Indirect Role for COPI in the Completion of Fcγ Receptor-mediated Phagocytosis
2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês
10.1074/jbc.m102009200
ISSN1083-351X
AutoresDavid J. Hackam, Roberto J. Botelho, Carola Sjölin, Ori D. Rotstein, John M. Robinson, Alan D. Schreiber, Sergio Grinstein,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoRecent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of εCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcγRIIA). In the presence of functional COPI, FcγRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcγRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus. Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of εCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcγRIIA). In the presence of functional COPI, FcγRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcγRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus. endoplasmic reticulum brefeldin A Chinese hamster ovary electron microscopy Fc receptors type IIA green fluorescent protein transferrin ADP-ribosylation factor Phagocytosis of microbial pathogens by leukocytes is an essential component of the host defense against infection. Microorganisms become internalized into a membrane-bound vacuole called a phagosome, which subsequently matures upon fusion with endosomes and lysosomes into a powerful microbicidal organelle (1Beron W. Alvarez-Dominguez C. Mayorga L. Stahl P.D. Trends Cell Biol. 1995; 5: 100-104Abstract Full Text PDF PubMed Scopus (113) Google Scholar). The phagocytic capacity of neutrophils and macrophages is remarkable; individual cells can take up multiple and/or very large particles. As a result, the area of membrane internalized is significant and has in some cases been estimated to approach or exceed the total initial surface area of the phagocyte (2Werb Z. Cohn Z.A. J. Biol. Chem. 1972; 247: 2439-2446Abstract Full Text PDF PubMed Google Scholar). Despite the internalization of a considerable membrane expanse, no net loss of surface has been detected, and, in fact, surface gains have been documented electrophysiologically (3Holevinsky K.O. Nelson D.J. Biophys. J. 1998; 75: 2577-2586Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and by spectroscopic means (4Gonzalez-Rothi R.J. Straub L. Cacace J.L. Schreier H. Exp. Lung Res. 1991; 17: 687-705Crossref PubMed Scopus (47) Google Scholar). These observations therefore suggest that active exocytosis of endomembranes accompanies phagocytosis. Pharmacological studies have in fact suggested that secretion of endomembranes is essential for optimal phagocytosis. First, inhibitors of phospholipase A2 precluded phagocytosis, with a concomitant accumulation of clear vesicles under the site of particle attachment (5Lennartz M.R. Yuen A.F. Masi S.M. Russell D.G. Buttle K.F. Smith J.J. J. Cell Sci. 1997; 110: 2041-2052Crossref PubMed Google Scholar). Second, Cox et al. (6Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar) found that inhibition of phosphatidylinositol 3′-kinase by wortmannin, which can inhibit exocytosis, prevented phagocytosis of large particles, with comparatively minor effects on the ingestion of smaller ones. Third, botulinum and tetanus toxins, which interfere with SNARE-mediated membrane fusion, were found to induce partial inhibition of phagocytosis (7Hackam D.J. Rotstein O.D. Sjolin C. Schreiber A.D. Trimble W.S. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11691-11696Crossref PubMed Scopus (115) Google Scholar). Finally, it was shown that exocytic vesicles enriched in VAMP3, a v-SNARE that mediates secretion of recycling vesicles (8Galli T. Chilcote T. Mundigl O. Binz T. Niemann H. De Camilli P. J. Cell Biol. 1994; 125: 1015-1024Crossref PubMed Scopus (196) Google Scholar), were locally secreted at sites of phagocytosis (9Bajno L. Peng X.R. Schreiber A.D. Moore H.P. Trimble W.S. Grinstein S. J. Cell Biol. 2000; 149: 697-706Crossref PubMed Scopus (265) Google Scholar). Jointly, these studies suggest that extension of pseudopods during particle engulfment involves the focal exocytosis of endomembrane vesicles. The mechanisms mediating the formation and traffic of these putative vesicles are not understood. However, existing information regarding endomembrane traffic may be applicable to the genesis of phagosomes. In particular, COPI has been convincingly shown to participate in the budding and traffic of vesicles between the Golgi complex and the endoplasmic reticulum (ER)1(10Hobbie L. Fisher A.S. Lee S. Flint A. Krieger M. J. Biol. Chem. 1994; 269: 20958-20970Abstract Full Text PDF PubMed Google Scholar, 11Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar, 12Guo Q. Penman M. Trigatti B.L. Krieger M. J. Biol. Chem. 1996; 271: 11191-11196Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) as well as vesicles involved in recycling and endosome maturation (13Daro E. Sheff D. Gomez M. Kreis T. Mellman I. J. Cell Biol. 1997; 139: 1747-1759Crossref PubMed Scopus (118) Google Scholar, 14Gu F. Aniento F. