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• W2005221512 abstract "Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcγRIIA or both FcγRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs. Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcγRIIA or both FcγRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs. Phagocytosis plays an essential role in host-defense mechanisms (1.Jones S. Lindberg F.P. Brown E.J. Phagocytosis.in: Paul W.E. Fundamental Immunology. 1. Lippincott-Raven, Philadelphia, PA1999: 997-1020Google Scholar). Phagocytosis is often triggered by the interaction of target-bound opsonins with specific receptors on the surface of phagocytes. These receptors include the Fc receptors, which bind to the Fc portion of immunoglobulins (2.Ravetch J. Bolland S. IgG Fc receptors.Annu. Rev. Immunol. 2001; 19: 275-290Crossref PubMed Scopus (1387) Google Scholar), and the complement receptors (3.Brown E. Complement receptors, adhesions, and phagocytosis.Infect. Agents Dis. 1992; 1: 63-70PubMed Google Scholar), which bind to complement deposited on targets. Activation of these receptors leads to a reorganization of the plasma membrane that profoundly affects the function of phagocytes. The plasma membrane forms pseudopods that extend around an extracellular particle followed by their fusion to form a membrane-bound intracellular vesicle, the phagosome. As the process of phagocytosis proceeds, cytoplasmic granules fuse with the phagosome membrane to deliver hydrolytic and antibacterial enzymes to the phagosome (4.Beron W. Alvarez-Dominquez C. Mayorga L. Stahl P.D. Membrane trafficking along the phagocytic pathway.Trends Cell Biol. 1995; 5: 100-104Abstract Full Text PDF PubMed Scopus (113) Google Scholar). Phagolysosome formation is dependent upon intracellular Ca2+ and requires the activity of a complex containing docking and fusion proteins (5.Burgoyne R. Geisow M.J. The annexin family of calcium-binding proteins.Cell Calcium. 1989; 10: 1-10Crossref PubMed Scopus (218) Google Scholar, 6.Borregard N. Boxer L.A. Disorders of neutrophil function.in: Lichtman M. Beutler E. Kipps T.J. Seligsohn V. Kaushansky K. Prchal J.T. Williams Hematology. 7th edition. McGraw-Hill, New York2005: 921-957Google Scholar). Ca2+ is a ubiquitous intracellular messenger controlling a diverse range of cellular processes, such as gene transcription, muscle contraction, cell proliferation, and apoptosis (7.Mellström B. Naranjo R. Ca2+-dependent transcriptional repression and derepression: DREAM, a direct effector.Semin. Cell Dev. Biol. 2001; 12: 59-63Crossref PubMed Scopus (39) Google Scholar). Cytosolic Ca2+ signaling occurs through both Ca2+ release from intracellular stores and Ca2+ entry from the extracellular environment. Membrane Ca2+ channels can be 1) voltage operated channels (VOCs), 2) non-voltage-gated Ca2+-permeable channels (NVGCs), 3) receptor-operated Ca2+ channels (ROCs), 4) store-operated channels (SOCs), and 5) small molecule-operated channels (SMOCs). VOCs are used largely by excitable cell types such as muscle and neuronal cells, in which they are activated by plasma membrane depolarization. The NVGCs include ion channels activated by the binding of extracellular and intracellular messengers, mechanical stress, or the depletion of intracellular calcium stores. ROCs are structurally and functionally diverse channels found on secretory cells and nerve terminals. SMOCs are activated by a number of small messenger molecules. There is evidence that Ca2+ channels can be activated by intracellular lipid messengers, such as diacylglycerol (8.Hofmann T. Obukhov G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Direct activation of TRPC-6 and TRPC-3 channels by diacylglycerol.Nature. 