in inhibition of phagocytosis. Thus, impairment of the function of clathrin (19), dynamin II (15), or amphiphysin II (16) all markedly depressed the phagocytic efficiency.

Clathrin-dependent membrane budding and fission are also essential for the secretory and recycling pathways (20, 21). This raises the possibility that the seemingly paradoxical effects of clathrin impairment on phagocytosis may be indirect, caused by depletion of plasmalemmal components that are required for the phagocytic process. To address this possibility and to analyze the role of clathrin in phagosome maturation, we used an isoform of dynamin that is predominantly localized to the plasma membrane. Three isoforms of dynamin have been described. Dynamin I is abundant in the brain, and dynamin III is abundant in testis, lung, and neurons (22), whereas dynamin II is ubiquitous. Importantly, whereas dynamin II is found both in endomembranes and at the plasma membrane, dynamin I is predominantly plasmalemmal (23). We reasoned that transfection of cells with a dominant-negative form of dynamin I was less likely to result in an indirect inhibition of phagocytosis and would enable us to compare the requirement of clathrin in endocytosis versus phagocytosis and to assess the possible role of clathrin-dependent budding in phagosomal maturation. In addition, we used antisense technology to depress the level of clathrin. By controlling the expression of the heavy chain using an inducible vector, we were able to assess the role of clathrin in Fcgamma receptor-mediated endocytosis and phagocytosis with minimal nonspecific damage to the cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Reagents and Antibodies-- Dulbecco's modified Eagle's medium and fetal bovine serum were from Wisent. Sheep red blood cells (RBC)1 and rabbit anti-sheep RBC antibodies were from ICN Cappel (Aurora, OH). Rhodamine-conjugated human transferrin (Tf) and rhodamine-conjugated dextran were from Molecular Probes (Eugene, OR), and monoclonal anti-Tf receptor antibody was from Zymed Laboratories Inc. (San Francisco, CA). Cytochalasin D was from Calbiochem (San Diego, CA). Rat anti-mouse (ID4B) LAMP1 antibodies were from the Developmental Studies Hybridoma Bank, maintained by the University of Iowa and Johns Hopkins University. Cy3-conjugated anti-mouse, anti-rat, and anti-human antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Monoclonal anti-mouse CD18 (integrin beta 2 chain) antibody was from BD Biosciences. Human IgG was purchased from Sigma.

Cell Culture and Transfection-- RAW 264.7 murine macrophages obtained from the American Type Culture Collection (Manassas, VA) were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were grown at 37 °C in a humidified atmosphere of 95% air and 5% carbon dioxide. Transient transfection of RAW 264.7 cells with clathrin-GFP, dynamin (Dyn) I (K44A), Dyn II (K44A), Dyn I (S45N), wild-type (WT) dynamin, and Rab5(S34N)-GFP, a GFP-labeled dominant-negative (DN) form of Rab5, was performed utilizing FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's protocol. Cells were used 24 h after transfection of clathrin and DN-Rab5 and 48 h after dynamin transfection.

BHK21-tTA cells transfected with antisense clathrin heavy chain (24-26) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 2 mM L-glutamine, 1 µg/ml puromycin, 0.2 mg/ml geneticin, and 2 µg/ml tetracycline. Cells were used 48 h after inducing the expression of the antisense mRNA by removing tetracycline from the medium.

EGFP-clathrin light chain cDNA (27) was kindly provided by Dr. J. H. Keen (Kimmel Cancer Institute and Thomas Jefferson University, Philadelphia, PA). Plasmids encoding Dyn I K44A, Dyn S45N, and Dyn WT were generously provided by Dr. S. L. Schmid (Department of Cell Biology, Scripps Research Institute, La Jolla, CA). Dyn II K44A cDNA was prepared as described (16). Rab5(S34N)-GFP was kindly provided by Dr. C. Bucci (Universita degli Studi de Lecce, Lecce, Italy).

Receptor-mediated Endocytosis Assay-- Endocytosis was assessed by quantifying the uptake of Tf or aggregated human IgG. Prior to Tf uptake determinations RAW 264.7 cells grown on 25-mm coverslips were pre-incubated in serum-free medium for 1 h. Rhodamine-Tf (50 µg/ml) was added, and the cells were incubated at 37 °C for 30 min. Tf bound extracellularly was removed by washing cells sequentially in media of pH 5.5 and pH 7.4.

Human IgG (10 mg/ml) was aggregated by heating to 62 °C for 20 min. Insoluble complexes were sedimented, and 1 mg/ml of soluble aggregated IgG was added to the cells, which were incubated at 37 °C for 30 min. Subsequently, cells were washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 1 h, and permeabilized with 0.1% Triton X-100 in PBS. Internalized IgG complexes were identified by incubating cells with anti-human Cy3-conjugated antibodies (1:1000).

