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Recycling of the Membrane-anchored Chemokine, CX3CL1

2005; Elsevier BV; Volume: 280; Issue: 20 Linguagem: Inglês

10.1074/jbc.m413073200

1083-351X

Guangying Liu, Vathany Kulasingam, R. Todd Alexander, Nicolas Touret, Alan M. Fong, Dhavalkumar D. Patel, Lisa A. Robinson,

Glycosylation and Glycoproteins Research

CX3CL1 (fractalkine) plays an important role in inflammation by acting as both chemoattractant and as an adhesion molecule. As for other chemokines, expression of CX3CL1 is known to be regulated at the level of transcription and translation. The unique transmembrane structure of CX3CL1 raises the possibility of additional functional regulation by altering its abundance at the cell surface. This could be accomplished in principle by changes in traffic between subcellular compartments. To analyze this possibility we examined the subcellular distribution of CX3CL1 in human ECV-304 cells stably expressing untagged or green fluorescent protein-tagged forms of the chemokine. CX3CL1 was present in two distinct compartments, diffusely on the plasma membrane and in a punctate juxtanuclear compartment. The latter shared some features with, yet was distinct from the conventional endocytic pathway and may represent a specialized recycling subcompartment. Accordingly, surface CX3CL1 was found to be in dynamic equilibrium with the juxtanuclear vesicular compartment. Intracellular CX3CL1 co-localized with the SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins syntaxin-13 and VAMP-3. Cleavage of VAMP-3 by tetanus toxin or impairment of syntaxin-13 function by expression of a dominant-negative allele inhibited the ability of internalized CX3CL1 to traffic back to the plasma membrane. These data demonstrate the existence of a dynamic, SNARE-mediated recycling of CX3CL1 from the cell surface to and from an endomembrane storage compartment. The intracellular storage depot may serve as a source of the chemokine that could be rapidly mobilized by stimuli. CX3CL1 (fractalkine) plays an important role in inflammation by acting as both chemoattractant and as an adhesion molecule. As for other chemokines, expression of CX3CL1 is known to be regulated at the level of transcription and translation. The unique transmembrane structure of CX3CL1 raises the possibility of additional functional regulation by altering its abundance at the cell surface. This could be accomplished in principle by changes in traffic between subcellular compartments. To analyze this possibility we examined the subcellular distribution of CX3CL1 in human ECV-304 cells stably expressing untagged or green fluorescent protein-tagged forms of the chemokine. CX3CL1 was present in two distinct compartments, diffusely on the plasma membrane and in a punctate juxtanuclear compartment. The latter shared some features with, yet was distinct from the conventional endocytic pathway and may represent a specialized recycling subcompartment. Accordingly, surface CX3CL1 was found to be in dynamic equilibrium with the juxtanuclear vesicular compartment. Intracellular CX3CL1 co-localized with the SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins syntaxin-13 and VAMP-3. Cleavage of VAMP-3 by tetanus toxin or impairment of syntaxin-13 function by expression of a dominant-negative allele inhibited the ability of internalized CX3CL1 to traffic back to the plasma membrane. These data demonstrate the existence of a dynamic, SNARE-mediated recycling of CX3CL1 from the cell surface to and from an endomembrane storage compartment. The intracellular storage depot may serve as a source of the chemokine that could be rapidly mobilized by stimuli. Inflammation is marked by the migration of circulating leukocytes into sites of injury. The inflammatory process involves a series of coordinated interactions between leukocytes and endothelial or epithelial cells. Central to this sequence of events are chemokines, a family of low molecular weight proteins that function as attractants of leukocytes bearing the complementary receptors. When engagement of chemokine receptors occurs, leukocytes become activated and are induced to adhere firmly to the inflamed endothelium. These initial steps culminate in diapedesis of the leukocyte across the endothelium and migration into the injured tissue (1Olson T. Ley K. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002; 283: 7-28Crossref PubMed Scopus (567) Google Scholar). The local complement of chemokines elaborated by each organ is specific and varies with the type of inflammation present (1Olson T. Ley K. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002; 283: 7-28Crossref PubMed Scopus (567) Google Scholar). In addition, specific subsets of leukocytes bear distinct chemokine receptors. In this manner chemokines and their cognate receptors confer organ specificity to leukocyte migration and help to fine-tune the nature of the observed inflammatory response. One such chemokine, CX3CL1 (fractalkine) and its receptor, CX3CR1, have been shown to be of central importance in diverse inflammatory and infectious disease processes. Interrupting this pathway in vivo has a highly protective effect in animal models of renal inflammation (2Feng L. Chen S. Garcia G. Xia Y. Siani M. Botti P. Wilson C. Harrison J. Bacon K. Kidney Int. 1999; 56: 612-620Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), rejection of transplanted organs (3Haskell C. Hancock W. Salant D. Gao W. Csizmadia V. Peters W. J. Clin. Investig. 2001; 108: 679-688Crossref PubMed Scopus (146) Google Scholar, 4Robinson L. Nataraj C. Thomas D. Howell D. Griffiths R. Bautch V. Patel D. Feng L. Coffman T. J. Immunol. 2000; 165: 6067-6072Crossref PubMed Scopus (155) Google Scholar), and atherosclerosis (5Combadiere C. Potteaux S. Gao J. Esposito B. Casanova S. Lee E. Debre P. Tedgui A. Murphy P. Mallat Z. Circulation. 2003; 107: 1009-1016Crossref PubMed Scopus (410) Google Scholar, 6Lesnik P. Haskell C. Charo I. J. Clin. Investig. 2003; 111: 333-340Crossref PubMed Scopus (417) Google Scholar). Unlike most other chemokines, CX3CL1 exists in two forms; as a soluble chemotactic polypeptide and as a transmembrane chemokine/mucin hybrid protein. In its soluble form, CX3CL1 acts as a chemoattractant for leukocytes bearing its unique receptor, CX3CR1 (7Bazan J. Bacon K. Hardiman G. Wang W. Soo K. Rossi D. Greaves D. Zlotnick A. Schall T. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1711) Google Scholar, 8Imai T. Hieshima K. Haskell C. Baba M. Nagira M. Nishimura M. Kakizaki M. Takagi S. Nomiyama H. Schall T. Yoshie O. Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1171) Google Scholar). In the membrane-bound form, the mucin stalk allows the chemokine domain of CX3CL1 to be efficiently presented to circulating leukocytes (9Fong A. Robinson L. Steeber D. Tedder T. Yoshie O. Imai T. Patel D. J. Exp. Med. 1998; 188: 1413-1419Crossref PubMed Scopus (595) Google Scholar, 10Fong A. Erickson H. Zachariah J. Poon S. Schamberg N. Imai T. Patel D. J. Biol. Chem. 2000; 275: 3781-3786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Membrane-associated CX3CL1 has been shown to mediate multiple steps of the leukocyte adhesion cascade, including capture of circulating leukocytes, either alone or in concert with other adhesion molecules such as VCAM-1 (9Fong A. Robinson L. Steeber D. Tedder T. Yoshie O. Imai T. Patel D. J. Exp. Med. 1998; 188: 1413-1419Crossref PubMed Scopus (595) Google Scholar, 11Kerfoot S. Lord S. Bell R. Gill V. Robbins S. Kubes P. Eur. J. Immunol. 