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (140) Google Scholar). COPI exists as a protein complex in the cytosol and can assemble on docking sites of membranes, where it promotes the fission of vesicles (15Scales S.J. Gomez M. Kreis T.E. Int. Rev. Cytol. 2000; 195: 67-144Crossref PubMed Google Scholar, 16Wieland F. Harter C. Curr. Opin. Cell Biol. 1999; 11: 440-446Crossref PubMed Scopus (91) Google Scholar). We therefore considered the possibility that COPI may participate in the process of phagosome formation. To this end, we used brefeldin A (BFA), which inactivates the GTP exchange factors of adenosine ribosylation factor, to inhibit COPI or the Chinese hamster ovary (CHO) mutant cell line called ldlF, originally isolated by Hobbie et al. (10Hobbie L. Fisher A.S. Lee S. Flint A. Krieger M. J. Biol. Chem. 1994; 269: 20958-20970Abstract Full Text PDF PubMed Google Scholar). These cells harbor a temperature-sensitive mutation in the gene encoding εCOP, a component of COPI (10Hobbie L. Fisher A.S. Lee S. Flint A. Krieger M. J. Biol. Chem. 1994; 269: 20958-20970Abstract Full Text PDF PubMed Google Scholar, 11Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar). When incubated at the permissive temperature (34 °C), such cells express functional εCOP, while incubation at the restrictive temperature (≥39 °C) results in destabilization and rapid degradation of the mutant εCOP, thereby inactivating COPI (10Hobbie L. Fisher A.S. Lee S. Flint A. Krieger M. J. Biol. Chem. 1994; 269: 20958-20970Abstract Full Text PDF PubMed Google Scholar, 11Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar). While extremely useful for the study of COPI function, ldlF cells are not phagocytic. In order to analyze the role of COPI in phagocytosis, ldlF cells were stably transfected with Fc receptors (FcγRIIA). Such heterologous transfection of opsonin receptors was shown earlier to confer phagocytic capacity to nonmyeloid cells, including CHO cells (17Botelho R.J. Hackam D.J. Schreiber A.D. Grinstein S. J. Biol. Chem. 2000; 275: 15717-15727Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 18Hackam D.J. Rotstein O.D. Schreiber A. Zhang W. Grinstein S. J. Exp. Med. 1997; 186: 955-966Crossref PubMed Scopus (148) Google Scholar, 19Park J.G. Isaacs R.E. Chien P. Schreiber A.D. J. Clin. Invest. 1993; 92: 1967-1973Crossref PubMed Scopus (43) Google Scholar). We found that, like CHO cells, ldlF cells transfected with FcγRIIA (called FcR-ldl hereafter) effectively internalized IgG-opsonized particles when grown at the permissive temperature. However, following down-regulation of COPI, phagocytosis was progressively and drastically inhibited. Evidence is provided that COPI is indirectly required for phagocytosis by maintaining a VAMP3-containing endomembrane pool that appears to be required for pseudopod extension during FcγR-mediated phagocytosis. Fura-2 acetoxymethyl ester, zymosan, FM1–43, rhodamine-123, and rhodamine-phalloidin were from Molecular Probes, Inc. (Eugene, OR). Hepes-buffered medium RPMI 1640, 0.8-μm dyed latex beads, BFA, and cycloheximide were obtained from Sigma. 125I-Diferric human transferrin (125I-Tfn) was from PerkinElmer Life Sciences, and [35S]methionine/cysteine was from Amersham Pharmacia Biotech. pEGFP, the plasmid encoding GFP, was fromCLONTECH. The VAMP3-GFP chimera was previously described (9Bajno L. Peng X.R. Schreiber A.D. Moore H.P. Trimble W.S. Grinstein S. J. Cell Biol. 2000; 149: 697-706Crossref PubMed Scopus (265) Google Scholar). Human IgG was from Baxter Healthcare Corp. (Glendale, CA). Rabbit anti-GFP and anti-catalase antibodies were from Molecular Probes, Inc. and Calbiochem (La Jolla, CA), respectively. The rabbit polyclonal antibodies to εCOP, α-mannosidase II, and calnexin were generous gifts from Drs. M. Krieger (Massachusetts Institute of Technology, Cambridge, MA), K. Moremen (Emory University, Atlanta, GA), and