Phagocytosis Assay-- Sheep RBC were opsonized with rabbit anti-sheep RBC antibody (1:50) and latex beads with 1 mg/ml human IgG at 37 °C for 1 h. Opsonized RBC or latex beads were added to RAW 264.7 macrophages grown on coverslips (30-50 RBC or beads per RAW 264.7 cell) and incubated at 37 °C for 30 min. External RBC were lysed by hypotonic shock with distilled water, lysis was terminated by addition of 10× PBS, and the cells were washed and resuspended in Hepes-buffered RPMI 1640 medium. Unbound particles were washed, and external beads were labeled in the cold with Cy3-conjugated anti-human antibody. Cells were fixed and permeabilized, and the beads were labeled with Cy5-conjugated anti-human antibody. Those beads that were Cy5-positive but Cy3-negative were considered to be internalized.

Fluid Phase Assay-- RAW 264.7 cells were incubated with rhodamine-conjugated dextran (500 µg/ml) at 37 °C for 30 min, washed with PBS, and either visualized immediately or fixed in 4% paraformaldehyde for 1 h prior to visualization.

Assessment of Phagosomal Maturation-- Phagocytosis of opsonized RBC by RAW 264.7 cells were induced as described above. After 5 min at 37 °C the external RBC were lysed, and maturation of the phagosomes containing internalized RBC were allowed to proceed for 1 h. Cells were fixed in 4% paraformaldehyde for 1 h followed by permeabilization and blocking in PBS with 0.1% Triton X-100 and 5% milk. The cells were next immunostained with anti-human Tf receptor (1:500), anti-mouse CD18 (1:500), or anti-mouse LAMP1 (1:4) antibodies. Cy3-conjugated secondary anti-mouse and anti-rat antibodies were used as appropriate, at 1:1500 dilution.

Samples were analyzed using a Leica fluorescence microscope (model DMIRB) with a ×100 oil immersion objective and appropriate filter sets. All cells and phagosomes were identified under DIC optics. Images were acquired digitally utilizing a cooled charge-coupled device camera (Micromax; Princeton Instruments, Trenton, NJ) controlled by the Winview software. Confocal microscopy was performed with a Zeiss LSM 510 laser scanning confocal microscope. The presence of Tf receptor, CD18, or LAMP1 in the phagosomal membrane was scored in individual phagosomes in an all-or-none basis.

Measurements and Statistical Analysis-- All experiments were performed at least in triplicate, and data are expressed as means ± S.E. The significance of differences between means was assessed utilizing Student's t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EGFP-Clathrin Expressed in RAW 264.7 Cells Localizes to Coated Pits and Phagocytic Cups-- Upon binding to Fcgamma receptors, IgG-containing immune complexes are internalized and travel along the endocytic pathway where they are targeted for degradation. This is part of a key process involved in antigen processing and presentation (6, 7). Transient expression of a fusion of the light chain of clathrin with EGFP was used to examine whether clathrin is involved in the receptor-mediated endocytosis of aggregated IgG by RAW 264.7 cells. As illustrated in Fig. 1, A-C, EGFP-clathrin was found to colocalize with some, but not all, of the immune complexes undergoing internalization. The partial colocalization may be attributed to asynchrony of the internalization process, or to the co-existence of clathrin-dependent and clathrin-independent uptake pathways. It is noteworthy that another immunoreceptor, the interleukin 2 receptor, is internalized primarily via clathrin-independent endocytosis (28).


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Fig. 1.   Localization of EGFP-clathrin during endocytosis and phagocytosis. RAW 264.7 macrophages were transiently transfected with EGFP-clathrin 24 h before analysis. A-C, endocytosis was initiated by addition of aggregated human IgG. After 10 min, the cells were fixed, permeabilized, and stained with Cy3-conjugated anti-IgG and finally analyzed by confocal microscopy. A, green (clathrin-EGFP) fluorescence; B, red (Cy3-stained IgG) fluorescence; C, overlay of A and B. Arrows indicate sites of colocalization. D and E, phagocytosis was initiated by addition of IgG-opsonized RBC. After 10 min, the cells were fixed and analyzed by DIC (D) or by fluorescence microscopy to detect clathrin-EGFP (E). Arrows point to clathrin on phagocytic cups. Images are representative of three similar experiments. F, localization of dynamin I during phagocytosis was carried out in RAW 264.7 cells transfected with hemagglutinin-tagged wild-type dynamin I. Following phagocytosis of IgG-opsonized RBC for 5 min, the cells were fixed, permeabilized, and stained with fluorescein isothiocyanate-conjugated anti-hemagglutinin and analyzed by DIC (inset) or fluorescence confocal microscopy.