1999; 397: 259-263Crossref PubMed Scopus (1256) Google Scholar) and arachidonic acid (9.Mignen O. Shuttleworth T. IARC, a novel arachidonate-regulated, noncapacitative Ca2+ entry channel.J. Biol. Chem. 2000; 275: 9114-9119Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). SOCs are opened after the depletion of internal Ca2+ stores. As many cell types have been shown to have enhanced Ca2+ entry after Ca2+ pool depletion, SOCs are one of the most frequently encountered plasma membrane Ca2+ channels. At present, the best candidates for the molecular identity of SOCs are homologs of the transient receptor potential channel (TRPC) that functions in Drosophila photoreception (10.Boulay G. Brown M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-triphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for the roles of TRP and IP3R in store depletion activated Ca2+ entry.Proc. Natl. Acad. Sci. USA. 1999; 96: 14955-14960Crossref PubMed Scopus (348) Google Scholar). The mechanisms by which SOCs operate are largely unknown. Recent studies have indicated that members of the TRP family of ion channels can function as Ca2+ influx channels in excitable and nonexcitable tissues (10.Boulay G. Brown M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-triphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for the roles of TRP and IP3R in store depletion activated Ca2+ entry.Proc. Natl. Acad. Sci. USA. 1999; 96: 14955-14960Crossref PubMed Scopus (348) Google Scholar). Sphingolipids, including sphingosine, sphingosine-1-phosphate, and sphingosylphosphorylcholine, have diverse effects on the regulation of intracellular free calcium concentration in nonexcitable and excitable cells (11.Spiegel S. Milstein S. Sphingosine-1-phosphate, a key signaling molecule.J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 12.Mayer zu Heringdort D. van Koppen C.J. Jacobs K.H. Molecular diversity of sphingolipid signaling.FEBS Lett. 1997; 410: 34-38Crossref PubMed Scopus (120) Google Scholar). Many of these effects are mediated by G-protein-coupled plasma membrane receptors for sphingolipids (12.Mayer zu Heringdort D. van Koppen C.J. Jacobs K.H. Molecular diversity of sphingolipid signaling.FEBS Lett. 1997; 410: 34-38Crossref PubMed Scopus (120) Google Scholar, 13.Xu Y. Zhu K. Hong G. Wu W. Baudthuin L.M. Xiao Y. Damron D.C. Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1.Nat. Cell Biol. 2000; 2: 261-267Crossref PubMed Scopus (176) Google Scholar). Ceramide-1-phosphate (C1P) has also emerged as a putative modulator of cellular functions (14.Gomez-Munoz A. Kong J.Y. Salh B. Steinbrecher U.P. Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages.J. Lipid Res. 2004; 45: 99-105Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). According to some reports, C1P itself does not modulate intracellular free calcium concentration (15.Pettus B. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. et al.Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2.J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 16.Gomez-Munoz A. Waggoner D.W. O'Brien L. Brindley D.N. Interaction of ceramides, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and phospholipase D activity.J. Biol. Chem. 1995; 270: 26318-26325Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Others have shown that C1P enhances store-operated Ca2+ entry into thyroid cells (17.Törnquist K. Ramström C. Rudnäs B. Klika K. Dugué B. Adams J. Zipkin R. Pihlaja K. Pasternack M. Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca2+ entry in thyroid FRTL-5 cells.Eur. J. Biochem. 2002; 253: 1-11Google Scholar). C1P does not affect Ca2+ mobilization in mouse fibroblasts (18.Gomez-Munoz A. Duffy P.A. Martin A. O'Brien L. Byun H.S. Bittman R. Brindley D.N. Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides.Mol. Pharmacol. 1995; 47: 833-899PubMed Google Scholar, 19.Gomez-Munoz A. Frago L.M. Alvarez L. Varela-Nieto I. Stimulation of DNA synthesis by natural ceramide 1-phosphate.Biochem. J. 1997; 325: 435-440Crossref PubMed Scopus (122) Google Scholar), neutrophils (20.Rile G. Yatomi Y. Takafuta T. Ozaki Y. Ceramide 1-phosphate formation in neutrophils.Acta Haematol. 2003; 109: 76-83Crossref PubMed Scopus (36) Google Scholar), or A549 cells (15.Pettus B. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. et al.Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2.J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). A truncated C1P with a C2 ceramide induces Ca2+ mobilization in calf pulmonary artery endothelial cells (21.Gijsbers S. Mannaerts G.P. Himpens B. Van Veldhoven P.P. N-Acetyl-sphingenine-1-phosphate is a potent calcium mobilizing agent.FEBS Lett. 1999; 453: 269-272Crossref PubMed Scopus (25) Google Scholar). Therefore, the effect of C1P in Ca2+ regulation remains controversial. Most studies to date have examined the mechanism of action of C1P by exogenous addition to cells. The cloning of ceramide kinase (CERK) provides a new tool to study the role of C1P in cell signaling. We have found that transfection of COS-1 cells with human ceramide kinase (hCERK) affected Ca2+ signaling events. In this study, we identified calcium signals in the vicinity of phagosomes during phagocytosis by introducing the hCERK gene into COS-1 cells. To ascertain whether C1P was also formed during phagocytosis, hCERK transfected COS-1 cells were labeled with the ceramide precursor [3H]d-erythro-sphingosine. The amount of radioactive C1P increased by 3-fold in the hCERK transfected cells compared with the vector control cells and nontransfected FcγRIIA cells. These transfections did not have any significant effect on cell viability and growth and, as stated above, led to an increase in CERK activity and C1P levels during stimulation with EIgG (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Our studies suggest a novel role for C1P in Ca2+ signaling and contribute to our understanding of the role of increased Ca2+ signals in promoting phagocytosis and phagolysosomal fusion. The proteinase inhibitors pepstatin and leupeptin, mibefradil, CdCl2, and rabbit anti-goat antibody were obtained from Sigma. LysoTracker Red DND-99 Indo-1-AM was obtained from Molecular Probes, Inc. (Eugene, OR). Carboxyamido-triazole (CAI) was the generous gift of Dr. R. Schultz (Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD). The SOC antagonist 1-(β-[3-(4-methoxyphenyl)propoxy]4-methoxyphenethyl)-1H-imidazole (SKF96365) was purchased from Biomol (Plymouth Meeting, PA). Western blotting detection reagents and horseradish peroxidase-conjugated sheep anti-mouse antibody were acquired from Amersham Biosciences (Piscataway, NJ). Goat anti-rabbit IgG horseradish peroxidase and FITC-conjugated caveolin-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody against caveolin-1 and rabbit anti-TRPC-1 antibody were purchased from Chemicon International (Temecula, CA). Purified rabbit CERK polyclonal antibody was purchased from Abgent (San Diego, CA). Dulbecco's modified Eagle's medium, opti-MEM medium, trypsin EDTA, l-glutamine, penicillin/streptomycin, and geneticin (G-418 sulfate) were from Gibco BRL (Grand Island, NY). Zeocin and LipofectAMINE™ reagent were from Invitrogen (Carlsbad, CA). Sheep erythrocytes were purchased from BioWhittaker (Walkersville, MD) and were opsonized with anti-sheep erythrocyte IgG from Cappel Organon Teknika (Durham, NC). Polyvinylidene difluoride membranes were from Schleicher and Schuell (Keene, NH). COS-1 cells, stably transfected with FcγRIIA receptor cDNA, were maintained in Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose, 25 mg/ml glutamine, 100 U/ml streptomycin, 100 mg/ml penicillin, and 10% heat-inactivated fetal calf serum. COS-1 cells transfected stably with FcγRIIA were cultured in 35 mm dishes to create double expression transfectants with FcγRIIA and hCERK. The cells were transfected with 1 μg/ml pcDNA-hCERK expression vector using LipofectAMINE Plus™ in 1 ml of opti-MEM medium at 80% confluence. One milliliter of DMEM containing 20% fetal bovine serum was added after 3 h of incubation at 37°C and 5% CO2. To establish stable transfectants, cells were cultured with medium containing 200 μg/ml G-418 and 200 μg/ml Zeocin to select double transfectants. Sheep red blood cells were sensitized with rabbit IgG anti-sheep erythrocyte antibody as described (EIgG) (23.Suchard S. Hinkovska-Galcheva V. Mansfield P. Boxer L. Shayman J. Ceramide inhibits IgG-dependent phagocytosis in human polymorphonuclear leukocytes.Blood. 