2003; 33: 729-739Crossref PubMed Scopus (37) Google Scholar). After the initial recruitment step, CX3CL1 interacts with CX3CR1 to further promote activation and firm the arrest of monocytes, NK cells, and CD8+ T lymphocytes (9Fong A. Robinson L. Steeber D. Tedder T. Yoshie O. Imai T. Patel D. J. Exp. Med. 1998; 188: 1413-1419Crossref PubMed Scopus (595) Google Scholar). Thus, CX3CL1 has dual roles as a chemoattractant and a cellular adhesion molecule. Like other chemokines, CX3CL1 biosynthesis is regulated at the transcriptional and translational levels (7Bazan J. Bacon K. Hardiman G. Wang W. Soo K. Rossi D. Greaves D. Zlotnick A. Schall T. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1711) Google Scholar, 9Fong A. Robinson L. Steeber D. Tedder T. Yoshie O. Imai T. Patel D. J. Exp. Med. 1998; 188: 1413-1419Crossref PubMed Scopus (595) Google Scholar, 12Harrison J. Jiang Y. Wees E. Salafranca M. Liang H. Feng L. Belardinelli L. J. Leukocyte Biol. 1999; 66: 937-944Crossref PubMed Scopus (78) Google Scholar, 13Ludwig A. Berkhout T. Moores K. Groot P. Chapman G. J. Immunol. 2002; 168: 604-612Crossref PubMed Scopus (129) Google Scholar, 14Muehlhoefer A. Saubermann L. Gu X. Luedtke-Heckenkamp K. Xavier R. Blumberg R. Podolsky D. MacDermott R. Reinecker H. J. Immunol. 2000; 164: 3368-3376Crossref PubMed Scopus (211) Google Scholar). However, because of its unique transmembrane disposition, CX3CL1 activity may be controlled by additional mechanisms. Indeed, it is conceivable that the surface exposure of the membrane-associated chemokine is regulated by traffic between subcellular compartments, as has been reported for a variety of membrane receptors and transporters (15Brown D. Am. J. Physiol. Renal Physiol. 2003; 284: 893-901Crossref PubMed Scopus (222) Google Scholar, 16Gentzsch M. Chang X. Cui L. Wu Y. Ozols V. Choudhury A. Pagano R. Riordan J. Mol. Biol. Cell. 2004; 15: 2684-2696Crossref PubMed Scopus (181) Google Scholar, 17Liu L. Omata W. Kojima I. Shibata H. J. Biol. Chem. 2003; 278: 30157-30169Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 18Sharma M. Pampinella F. Nemes C. Benharouga M. So J. Du K. Bache K. Papsin B. Zerangue N. Stenmark H. Lukacs G. J. Cell Biol. 2004; 164: 923-933Crossref PubMed Scopus (280) Google Scholar, 19Zeigerer A. Lampson M. Karylowski O. Sabatini D. Adesnik M. Ren M. McGraw T. Mol. Biol. Cell. 2002; 13: 2421-2435Crossref PubMed Scopus (161) Google Scholar). Remarkably, very little is known about the subcellular distribution and traffic of CX3CL1. The objective of the present study was, therefore, to characterize the subcellular distribution of CX3CL1, its ability to translocate between cellular compartments, and the molecular determinants of this traffic. We found that CX3CL1 actively cycles between the plasma membrane and an internal, vesicular pool and also characterized the soluble N-ethylmaleimide factor attachment protein receptors (SNARE) 1The abbreviations used are: SNARE, soluble N-ethylmaleimide factor attachment protein receptor; GFP, green fluorescent protein; EGFP, enhanced GFP; PAEC, primary porcine aortic endothelial cells; Ab, antibody; FRAP, fluorescence recovery after photobleaching; DN, dominant negative; TACE, tumor necrosis factor-converting enzyme. proteins involved in this process. The implications of these findings for the regulation of CX3CL1 function are discussed. Reagents and Antibodies—The following antibodies were used: goat polyclonal anti-CX3CL1 (R&D Systems, Inc., Minneapolis, MN), anti-CD63 (Caltag Laboratories, Burlingame, CA), anti-GM130 (Transduction Laboratories, Lexington, KY), Cy3-, peroxidase-, and Alexa 488-conjugated anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; Molecular Probes, Inc., Eugene, OR), and Fab fragment of fluorescein isothiocyanate-conjugated anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Rhodamine-conjugated transferrin and nigericin were obtained from Molecular Probes, and colchicine, brefeldin, and o-phenylenediamine dihydrochloride were from Sigma. DNA expression constructs encoding GFP-tagged VAMP-3, VAMP-2, and syntaxin-13 as well as dominant negative-syntaxin-13 and mammalian tetanus toxin were generously provided by Dr. William S. Trimble (The Hospital for Sick Children Research Institute, Toronto, Ontario) and Dr. R. Scheller (Stanford University, Stanford, CA) (20Advani R. Bae H.