Next, we assessed whether clathrin is associated with phagosomes during engulfment of IgG-opsonized particles. RAW 264.7 cells transiently transfected with EGFP-clathrin were incubated with IgG-opsonized RBC for 5 min and analyzed by fluorescence microscopy. In agreement with the report by Aggeler and Werb (14), we were able to detect clathrin on the membrane of nascent phagosomes (Fig. 1, D and E).

We also analyzed whether dynamin I associated with the phagosomal membrane. RAW 264.7 cells were transfected with epitope-tagged wild-type dynamin I and analyzed by confocal microscopy following immunostaining. It is noteworthy that, prior to phagocytosis, dynamin I was associated with the plasma membrane but not with endomembranes (not shown). Upon addition of opsonized particles, dynamin I accumulated markedly in the phagosomal cup (Fig. 1F), resembling the distribution of dynamin II reported by Gold et al. (16).

Effect of Dyn I K44A on Tf Endocytosis-- Severing of clathrin-coated pits to form intracellular vesicles requires functional dynamin (29-31). To assess the role of clathrin-dependent vesiculation in Fcgamma receptor-mediated endocytosis and phagocytosis, we transfected cells with an inactive form of dynamin I (Dyn I K44A), which has been reported to exert a dominant-negative effect (29, 31). To identify the transfected cells, Dyn I K44A was cotransfected with soluble EGFP (10:1 cDNA ratio). Because Dyn I K44A was tagged with the hemagglutinin epitope, we were able to verify the effectiveness of co-transfection (>90%) and, in addition, confirmed that the dominant-negative protein was largely associated with the plasma membrane (not illustrated).

Fig. 2 illustrates the effect of Dyn I K44A on receptor-mediated endocytosis in RAW 264.7 cells. Uptake of Tf was greatly diminished in cells expressing the dominant-negative construct, identified by expression of co-transfected EGFP (Fig. 2, A-C). For these and all subsequent experiments, cells were incubated 48 h post-transfection, because inhibition at this time was greater than after 24 h (data not shown). The inhibition was not a consequence of the transfection process per se, because expression of wild-type dynamin I was without effect. Moreover, expression of a second dynamin I mutant (Dyn I S45N), also reported to be inhibitory (32), similarly obliterated Tf uptake (Fig. 2D). These findings are in good agreement with the effects of dynamin I mutants in other cell types (29-31, 33).


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Fig. 2.   Effect of dominant-negative dynamin I on Tf endocytosis. RAW 264.7 cells were transiently co-transfected with wild-type or mutant dynamin and EGFP (10:1 cDNA ratio). After 48 h, the cells were allowed to internalize rhodamine-labeled Tf (Tf-rhod) for 30 min before analysis. A, DIC image; B, green (EGFP) fluorescence; C, red (Tf) fluorescence. Images are representative of three similar experiments. D, quantification of Tf uptake in untransfected cells (Control) and in cells transfected with WT or dominant-negative forms of dynamin I (Dyn K44A and S45N). Ordinate, percent Tf-positive cells. Data are means ± S.E. of three experiments. A minimum of 100 cells were counted per experiment.

Effect of Dyn I K44A on Fcgamma Receptor-mediated Endocytosis-- The effect of dominant-negative dynamin I on Fcgamma receptor-mediated endocytosis was studied next (Fig. 3). RAW 264.7 cells were transfected with Dyn I K44A, and their ability to internalize aggregated IgG was quantified. As before, transfected cells were identified by co-expression of EGFP (Fig. 3B), and uptake of IgG was determined following permeabilization and addition of labeled anti-IgG antibody (Fig. 3C). As summarized in Fig. 3D, inhibition of dynamin function reduced IgG uptake by >80%.


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Fig. 3.   Effect of dominant-negative dynamin I on endocytosis of aggregated IgG. RAW 264.7 cells were transiently co-transfected with Dyn I K44A and EGFP (10:1 cDNA ratio). After 48 h, the cells were allowed to internalize aggregated IgG for 30 min before fixation. After permeabilization, IgG was stained using Cy3-conjugated secondary antibodies. A, DIC image; B, green (EGFP) fluorescence; C, red (IgG) fluorescence. Images are representative of three similar experiments. D, quantification of IgG uptake in untransfected cells (Control) and in cells transfected with dominant-negative dynamin I (Dyn K44A). Ordinate, percent IgG-positive cells. Data are means ± S.E. of three experiments. A minimum of 100 cells were counted per experiment.