1997; 89: 2139-2147Crossref PubMed Google Scholar). Phagocytosis assays were conducted as described previously (24.Hinkovska-Galcheva V. Kjeldsen L. Mansfield P. Boxer L. Shayman J.A. Suchard S. Activation of a plasma membrane-associated neutral sphingomyelinase and concomitant ceramide accumulation during IgG-dependent phagocytosis in human polymorphonuclear leukocytes.Blood. 1998; 91: 4761-4769Crossref PubMed Google Scholar). A plasmid carrying the hCERK gene was provided by Dr. Takafumi Kohama (Sankyo Co., Ltd.) (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Construction of catalytically inactive hCERK (G198DhCERK) was done as described previously (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). A C-terminal fusion of human CERK and DsRed monomer was constructed by PCR amplification of hCERK and cloning into plasmid pDsRed-Monomer-N1. hCERK was amplified by PCR from the existing construct, pcDNA-hCERK, using the following primers: CERK 5′XhoI (5′-CTCGAGATGGGGCGACGGGGGCG-3′) and CERK 3′HindIII (5′-AAGCTTGCTGTGTGAGTCTGGCTTCG-3′). The resulting PCR product was first cloned into pCR2.1-TOPO (Invitrogen), and recombinants were subsequently digested with XhoI and HindIII to release a 1.6 kb fragment. The XhoI-HindIII CERK-containing fragment was gel-purified and ligated to pDsRed-Monomer-N1, also digested with XhoI and HindIII, creating an in-frame N-terminal fusion between CERK and the DsRed monomer. The fusion protein was tested for CERK activity and found to be biologically active. Transfected COS-1 cells were fractionated as described previously (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). FcγRIIA, FcγRIIA/vector, and FcγRIIA/hCERK transfected COS-1 cells were challenged with opsonized erythrocytes (EIgG), and the EIgG not ingested were lysed. COS-1 cells were harvested by scraping them into a buffer containing 20 mM Tricine (pH 7.8), 0.25 M sucrose, and 1 mM EDTA. The cells were washed and then disrupted in the same buffer with 30 strokes in a Wheaton tissue grinder. The postnuclear supernatant fraction was obtained by centrifugation of the cell lysates at 100 g for 10 min. The plasma membrane fraction was removed from 30% Percoll and 0.25 M sucrose buffer after centrifugation at 84,000 g for 30 min. Caveolae membrane fractions were isolated from the purified plasma membrane fractions using OptiPrep gradients as described (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Aliquots of membrane fractions and the caveolae/lipid raft fractions were analyzed by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes and subjected to Western blotting using anti-caveolin-1, anti-c-Myc, and anti-TRPC-1 antibodies. To measure phagolysosomal fusion, cells were imaged on a Zeiss Axiovert 135 inverted microscope coupled to a cooled digital charge-coupled device camera (QImaging, Barnaby, British Columbia, Canada). Cover slips were observed using bright-field microscopy, and fluorescence was visualized using narrow band-pass discriminating filters with excitation at 535 nm and emission at 590 nm for tetramethylrhodamine. Images were obtained using Volumescan (Vaytek, Inc., Fairfield, IA) and were processed using Image-Pro Plus (Media Cybernetics, Silver Spring, MD). In some studies, mibefradil and SKF96365, inhibitors of SOCs, were used to evaluate their effect on phagocytosis and phagolysosomal fusion in the concentrations stated in each figure. Confocal scanning fluorescence microscopy was performed with an Olympus Fluoview FV 500. This microscope was used to obtain z series image stacks of cells. Cells were transfected with hCERK or red fluorescent protein (RFP)-tagged hCERK. Cover slips with adherent transfectants that had undergone phagocytosis were washed once with cold PBS and then fixed with 3% paraformaldehyde in PBS for 10 min at room temperature. After the first fixation, the cells were rinsed three times with PBS and permeabilized for 2 min at 25°C with 0.1% Triton X-100 in PBS. The cells were washed extensively in PBS and fixed again in 3% paraformaldehyde for 5 min. After blocking for 30 min with 3% BSA in PBS, the cells were incubated with anti-caveolin-1, anti-hCERK, or anti-TRPC-1 antibody at a dilution of 1:200 for 1 h at room temperature. Samples were washed and then labeled with the