-R. Bock J. Chao D. Doung Y.-C. Prekeris R. Yoo J.-S. Scheller R. J. Biol. Chem. 1998; 273: 10317-10324Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 21Chao D. Hay J. Winnick S. Prekeris R. Klumperman J. Scheller R. J. Cell Biol. 1999; 144: 869-881Crossref PubMed Scopus (61) Google Scholar, 22Collins R. Schreiber A. Grinstein S. Trimble W. J. Immunol. 2002; 169: 3250-3256Crossref PubMed Scopus (75) Google Scholar, 23Huang X. Kang Y. Pasyk E. Sheu L. Wheeler M. Trimble W. Salapatek A. Gaisano H. Am. J. Physiol. Cell Physiol. 2001; 281: 740-750Crossref Google Scholar, 24Randhawa V. Bilan P. Khayat Z. Daneman N. Liu Z. Ramlal T. Volchuk A. Peng X. Coppola T. Regazzi R. Trimble W. Klip A. Mol. Biol. Cell. 2000; 11: 2403-2417Crossref PubMed Scopus (99) Google Scholar). ECV-CX3CL1 cells were transiently transfected with DNA encoding the above GFP-tagged proteins using FuGENE (Roche Diagnostics), and transfected cells were analyzed 24 h later. In separate experiments ECV-CX3CL1 cells were transfected with EGFP alone or in combination with dominant negative-syntaxin-13 or tetanus toxin at a ratio of 1:10. Cell Culture—ECV-304 cells were obtained from the American Type Culture Collection (Manassas, VA), and the generation of CX3CL1-expressing ECV-304 cells (ECV-CX3CL1) has been previously described (9Fong A. Robinson L. Steeber D. Tedder T. Yoshie O. Imai T. Patel D. J. Exp. Med. 1998; 188: 1413-1419Crossref PubMed Scopus (595) Google Scholar). A plasmid encoding CX3CL1-GFP hybrid molecules was generated by PCR and subsequent ligation of the DNA fragments into pEGFP-N2 (BD Biosciences Clontech). DNA encoding the extracellular and transmembrane portions of CX3CL1 was synthesized using primers 5′-CGGGTCGACTCAGCCATGGCTCCGATA and 5′-CTGAGGATCCCCACGGGCACCAGGAC and digested with Sal1 and BamH1. These fragments were ligated together into the pEGFP-N2 expression vector digested with SalI and BamH1. The nucleotide sequence of both strands of the new construct (pCX3CL1-GFP) was determined to verify its identity. ECV-304 cells were grown in Medium 199 (Invitrogen) containing 10% fetal calf serum and transfected by electroporation (Gene Pulser II, Bio-Rad) and selected in 500 μg/ml G418 (Invitrogen). CX3CL1 expression was determined by flow cytometry. For some experiments ECV-CX3CL1-GFP cells were grown on glass coverslips and incubated at 37 °C with either colchicine (10 μm) or brefeldin A (100 μm) for 30 min. COS-7 fibroblast cells were obtained from ATCC and transiently transfected with pCX3CL1-GFP (COS-CX3CL1-GFP) using FuGENE (Roche Diagnostics) according to the manufacturer's specifications. Primary porcine aortic endothelial cells (PAEC), a gift from Dr. Aleksander Hinek (The Hospital for Sick Children Research Institute, Toronto, Ontario), were grown in M199 containing 10% fetal calf serum and transfected with the pCX3CL1-GFP construct by electroporation (PAEC-CX3CL1-GFP). Immunofluorescence Staining—ECV-CX3CL1 cells were grown on glass coverslips, fixed using 4% paraformaldehyde, washed, and blocked with 5% donkey serum at room temperature for 1 h. Cells were incubated with anti-CX3CL1 antibody (Ab) (2.5 μg/ml) at room temperature for 1 h followed by Alexa 488-conjugated anti-goat IgG. After washing again, the cells were permeabilized using 0.1% Triton. Cells were incubated again with anti-CX3CL1 Ab, this time followed by Cy3-conjugated anti-goat IgG. In other experiments ECV-CX3CL1-GFP or SNARE-transfected ECV-CX3CL1 cells were grown on glass coverslips, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton. Cells were washed and labeled with anti-CX3CL1 Ab (1 μg/ml) followed by Cy3-conjugated anti-goat IgG. Immunofluorescence was examined using a Leica DMIRE2 microscope and OpenLab software (Improvision Inc., Lexington, MA). Co-localization was determined using OpenLab 4.0.2 co-localization software. The degree of co-localization was expressed using the Pearson's correlation coefficient (r), as previously described (25Manders M. Verbeek F. Aten J. J. Microsc. (Oxf.). 