Effect of Dyn I K44A on Fcgamma Receptor-mediated Phagocytosis of Red Cells-- We next tested the effect of Dyn I K44A on phagocytosis. We anticipated that the preferential association of this isoform with the plasma membrane would resolve whether the reported inhibitory effects of dynamin II and other clathrin-disrupting maneuvers reflect a direct role of plasmalemmal clathrin in phagocytosis or are indirect (see Introduction). As shown in Fig. 4A, phagocytosis was scored by DIC microscopy following hypotonic lysis of adherent extracellular RBC. In the transfected cells, phagocytosis was also apparent from the exclusion of EGFP from areas of the cytosol (Fig. 4B). Remarkably, Dyn I K44A had only a modest, statistically insignificant effect on phagocytosis, which was not different from that of the wild-type protein (Fig. 4C). Similar negative results were obtained using Dyn I S45N (Fig. 4C), under conditions where IgG endocytosis was largely eliminated.


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Fig. 4.   Effect of dominant-negative dynamin I on phagocytosis. RAW 264.7 cells were transiently co-transfected with wild-type or mutant dynamin and EGFP (10:1 cDNA ratio). After 48 h, the cells were allowed to internalize IgG-opsonized RBC for 30 min before analysis. A, DIC image; B, green (EGFP) fluorescence; C, quantification of the phagocytic efficiency in untransfected cells (Control) and in cells transfected with WT or dominant-negative forms of dynamin I (Dyn K44A and S45N). Ordinate, phagocytic index (RBC per 100 macrophages). Data are means ± S.E. of three experiments. A minimum of 100 cells were counted per experiment. NS, p > 0.05.

These results contrast sharply with those obtained earlier by Gold et al. (16) using the equivalent dominant-negative form of dynamin II (Dyn II K44A). To ensure that these results reflect distinct properties of the two isoforms and not differences in the experimental system we tested the effect of transient transfection of Dyn II K44A on RAW 264.7 cells. Under conditions like those of Fig. 4, transiently transfected Dyn II K44A inhibited phagocytosis of IgG-coated sheep RBC by >= 85%.

Effect of Clathrin Antisense mRNA on Endocytosis and Phagocytosis-- In view of the apparent discrepancy between the data obtained with dynamin I and II, we sought alternative ways of assessing the involvement of the clathrin complex in phagocytosis. To this end, we used a cell line in which the expression level of the heavy chain of clathrin can be controlled by inducing the expression of antisense mRNA (24-26). The BHK cells into which the tetracycline-regulated expression of antisense mRNA was engineered, however, are normally unable to internalize IgG-coated particles. This problem was circumvented by transiently expressing GFP-tagged human Fcgamma RIIA receptors in these cells, 24 h prior to the assay. Prior to stimulation, these receptors are largely localized to the plasmalemma, with a fraction found in juxtanuclear recycling endosomes (not shown). As shown in Fig. 5, A and B, addition of aggregated IgG displaced most of the receptors to an intracellular compartment, where they co-localized with the ligand. Partial depletion of the heavy chain of clathrin by expression of antisense mRNA resulted in a pronounced (>= 90%) inhibition of endocytosis of aggregated IgG and prevented the internalization of plasmalemmal receptors (Fig. 5, C, D, and G).


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Fig. 5.   Effect of clathrin antisense mRNA on endocytosis and phagocytosis. BHK21-tTA cells transfected with antisense clathrin heavy chain were incubated in the presence or absence of tetracycline, conditions that prevent (A and B) or induce (C-F) the expression of the antisense mRNA, respectively. After 24 h, the cells were transiently transfected with Fcgamma RIIA-GFP, and after a further 24 h they were assayed for endocytosis of aggregated IgG (A-D) or phagocytosis of IgG-opsonized beads (E and F). A-D, measurement of endocytosis of aggregated IgG. Cells were allowed to internalize soluble aggregated IgG for 30 min and were fixed. Surface-adherent IgG was labeled with Cy3-conjugated anti-IgG antibodies in the cold. The cells were then permeabilized, and total IgG was stained using Cy5-conjugated antibodies. In A and C, total IgG (blue) and Fcgamma receptor (green) are identified. In B and D, extracellular adherent IgG (pink) and total IgG (blue) are identified. Corresponding DIC images are shown in the insets. E and F, measurement of phagocytosis of 3.1 µm (E) and 0.8 µm (F) beads. Cells were allowed to internalize opsonized beads for 30 min and were fixed. Extracellularly exposed beads were labeled with Cy3-conjugated anti-IgG antibodies in the cold (pink). The cells were then permeabilized, and all the beads were stained using Cy5-conjugated antibodies (blue). Corresponding DIC and fluorescence images were overlaid. G, quantification of the uptake of soluble aggregated IgG and IgG-coated polystyrene beads in normal (open bars) or clathrin-depleted (solid bars) cells. Ordinate, uptake of soluble aggregated IgG or IgG-coated beads, normalized relative to clathrin-expressing control. Data are means ± S.E. of three experiments. A minimum of 100 cells were counted per experiment. CHC, clathrin heavy chain.