David Williams (University of Toronto), respectively. Mouse anti-phosphotyrosine antibody mixture, containing equivalent amounts of monoclonal antibodies PY-7E1, PY-1B2, and PY-20 was fromZymed Laboratories Inc. (San Francisco, CA). Mouse anti-FcγRIIA monoclonal antibody IV.3 and the anti-β1-integrin monoclonal antibody, 7E2, were from Medarex (Annandale, NJ) and from the Developmental Hybridoma Studies Bank (Iowa City, IA), respectively. Cy3-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Sheep erythrocytes (SRBC) and goat and rabbit anti-SRBC antibodies were from ICN/Cappel (Aurora, OH). The murine macrophage cell lines, J774 and RAW 264.7, were obtained from the ATCC (Manassas, VA). RAW macrophages were transfected with Fugene 6, as described by the manufacturer (Roche Molecular Biochemicals). ldlF cells, bearing a thermolabile mutation in the εCOP, were the generous gift of Dr. M. Krieger. Wild-type CHO and ldlF cells were stably transfected with FcγRIIA cDNA using calcium phosphate, yielding FcR-CHO and FcR-ldl cells, respectively. All cell lines were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% penicillin-streptomycin (Life Technologies, Inc.) and maintained under 5% CO2. Unless otherwise indicated, FcR-CHO cells were grown at 37 °C, and FcR-ldl cells were grown at 34 °C. For down-regulation of COPI, FcR-ldl cells were placed at the restrictive temperature of 39 °C for the specified periods. Alternatively, cells were incubated with 100 μm BFA for the indicated periods, followed by phagocytosis in the continuous presence of BFA. To assess phagocytosis, SRBC were opsonized with goat or rabbit anti-SRBC IgG (1:40 for 1 h at 37 °C) and then washed in cold phosphate-buffered saline to remove unbound IgG. The opsonized SRBC were added to plated phagocytic cells (∼10 SRBC/cell) and incubated for 1 h at 37 °C. Extracellular SRBC were then removed by a brief (30-s) hypotonic lysis in water, and internalized SRBC, which resist lysis, were quantified under Nomarski optics. To assess particle adherence, FcR-CHO cells or FcR-ldl cells were incubated with opsonized SRBC for 30 min at 4 °C. Nonadherent SRBC were removed by washing in phosphate-buffered saline, and the number of bound SRBC/cell was quantified under light microscopy. Zymosan particles and 0.8-μm latex beads were opsonized with 1 mg/ml human IgG and washed as described above. To label F-actin, cells were fixed for up to 3 h with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 and incubated with rhodamine-phalloidin (0.01 unit/ml phosphate-buffered saline). To stain for α-mannosidase II, calnexin, catalase, and phosphotyrosines, fixed and permeabilized cells were incubated with a 1:100, 1:1000, 1:500, or 1:100 dilution of the respective antibodies for 1 h and subsequently incubated for 1 h with fluorochrome-conjugated secondary antibodies used at 1:1000. To stain for GFP, fixed, nonpermeabilized VAMP3-GFP-transfected cells were incubated with a 1:600 dilution of rabbit anti-GFP antibody followed by Cy3-conjugated anti-rabbit at 1:1000. FcR-ldl (not permeabilized) cells were stained with a 1:50 dilution of anti-FcγRIIA and undiluted anti-β1-integrin antibodies to quantitate surface expression of FcγRIIA and β1-integrin, respectively. Fluorescence was analyzed using either a Leica model TCS4D or a Zeiss LSM 510 laser confocal microscope. Composites of confocal images were assembled and labeled using Photoshop (Adobe, Mountain View, CA) and Microsoft Powerpoint software. Quantification of immunofluorescence was performed using Scion Image (Scion Image, MA). Surface β1-integrin was quantified using fixed, nonpermeabilized cells attached to glass coverslips by confocal microscopy. This was accomplished by acquiring confocal optical slices at a constant interval and integrating the total cellular fluorescence by stacking the collected slices. Fluorescence intensity of the reconstructed images was quantified using Scion Image. For flow cytometry, the cells were immunostained as above and then scraped off the coverslips in ice-cold divalent cation-free phosphate-buffered saline. After washing, the cells were analyzed as in Ref. 7Hackam D.J. Rotstein O.D. Sjolin C. Schreiber A.D. Trimble W.S. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11691-11696Crossref PubMed Scopus (115) Google Scholar. Cells grown on glass coverslips were incubated overnight at either 34 or 39 °C and then loaded with fura-2 by incubation with 10 μm of the parental acetoxymethyl ester for 30 min. Coverslips were then mounted in a thermostatted Leiden holder on the stage of a Zeiss IM-35 microscope, equipped with a × 63 oil immersion objective. The microscope set-up has been previously described in detail (18Hackam D.J. Rotstein O.D. Schreiber A. Zhang W. Grinstein S. J. Exp. Med. 1997; 186: 955-966Crossref PubMed Scopus (148) Google Scholar). Calibration of fluorescence ratio versus[Ca2+]i was performed as described (20Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). All measurements were at 37 °C. For scanning electron microscopy, cells were fixed in 2% glutaraldehyde and postfixed with 1% OsO4. Following washing, the cells were dehydrated in a graded series of ethanol. The samples were then critical point-dried in a Pelco CPD-2 critical point drying device and mounted on EM stubs with colloidal silver glue. They were then coated with evaporated gold/palladium with a Pelco sputter coater model 3 for 50 s at 18 mA. The samples were then examined with a Philips XL 30 scanning electron microscope. For transmission EM, cells were fixed as for scanning EM. The cells were washed extensively and then en bloc stained with 1% aqueous uranyl acetate for 30 min. Following washing, the samples were dehydrated through a graded series of ethanol and then embedded in Epon as we have described previously (21Robinson J.M. Okada T. Castellot Jr., J.J. Karnovsky M.J. J. Cell Biol. 1986; 102: 1615-1622Crossref PubMed Scopus (54) Google Scholar). Thin sections were cut and stained with lead citrate and uranyl acetate and observed with a Philips CM-12 electron microscope. Samples were solubilized in Laemmli's sample buffer (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar), resolved by SDS-polyacrylamide gel electrophoresis using the Protean II minigel system (Bio-Rad), and transferred onto polyvinylidene difluoride membranes. Membranes were then immersed in blocking buffer (5% milk and 0.05% Tween 20) overnight at 4 °C. Blots were incubated with anti-εCOP antibody (1:8000) and anti-tubulin antibody (1:500) for 1 h at room temperature. The blots were then washed three times for 10 min each in antibody buffer (50 mm Tris/HCl, 150 mm NaCl, 0.05% Tween 20, 0.04% Nonidet P-40, pH 7.5) and next incubated with peroxidase-conjugated anti-rabbit IgG (1:5000) for 1 h. Membranes were washed and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech). FcR-ldl cells were either maintained at 34 °C or incubated at 39 °C for ≥12 h and then serum-starved for 1 h. The rate of receptor-mediated endocytosis of125I-Tfn was then measured by incubating cells with 0.4 μCi/ml of 125I-Tfn for either 10 or 30 min. Extracellular125I-Tfn was then washed and stripped with phosphate-buffered saline solution at pH 3.0. To measure the rate of125I-Tfn recycling, cells were incubated for 15 or 30 min in the absence of 125I-Tfn after a pulse of 30 min. All steps were performed at 37 °C. The cells were then lysed, and the amount of internalized 125I-Tfn was quantified with a γ counter. To study the role of COPI in phagocytosis, ldlF cells were stably transfected with FcγRIIA, yielding FcR-ldl cells. To verify that the mutation characteristic of ldlF cells persisted in FcR-ldl cells, the εCOP content of both cell types was assessed by immunoblotting following incubation at the permissive (34 °C) or restrictive (39 °C) temperature. For comparison, wild-type CHO stably transfected with FcγRIIA (FcR-CHO cells) were also analyzed. Tubulin, which is constitutively expressed in these cells in a temperature-independent manner, was used as a reference. When grown at 34 °C, both ldlF and FcR-ldl express εCOP, although at levels that are 2–4 times lower than that found in FcR-CHO cells (Fig.1 A). A similar differential expression of εCOP at 34 °C was reported earlier between ldlF and wild-type CHO cells (12Guo Q. Penman M. Trigatti B.L. Krieger M. J. Biol. Chem. 1996; 271: 11191-11196Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Incubation at the restrictive temperature for ≤18 h resulted in the complete disappearance of εCOP in ldlF and FcR-ldl cells but had no detectable effect on the expression of this protein in FcR-CHO cells (Fig. 1 A), as found earlier for wild-type CHO cells (12Guo Q. Penman M. Trigatti B.L. Krieger M. J. Biol. Chem. 1996; 271: 11191-11196Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The disappearance of εCOP was also apparent by analyzing the functional consequences of incubation at 39 °C. As illustrated in Fig. 1 B, i, the Golgi complex normally displays a tight juxtanuclear structure composed of cisternae and vesicles. Maintenance of this structure is known to depend on the continued function of COPI, which in turn requires the presence of εCOP (13Daro E. Sheff D. Gomez M. Kreis T. Mellman I. J. Cell Biol. 1997; 139: 1747-1759Crossref PubMed Scopus (118) Google Scholar,14Gu F. Aniento F. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (140) Google Scholar). Degradation of εCOP in FcR-ldl cells was associated with dispersal of the juxtanuclear complex, resulting in a diffuse punctate staining of α−mannosidase II-reactive vesicles (Fig.1 B, iv). Dispersal of the Golgi complex was due to disappearance of εCOP and not to the temperature shift itself, since FcR-CHO cells were found to preserve their juxtanuclear Golgi cisternae after overnight incubation at 39 °C (Fig. 1 B,iii). In contrast to the Golgi complex, incubating cells at 39 °C did not appear to cause detectable changes to peroxisomes, mitochondria, and the ER. The punctate distribution of catalase, a marker of peroxisomes, was indistinguishable in cells incubated at 34 and 39 °C (not shown), implying that peroxisomes are able to import and retain this soluble protein in their lumen. Furthermore, rhodamine-123 accumulated normally in the mitochondria of cells incubated at 39 °C (Fig.1 C), indicating that the mitochondrial membrane potential was unaffected, which implies that the activity of the respiratory chain is normal. In addition, the reticulate morphology of the ER, revealed by immunostaining of calnexin, was similarly unaltered by prolonged incubation at the restrictive temperature (not illustrated). These results were consistent with ultrastructural analysis of thin sections by transmission EM, which showed no alterations in the structure or distribution of mitochondria or ER in cells treated at 39 °C (100 sections from three different experiments; not illustrated). Together, these observations imply that treatment of the cells at the restrictive temperature for ≤15 h does not induce wholesale, nonspecific disorganization of the cellular ultrastructure and that only organelles dependent on COPI for their homeostasis, such as the Golgi apparatus, undergo visible alterations, as shown previously (11Guo Q. Vasile E. Krieger M. J. Cell Biol. 1994; 125: 1213-1224Crossref PubMed Scopus (129) Google Scholar, 13Daro E. Sheff D. Gomez M. Kreis T. Mellman I. J. Cell Biol. 1997; 139: 1747-1759Crossref PubMed Scopus (118) Google Scholar, 14Gu F. Aniento F. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (140) Google Scholar). Previously, it had been shown that ldlF cells stably transfected with FcγRIIA were able to internalize IgG-opsonized particles (17Botelho R.J. Hackam D.J. Schreiber A.D. Grinstein S. J. Biol. Chem. 2000; 275: 15717-15727Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Therefore, we next investigated whether COPI is required for phagocytosis. FcR-ldl cells were maintained at either 34 or 39 °C and then exposed to IgG-opsonized SRBC to initiate phagocytosis. As illustrated in Fig.2 A and quantified in Fig.2 C, FcR-ldl cells maintained at the permissive temperature internalize SRBC effectively; phagocytosis occurred in upwards of 50% of the cells. This compares favorably with the phagocytosis efficiency of FcR-CHO cells (∼30%; Fig. 2 C). The greater phagocytic ability of FcR-ldl cells is most likely attributable to clonal differences. Depletion of εCOP by overnight incubation at 39 °C profoundly reduced the ability of FcR-ldl cells to perform phagocytosis. SRBC were observed in only 4 ± 3% of the εCOP-depleted cells. By contrast, FcR-CHO cells internalized SRBC slightly more effectively following incubation at 39 °C than when maintained at 34 °C. These observations imply that the effect noted in FcR-ldl cells is not due to the temperature per se but instead that the presence of COPI is essential for optimal phagocytosis. Consistent with the findings of Daro et al. (13Daro E. Sheff D. Gomez M. Kreis T. Mellman I. J. Cell Biol. 1997; 139: 1747-1759Crossref PubMed Scopus (118) Google Scholar) in ldl cells, the cells stably transfected with Fc receptors (FcR-ldl) were found to take up and recycle Tfn, albeit at reduced rates. As shown in Fig. 2 D, the initial rate of uptake was >2-fold lower in εCOP-depleted cells, although the extent of uptake after 