1993; 169: 375-382Crossref PubMed Scopus (1550) Google Scholar). In some experiments cells were examined by confocal microscopy using a Zeiss LSM 510 laser scanning confocal microscope with a 100× oil immersion objective and pinhole size 1.00 airy units (26Terebiznik M. Vieira O. Marcus S. Slade A. Yip C. Trimble W. Meyer T. Finlay B. Grinstein S. Nat. Cell Biol. 2002; 4: 766-773Crossref PubMed Scopus (231) Google Scholar). For some experiments confocal images were deconvolved by importing the LSM file into Volocity 3.0.2 and applying the appropriate point spread function to 95% confidence limit. GFP was examined using conventional laser excitation lines and filter sets. Quantitation of CX3CL1 Using Peroxidase-coupled Ab Labeling—To quantify the fraction of CX3CL1 at the cell surface, cells were grown to confluence in 24-well plates and fixed, and exposed epitopes were saturated by incubating with anti-CX3CL1 Ab (2.5 μg/ml) at 4 °C for 1 h. To quantify the total amount of CX3CL1 within the cell, cells were fixed and permeabilized before incubation with anti-CX3CL1 Ab. Cells were incubated with a blocking solution of 5% donkey serum followed by secondary peroxidase-coupled anti-goat IgG at room temperature for 1 h. Cells were washed, and the reaction was developed using the peroxidase substrate, o-phenylenediamine dihydrochloride. The optical density was read by spectrophotometry at 492 nm. Endocytosis Assays—ECV-CX3CL1, ECV-CX3CL1-GFP, COS-CX3CL1-GFP, or primary PAEC-CX3CL1-GFP cells were grown on glass coverslips and incubated with anti-CX3CL1 Ab at 37 or 4 °C for 1 h. Cells were washed, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton. Cells were incubated with Cy3-conjugated anti-goat IgG, washed, and mounted on glass slides using DAKO fluorescent mounting medium (DAKO Corp., Carpinteria, CA). In other experiments, after a 1-h serum-free period, ECV-CX3CL1-GFP cells were incubated with transferrin-rhodamine (30 μg/ml) at 37 °C for 1 h before fixing and mounting. In some experiments, after cells were incubated with anti-CX3CL1 Ab for 1 h, membrane-associated Ab was removed by acid wash (0.15 m NaCl, 50 mm glycine, 0.1% bovine serum albumin, pH 2.5) on ice using published methods (27van Kerkhof P. Sachse M. Klumperman J. Strous G. J. Biol. Chem. 2001; 276: 3778-3784Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cells were allowed to recover for various time periods, then washed, fixed, and incubated with Cy3-conjugated secondary Ab. Cell surface immunofluorescence intensity was measured using MetaMorph imaging software (Universal Imaging Corp., Westchester, PA). Fluorescence Recovery After Photobleaching (FRAP)—Analysis of FRAP was performed as previously described (28Lippincott-Schwartz J. Presley J. Zaal K. Hirschberg K. Miller C. Ellenberg J. Methods Cell Biol. 1999; 58: 261-281Crossref PubMed Google Scholar, 29Reits E. Neefjes J. Nat. Cell Biol. 2001; 3: 145-147Crossref PubMed Scopus (515) Google Scholar). Briefly, ECV-CX3CL1-GFP or PAEC-CX3CL1-GFP cells were incubated in medium RPMI containing 10% fetal calf serum and 25 mm HEPES (Invitrogen) and maintained at 37 °C. Live cells were analyzed by confocal microscopy as described above using LP505 filter. A 30-milliwatt LASOS argon laser, set to 25% intensity, was used to irreversibly photobleach a region encompassing the entire CX3CL1-associated juxtanuclear compartment (≈5-μm diameter). For