The clathrin dependence of phagocytosis was tested next. As shown in Fig. 5, E and F, engulfment of IgG-coated beads persisted in cells expressing clathrin antisense mRNA, under conditions where endocytosis was virtually obliterated. No significant difference in the phagocytic efficiency was noted in control versus antisense-expressing cells (Fig. 5G). These results are consistent with the observations made in cells expressing Dyn I K44A and indicate that, although essential for Fcgamma receptor endocytosis, normal clathrin/dynamin function is not required for phagocytosis.

Effect of Dyn I K44A and Cytochalasin D on Phagocytosis of Small and Large Beads-- The diametrically opposed effects of Dyn I K44A on Fcgamma receptor-mediated endocytosis and phagocytosis suggests vastly different underlying processes, a notion supported by the effects of cytochalasin. This antagonist of actin assembly precludes phagocytosis of opsonized RBC but has no discernible effect on endocytosis (not illustrated). It is of interest, however, that the sensitivity of phagocytosis to cytochalasins has been reported to vary directly with the size of the particle (34). Particles <= 1 µm in diameter can be internalized effectively in the presence of the actin antagonist. This range is of particular importance, as it encompasses the size of most pathogenic bacteria. The sensitivity of endocytosis to Dyn I K44A suggests that the actin-independent uptake of small particles may be mediated by clathrin/dynamin. To test this notion, we compared the effects of cytochalasin D and Dyn I K44A on the uptake of small (0.8 µm) and large (3.1 µm) IgG-opsonized latex particles. Particle internalization was scored by immunolabeling the opsonizing IgG before and after permeabilization of the cells. In accordance with the results obtained using RBC, phagocytosis of large beads was unaffected by Dyn I K44A (Fig. 6, A and E). Conversely, cytochalasin D inhibited the uptake of large beads completely, and no further effect was seen by expression of Dyn I K44A (Fig. 6, B and E). As reported earlier (34), we found that cytochalasin had only a partial (~40%) inhibitory effect on the phagocytosis of small (0.8 µm) beads (Fig. 6, D and E). Unexpectedly, expression of Dyn I K44A had no significant effect on the uptake of small particles and only marginally enhanced the inhibition induced by cytochalasin (Fig. 6, C-E). In accordance with this observation, we also found that the uptake of small beads was essentially unaffected by clathrin antisense mRNA (Fig. 5, F and G). Jointly, these observations imply that small particles are ingested, in part, by a mechanism that differs from that used by both large particles and immune complexes.


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Fig. 6.   Effect of dominant-negative dynamin I and cytochalasin D on the phagocytosis of small and large latex beads. RAW 264.7 cells were transiently co-transfected with mutant dynamin and EGFP (10:1 cDNA ratio). After 48 h, the cells were allowed to internalize either 3.1 µm (A and B) or 0.8 µm (C and D) IgG-opsonized beads for 30 min before analysis. Exposed beads were labeled with Cy3-conjugated anti-IgG antibodies in the cold. The cells were then permeabilized, and all the beads were stained using Cy5-conjugated anti-IgG antibodies. A-D, main panels are DIC images overlaid with fluorescence identifying extracellular adherent beads (pink) and intracellular beads (blue). Insets are green (EGFP) fluorescence, identifying transfected cells. In B and D cells were treated with 10 µM cytochalasin D. E, quantification of the phagocytic efficiency in untransfected cells (Control) and in cells transfected with dominant-negative dynamin I (Dyn K44A) in the presence or absence of cytochalasin D. Left ordinate, number of beads ingested per cell (black bars). Right ordinate, relative proportion of ingested beads, normalized to control (open bars). Data are means ± S.E. of three experiments. A minimum of 100 cells were counted per experiment.

Effect of DN-Rab5 on Endocytosis and Phagocytosis-- Unlike most receptor-associated solutes, extracellular fluids are taken up by cells by a pinocytic process that is independent of clathrin, yet exquisitely sensitive to inhibitory forms of Rab5 (35, 36). We considered the possibility that an analogous Rab5-dependent process could contribute to small particle uptake. We therefore tested the effects of a dominant-negative form of Rab5 on Fcgamma receptor-mediated phagocytosis of small beads. To ensure the effectiveness of the construct we also assessed the pinocytic uptake of the fluid phase marker rhodamine-dextran. As shown in Fig. 7, A, B, and G, expression of DN-Rab5 obliterated fluid phase uptake in macrophages. In contrast, DN-Rab5 had no discernible effect on the ingestion of small particles (Fig. 7, E-G). Therefore, the actin- and dynamin-resistant ingestion of small IgG-coated particles is not akin to pinocytosis.