secondary FITC-conjugated rabbit anti-goat antibody (1:200 dilutions for 30 min at room temperature). To minimize nonspecific binding, 1% BSA was included in these solutions. Images from multiple confocal planes were collected. Opsonized red blood cells were incubated with 30 μg of FITC per 1 × 106 cells at room temperature for 4 h. The fluorescent conjugates were separated from unreacted fluorochromes by Sephadex G-25 column chromatography. Purified conjugates were dialyzed against PBS at pH 7.4 overnight at 4°C. To study dynamic changes in calcium during phagocytosis, microscopy was performed using a Nikon Eclipse TE2000-U inverted microscope. To detect time-lapse changes in fluorescence in the short wavelength emission region of Indo-1, a 355HT15 exciter, a 390 LP dichroic reflector, and a 405DF43 emission filter were used. An iXon model DV8 16-bit electron multiplying charge coupled device camera cooled to −100°C (Andor Technologies, South Windsor, CT) was used. To examine the high-speed dynamics of calcium signaling, a Perkin-Elmer FX-4400 flash lamp was used for excitation (25.Petyy H. Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology.Microsc. Res. Tech. 2007; 70: 687-709Crossref PubMed Scopus (81) Google Scholar). This flash lamp produces pulses that are 6 μs in duration and up to 1 J in energy, which is sufficient to saturate the fluorescent labels. The excitation light is passed through a water filter to attenuate ultraviolet and infrared components (∼109- to 1010-fold) relative to visible wavelengths. The light can be further filtered using KG-1 and interference filters before entering the epifluorescence microscope. A high-numerical aperture objective (1.45) was used to maximize sample brightness. To provide the greatest sensitivity to weak fluorescent signals, a Princeton Instruments PI-MAX2 intensified charge coupled device camera with a 5 MHz controller was used to collect images. The 1 × 1 K images were 2 × 2 binned to form the raw images shown. To reduce the amount of noise in the processed micrographs, previously described wavelet image filtration software was used before the application of a pseudocolor look-up table (26.Moss W. Haade S. Lyle J.M. Agard D.A. Sedat J.W. A novel 3D wavelet-based filter for visualizing features in noisy biological data.J. Microsc. 2005; 219: 43-49Crossref PubMed Scopus (16) Google Scholar). In a previous study (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), we used an in vitro cell culture system to evaluate the mechanism of C1P-potentiated phagocytosis. Stable transfectants of COS-1 cells expressing FcγRIIA or both FcγRIIA and hCERK were created. Those studies indicated that during FcγRIIA-mediated phagocytosis in cells overexpressing hCERK, CERK is activated and C1P is subsequently increased. Furthermore, the increase in C1P during phagocytosis led to a distinct gel-like ordered lipid phase within the cell membrane at sites of phagocytosis (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Furthermore, cell fractionation studies revealed that hCERK was enriched in plasma membrane caveolae fractions called lipid rafts, which are signaling domains enriched in sphingolipids. As sites of phagocytosis demonstrate physical and chemical properties of lipid rafts, the protein composition of lipid rafts with and without stimulation with EIgG was studied. Lipid rafts were obtained using cell fractionation methods, and their identity was confirmed using an anti-caveolin-1 monoclonal antibody (Fig. 1). As CERK and TRPC-1 have been associated with caveolin and/or lipid rafts (27.Lockwich T. Singh B.B. Liu X. Ambudkar I.S. Stabilization of cortical actin induces internalization of TRP3-associated caveolar Ca2+ signaling complex and loss of Ca2+ influx without disruption of TRP3-IP3R association.J. Biol. Chem. 2001; 27: 27-31Google Scholar, 28.Lockwich T. Liu X. Singh B. Jadlowiec J. Weiland S. Ambudkar I. Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains.J. Biol. Sci. 2000; 275: 11934-11942Google Scholar, 29.Brazer S. Singh B.B. Liu X. Swaim W. Ambudkar I.S. Caveolin-1 contributes to assembly of store operated Ca2+ influx channels by regulating plasma membrane localization of TRPC1.J. Biol. Chem. 2003; 278: 27208-27215Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), we speculate that TRPC-1 and its SOC-like activity may be relevant to calcium signaling in these transfectants. In the first series of experiments, we used an anti-TRPC-1 antibody to determine whether TRPC-1 channels are associated with lipid rafts in COS-1 transfectants. When unstimulated cells were studied, we were unable to detect TRPC-1 in lipid rafts (Fig. 1A). However, after challenging cells with EIgG, TRPC-1 was found in lipid rafts of all transfectants, although the strongest signal was found in hCERK transfected cells. TRPC-1 was also present in all nonraft fractions. Using an antibody for TRPC-1, we examined the kinetics of TRPC-1 localization to plasma membrane rafts during activation in cells transfected with hCERK (Fig. 1B). Cells were stimulated with opsonized erythrocytes (EIgG) for different periods of time and then subjected to fractionation. Western blotting revealed that TRPC-1 accumulated rapidly in raft fractions during activation but was barely detected in rafts during resting conditions. The primary antibody was either omitted or incubated with these blocking peptides as negative controls. Similarly, hCERK also appeared rapidly in lipid rafts after stimulation (22.Hinkovska-Galcheva V. Boxer L. Kindzelcki A. Hiraoka M. Abe A. Petty H.R. Shayman J.A. Ceramide-1-phosphate: a mediator of phagocytosis.J. Biol. Chem. 2005; 280: 26612-26621Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). To assess the cellular localization and appearance of hCERK, TRPC-1, and caveolin, confocal microscopy was performed. Unstimulated COS-1 transfectants were labeled with antibodies directed against hCERK, caveolin-1, TRPC-1, and HDL (Fig. 2A –D). As expected, cells were positively stained with these three reagents. When cells were incubated with EIgG for 30 min at 37°C, hCERK, caveolin-1, and TRPC-1 were redistributed at the cell surface (Fig. 2E–H). Cells labeled with HDL were used as negative controls; HDL did not redistribute after incubation with EIgG (Fig. 2H). Exposure to EIgG caused a more punctate appearance of these markers. To monitor the subcellular localization of hCERK during phagocytosis, RFP-tagged hCERK was used. When EIgG conjugated directly with FITC was incubated with COS-1 cells transiently transfected with RFP-hCERK for 30 min at 37°C, we observed that FITC-conjugated red blood cells and RFP-hCERK colocalized during phagocytosis (Fig. 3A). Cells transiently transfected with RFP-hCERK were incubated with EIgG for 30 min at 37°C. After lysis of uningested EIgG, cells were fixed and then labeled with anti-caveolin-1. Samples were washed and then labeled with the secondary FITC-conjugated rabbit anti-goat antibody. Optical images of CERK revealed the colocalization of hCERK and caveolin-1 (Fig. 3B). Coimmunoprecipitation or colocalization of caveolins with TRPC-1 and TRPC-3 have been reported previously (29.Brazer S. Singh B.B. Liu X. Swaim W. Ambudkar I.S. Caveolin-1 contributes to assembly of store operated Ca2+ influx channels by regulating plasma membrane localization of TRPC1.J. Biol. Chem. 2003; 278: 27208-27215Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 30.Lockwich T. Liu X. Singh B.B. Jadlowiec J. Wieland S. Ambudkar I.S. Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains.J. Biol. Chem. 2000; 275: 11934-11942Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). To measure phagocytosis-induced Ca2+ signaling, COS-1 cells stably transfected with hCERK were labeled with Indo-1 as described in Methods. Cells were placed on the microscope and EIgGs were added. The first image was acquired at time zero, and images were subsequently taken every 4 min for 3 h. Figure 4 illustrates the Indo-1 intensity over this period of time. To minimize photochemical damage, the light was shuttered between exposures. FcγRIIA transfected control cells and FcγRIIA/hCERK transfected cells were compared. As these kineti" @default.
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