30 min was only ∼30% lower. Also in agreement with Daro et al. (13Daro E. Sheff D. Gomez M. Kreis T. Mellman I. J. Cell Biol. 1997; 139: 1747-1759Crossref PubMed Scopus (118) Google Scholar), we found that the slower rate of Tfn uptake was compensated by a reduced rate of recycling (Fig. 2 D, lanes 5–8), accounting for the nearly normal Tfn content at steady state. The reduced recycling of transferrin is consistent with the proposed role of COPI in traffic along the endocytic pathway. During the course of the phagocytosis experiments described above, a striking morphological difference between cells incubated in different conditions became apparent. When incubated at the restrictive temperature, FcR-ldl cells were found to become more rounded (Fig. 2, compare A and B). As before, the effect was attributable to the depletion of COPI and not to the incubation at 39 °C, since FcR-CHO cells maintained their spread morphology at this temperature (not shown). Further evidence that this effect was specifically related to loss of COPI was obtained by treating FcR-CHO and FcR-ldl cells (maintained at 34 °C) with the fungal metabolite BFA. BFA associates with and blocks the activity of nucleotide exchange factors that regulate ARF, thereby preventing the association of coatomer subunits with membranes (23Helms J.B. Rothman J.E. Nature. 1992; 360: 352-354Crossref PubMed Scopus (585) Google Scholar, 24Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar). Treatment with 100 μm BFA for 30–60 min induced rounding of both cell lines (not shown). The cell rounding observed upon inactivation of COPI was associated with an apparent decrease in net surface area. This was determined by confocal microscopy, quantifying the amount of cell-associated FM1–43 (not shown), a dye that becomes fluorescent when it intercalates into the plasmalemma, thereby providing a measure of cell surface area (6Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar,25Smith C.B. Betz W.J. Nature. 1996; 380: 531-534Crossref PubMed Scopus (206) Google Scholar). The decrease in εCOP-depleted cells was reproducible and statistically significant, but since fluorescence is not a simple function of the area, the change in plasmalemmal surface was not quantified precisely. Several separate lines of evidence argue that rounding of COPI-deficient cells is not an indication of detrimental effects on cell function or viability. First, the cells remained impermeant to vital dyes and to the fluorescent dye FM1–43. Second, the cytosolic free calcium concentration, a sensitive measure of cell integrity and well being, was unaltered by depletion of εCOP (see below). Third, and as reported earlier for ldlF cells (13Daro E. Sheff D. Gomez M. Kreis T. Mellman I. J. Cell Biol. 1997; 139: 1747-1759Crossref PubMed Scopus (118) Google Scholar, 14Gu F. Aniento F. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1183-1195Crossref PubMed Scopus (140) Google Scholar), FcR-ldl cells treated overnight at 39 °C displayed endocytosis and recycling of transferrin (Fig. 2 D). Fourth, the rate of incorporation of [35S]methionine/cysteine into newly synthesized proteins was not significantly changed (not shown). Fifth, peroxisomes, the ER, and mitochondria in εCOP depleted cells resembled those of control FcR-ldl cells, and the mitochondrial potential appeared unaffected. Sixth, cells responded to the addition of stimuli with increased tyrosine phosphorylation and actin assembly (see below). Finally, the effects of incubation at 39 °C were reversible. These findings imply that within the time period examined, suppression of COPI function does not cause generalized detrimental effects on the cell. In view of the apparent loss of surface area, the effects of COPI depletion on phagocytosis could result from alterations in Fc receptor expression, distribution, or function. This possibility was considered in the experiments illustrated in Fig.3. At the permissive temperature, FcγRIIA receptors were clearly observable by immunostaining on the plasmalemma of FcR-ldl cells (Fig. 3 A). Neither the distribution (Fig. 3 B) nor the amount of receptors appeared to be altered following incubation at the restrictive temperature. Quantitation of receptors by flow cytometry indicated that similar levels of FcγRIIA were exposed after incubation at 34 or 39 °C for 18 h in both FcR-CHO and FcR-ldl cells (Fig. 3 E). The abilit
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