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Fig. 7.   Effect of dominant-negative Rab5 on endocytosis and phagocytosis. RAW 264.7 cells were transiently transfected with DN-Rab5-GFP. After 24 h, expression of DN-Rab5-GFP was confirmed by fluorescence microscopy (main panels in A, C, and E), and endocytosis or phagocytosis were assayed (B, D, and F). Insets show corresponding DIC images. A and B, measurement of fluid phase uptake. Cells were allowed to ingest rhodamine-dextran for 30 min before visualization of red fluorescence (B). C and D, measurement of endocytosis of aggregated IgG. Cells were allowed to internalize soluble aggregated IgG for 30 min and were fixed. Surface-adherent IgG was labeled with Cy3-conjugated anti-IgG antibodies in the cold. The cells were then permeabilized, and total IgG was stained using Cy5-conjugated antibodies. In D extracellular adherent IgG (pink) and intracellular IgG (blue) are identified. E and F, measurement of phagocytosis of 0.8-µm beads. Cells were allowed to internalize opsonized beads for 30 min and were fixed. Exposed beads were labeled with Cy3-conjugated anti-IgG antibodies in the cold. The cells were then permeabilized, and all the beads were stained using Cy5-conjugated antibodies. F, DIC image overlaid with fluorescence, identifying extracellular adherent beads (pink) and intracellular beads (blue). G, quantification of the uptake of rhodamine-dextran, soluble aggregated IgG, and IgG-coated 0.8-µm polystyrene beads in cells transfected with DN-Rab5. Ordinate, uptake of rhodamine-dextran, soluble aggregated IgG, or IgG-coated 0.8-µm beads, normalized relative to untransfected control. Where specified the cells were transfected with DN-Rab5. Data are means ± S.E. of three experiments. A minimum of 100 cells were counted per experiment.

Rab5 mediates the stimulation of epidermal growth factor receptor by its ligand (37). It is noteworthy that Fcgamma receptor endocytosis was unaffected by DN-Rab5 (Fig. 7, C and D), implying a different mode of activation.

Effect of Dyn I K44A on Phagosome Maturation-- The ability of RAW 264.7 cells to engulf IgG-opsonized particles despite the expression of dominant-negative dynamin I enabled us to analyze the role of clathrin-dependent fission in phagosome maturation. The transition between the early and intermediate stages of maturation is marked by the removal of Tf receptors from the phagosomal membrane, a process that in RAW cells is completed ~30 min after completion of phagocytosis (38). We therefore analyzed the rate of depletion of Tf receptors from phagosomes formed in cells expressing Dyn I K44A. Inhibition of Tf endocytosis by Dyn I K44A, as reported in Fig. 2, results in a concomitant accumulation of Tf receptors on the plasma membrane of the transfected cells (Fig. 8, A-C), facilitating detection of their rate of removal from the phagosomes. Despite the overabundance of Tf receptors in the membrane of Dyn I K44A-transfected cells, the receptors were rapidly cleared from the phagosomes shortly after internalization (Fig. 8, G-I). Quantification of the kinetics of removal from phagosomes showed that the half-life of Tf receptors in the phagosomes of Dyn I K44A-expressing cells was 10 min (Fig. 8J), which is similar to that we reported earlier for untreated RAW 264.7 cells (38). Because the endocytosis of Tf was demonstrably impaired in the plasma membrane of these cells, as indicated by the accumulation of receptors at the plasmalemma, we conclude that different processes mediate the budding and fission of Tf receptor-containing vesicles from the surface and phagosomal membranes.


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Fig. 8.   Effect of dominant-negative dynamin I on the association of Tf receptors with phagosomes. RAW 264.7 cells were transiently co-transfected with dynamin 1 K44A and EGFP (10:1 cDNA ratio). After 48 h, the cells were either fixed (A-C) or were allowed to internalize IgG-opsonized RBC for 5 min and then chased for either 5 (D-F) or 40 min (G-I) before fixation. The cells were then permeabilized, and the Tf receptors were immunostained as described under "Experimental Procedures." A, D, and G, DIC images; B, E, and H, green (EGFP) fluorescence; C, F, and I, red (Tf receptor) fluorescence. Images are representative of three similar experiments. J, quantification of the fraction of Tf receptor (TfR)-positive phagosomes as a function of time after phagosome formation. Data are means ± S.E. of three experiments. A minimum of 100 cells were counted for each time point.

Similar results were obtained when we measured the traffic of another plasmalemmal protein, CD18, a subunit of leukocyte integrins. In otherwise untreated cells, CD18 was found to be incorporated into newly formed phagosomes and was subsequently removed during the course of phagosomal maturation, becoming undetectable by 40 min. Essentially identical results were obtained in cells transfected with Dyn I K44A (data not shown).

Effect of Dyn I K44A on Acquisition of LAMP-1-- During the course of maturation, phagosomes sequentially fuse with early and late endosomes and eventually with lysosomes. The late stages of the maturation process are characterized by the acquisition of LAMP-1, a marker of late endosomes and lysosomes. Because clathrin assembles on endosomes (18) and is also important in the delivery of proteins to prelysosomes by the trans-Golgi (39), we studied whether recruitment of LAMP-1 to late phagosomes requires functional dynamin. In untreated cells, LAMP-1 acquisition by phagosomes becomes apparent by 10 min and proceeds to reach a plateau by ~30 min (Fig. 9). In Dyn I K44A-transfected cells the course of LAMP-1 recruitment to the phagosomes was virtually identical (Fig. 9D). Thus, the acquisition of LAMP-1 by phagosomes appears to be a dynamin I-independent process.


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Fig. 9.   Effect of dominant-negative dynamin I on acquisition of LAMP-1 by phagosomes. RAW 264.7 cells were transiently co-transfected with either WT or mutant (K44A) dynamin 1 and EGFP (10:1 cDNA ratio). After 48 h, the cells were allowed to internalize IgG-opsonized RBC for 5 min and chased for 60 min. The cells were then fixed, permeabilized, and immunostained for LAMP-1 as described under "Experimental Procedures." A, DIC image; B, green (EGFP) fluorescence; C, red (LAMP-1) fluorescence. Images are representative of three similar experiments. D, quantification of the fraction of LAMP-1-positive phagosomes as a function of time after phagosome formation. Open symbols and dotted line, wild-type dynamin. Solid symbols and line, K44A dynamin. Data are means ± S.E. of three experiments. A minimum of 100 cells were counted for each time point.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Dynamin has been reported to be an important mediator of budding and scission of clathrin-coated structures such as endocytic pits (29-31, 33) and those derived from the trans-Golgi network (40, 41). We found that endocytosis of aggregated IgG was virtually eliminated by Dyn I K44A (Fig. 3), adding Fcgamma receptor endocytosis to the list of dynamin-dependent processes. Because dynamin is intimately linked to clathrin-dependent endocytosis, this process is also likely to mediate the internalization of immune complexes. However, dynamin has also been implicated in the fission of non-clathrin coated vesicles such as caveolae (42, 43). Two observations argue against a role for caveolae in Fcgamma receptor internalization. First, at least some of the sites of Fcgamma receptor accumulation were decorated with clathrin (Fig. 1), and second, extraction of plasmalemmal cholesterol using methylcyclodextrin (10 mM for 30 min) did not prevent the endocytosis of aggregated IgG by RAW 264.7 cells (not illustrated). More importantly, we found that partial depletion of clathrin using antisense mRNA virtually eliminated endocytosis of aggregated IgG. We therefore concluded that a clathrin- and dynamin-dependent process is largely responsible for internalization of immune complexes.

Recently, Gold et al. (15) found that expression of dominant-negative dynamin II inhibited phagocytosis. Although we were able to reproduce the latter results, we also found that inhibitory forms of dynamin I were without effect on phagocytosis, under conditions where clathrin-dependent endocytosis was obliterated. To the extent that the two isoforms share a high degree of homology, it is unlikely that dynamin is required for pinching and scission of the phagosomal membrane. Indeed, Gold et al. (15) reported that in cells expressing dominant-negative dynamin II phagocytosis was arrested at an early stage, seemingly because of the inability of the cells to extend pseudopodia. This phenotype resembles the appearance of cells treated with wortmannin (12) and also of cells with impaired COP-I function (38), which are similarly defective in phagocytosis. Both of these conditions depress the delivery of endomembranes to the plasmalemma, whether from the secretory or recycling compartments. We therefore speculate that in cells expressing dominant-negative dynamin II, interference with the normal delivery of endomembranes to the surface or to a reserve pool may result in the depletion of components that are essential for pseudopod elongation. This effect is not observed in cells expressing inhibitory forms of dynamin I, likely because the latter are restricted to the surface membrane. Alternatively, dynamin II (but not dynamin I) may contribute to the assembly and remodeling of actin at the phagosomal cup.

The differential sensitivity of endocytosis and phagocytosis to clathrin antisense mRNA and to inhibitory forms of dynamin emphasizes the divergence in the underlying molecular mechanisms and may reflect the differential requirement for ubiquitination. It has recently become apparent that receptor-initiated ubiquitination can trigger the recruitment of clathrin and the associated coating and scission machinery (44). The involvement of clathrin in endocytosis is therefore not unexpected, inasmuch as Fcgamma receptor endocytosis depends on ubiquitination. By contrast, phagocytosis of large particles proceeds normally in ubiquitination-deficient cells (13). It remains to be defined how Fcgamma receptor-induced endocytosis and phagocytosis, two processes triggered by clustering of the same receptors, differ so drastically at the molecular level. We speculate that the more intense and prolonged stimulation elicited by large particles recruits additional signaling pathways that are not activated during endocytosis.

Although uptake of aggregated IgG and large IgG-opsonized particles occur by clearly different mechanisms, the distinction is not as obvious in the case of smaller particles, particularly those in the size range of most bacteria (<= 1 µm). We confirmed earlier observations (34) that such particles are only modestly sensitive to cytochalasin but also found that, unlike immune complexes and soluble aggregated IgG, small particle uptake was virtually unaffected by inhibition of dynamin or by reduction of clathrin with antisense mRNA. Of note, uptake of small particles also proceeded at near normal rates in cells with impaired ubiquitination (not illustrated). The existence of a cytochalasin- and clathrin/dynamin-insensitive fraction of ingestion implies that small particles can be internalized by a mechanism distinct from conventional clathrin-mediated endocytosis and actin-driven phagocytosis. We considered the possibility that a process akin to pinocytosis may be involved. However, this mechanism appears unlikely, because the uptake of small beads was not affected by DN-Rab5 under conditions where the uptake of a fluid phase marker was eliminated.

Although clathrin may not be involved directly in the particle engulfment process, it is nevertheless recruited to the phagocytic cup, albeit in modest amounts. This may simply be the result of frustrated endocytosis, triggered by receptor cross-linking yet unable to proceed because of the physical size of the opsonized particle. However, the recruitment of clathrin to the cup may have an ulterior purpose, namely, to participate in the fission of vesicles from the phagosome during maturation. Although we could not identify a role for dynamin I in the recycling of Tf receptors or of CD18, or in the acquisition of LAMP-1, it is nevertheless likely that clathrin-coated vesicles are responsible for some aspect of phagosomal maturation, which is an elaborate phenomenon.

It is noteworthy that Tf receptors are removed from the phagosomal membrane by a process that is insensitive to Dyn I K44A, whereas their budding and pinching from the plasma membrane is exquisitely sensitive to this dominant-negative mutant. This implies that the processes underlying the remodeling of the phagosomal membrane differ drastically from those of the plasma membrane wherefrom it originated. Little is currently known about the fission events that remove constituents from the early phagosome and that maintain near constant phagosomal size despite continued fusion of endomembranes throughout the maturation process. Earlier findings in epsilon COP-deficient cells and in cells treated with brefeldin A indicated that COP-1 plays a measurable yet comparatively minor role in phagosome remodeling (38). To test the cooperativity between the COP-I and clathrin pathways, we treated with brefeldin A (2 µg/ml) cells that had been transfected with Dyn I K44A. The expression of the dominant-negative dynamin did not accentuate the effects of brefeldin on the removal of Tf receptors and CD18, or on the acquisition of LAMP1 by the phagosomal membrane (data not shown). This implies that additional mechanisms operate during phagosome remodeling. Caveolae or other coating structures such as the nexin-retromer complex (45) may be primarily responsible for vesicular fission during phagosome maturation. Alternatively, clathrin-mediated fission that is insensitive to Dyn I K44A may be taking place.

    ACKNOWLEDGEMENTS

We thank Dr. J. H. Keen (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA) for advice and for providing clathrin light-chain cDNA. We also thank Dr. Otilia Vieira and Richard Collins for help.

    FOOTNOTES

* This work was supported in part by the Canadian Institutes of Health Research (CIHR), the Arthritis Society of Canada, the Arthritis Center of Excellence, and the Sanatorium Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Supported by a CIHR fellowship, the Arthritis Society of Canada, and the Arthritis Center of Excellence.

|||| Current holder of the Pitblado Chair in Cell Biology at The Hospital for Sick Children. Cross-appointed to the Department of Biochemistry, University of Toronto. To whom correspondence should be addressed: Dept. of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5727; Fax: 416-813-5028; E-mail: sga@sickkids.ca.

Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M207966200

    ABBREVIATIONS

The abbreviations used are: RBC, red blood cells; DIC, differential interference contrast; Dyn, dynamin; PBS, phosphate-buffered saline; Tf, transferrin; GFP, green fluorescent protein; WT, wild-type; DN, dominant-negative; EGFP, enhanced GFP.

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