photobleaching, maximal pinhole size and 100% of set laser intensity were used, whereas 22% intensity was used for image acquisition. The dwell time per pixel was 1.60 μs. The recovery of fluorescence was measured serially over time at 45-s intervals. Control, non-bleached areas of the plasma membrane of the same cell were also serially monitored to quantify the amount of bleaching caused by repeated image acquisition. At the end of each experiment, cells were imaged to ensure that no significant structural or positional changes had occurred during the course of the experiment. Measurement of pH in the Intracellular CX3CL1 Compartment— ECV-CX3CL1 cells were grown on coverslips and incubated for 2 h with anti-CX3CL1 Ab at 37 °C. Cells were washed then incubated with fluorescein isothiocyanate-conjugated Fab-fragment of anti-goat IgG (20 μg/ml) for 2 h at 37 °C. Coverslips were placed in a Leiden chamber and mounted on the stage of a Leica IRE microscope for ratio determination of emitted fluorescence at 2 excitation wavelengths, 440 and 490 nm, as previously described (30Touret N. Furuya W. Forbes J. Gros P. Grinstein S. J. Biol. Chem. 2003; 278: 25548-25557Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Excitation filters and image acquisition were controlled using a Lambda 10 filter-wheel controller (Sutter Instrument Company, Novato, CA) driven by the Metafluor software (Universal Imaging, Westchester, PA). A Dell Optiplex DGX 590 computer was interfaced with a Photometrics CCD camera via a 12-bit GPIB/IIA board (National Instruments, Foster City, CA). To generate standard calibration curves of fluorescence ratio versus pH, cells were perfused with a series of K+-rich solutions of known pH, each containing 5 μg/ml nigericin. The purpose of the initial studies was to examine the subcellular distribution and traffic of CX3CL1. Endogenous levels of CX3CL1 in primary cells are difficult to detect using currently available Ab. In light of this, we adopted another approach, namely expressing full-length CX3CL1 or GFP-tagged CX3CL1 in ECV-304 cells, a cell line with epithelial and endothelial characteristics (31Su T. Cariappa R. Stanley K. FEBS Lett. 1999; 453: 391-394Crossref PubMed Scopus (16) Google Scholar, 32Haller C. Kiessling F. Kubler W. Eur. J. Cell Biol. 1997; 75: 353-361Crossref Scopus (13) Google Scholar). In a previous study cell surface levels of CX3CL1 on CX3CL1-expressing ECV-304 cells (ECV-CX3CL1) have been shown to approximate those of primary human vascular endothelial cells, minimizing the risk of mistargeting due to overexpression (9Fong A. Robinson L. Steeber D. Tedder T. Yoshie O. Imai T. Patel D. J. Exp. Med. 1998; 188: 1413-1419Crossref PubMed Scopus (595) Google Scholar). CX3CL1 Is Expressed in Dual Locations within the Cell— ECV-CX3CL1 cells were fixed and labeled with an Ab directed against the extracellular domain of CX3CL1. As expected, the Ab labeled only the surface of intact cells, as determined using both conventional epifluorescence (Fig. 1A) and confocal microscopy (Fig. 1C). To assess whether CX3CL1 is also present intracellularly, the same cells were then permeabilized and labeled again with anti-CX3CL1 Ab followed by incubation with a different secondary Ab. Permeabilization revealed a sizable intracellular pool of CX3CL1 with juxtanuclear location (epifluorescence microscopy, Fig. 1B; confocal microscopy, Fig. 1D). The relative magnitude of the surface and endomembrane compartments was quantified using a spectroscopic immunoperoxidase labeling method (see "Experimental Procedures"). The plasmalemmal component of CX3CL1 was found to account for 57 ± 6% of the total cellular pool of CX3CL1. The existence of CX3CL1 in at least two distinct cellular compartments was verified in cells expressing GFP-tagged CX3CL1 (ECV-CX3CL1-GFP). In this construct GFP is attached to the cytosolic tail of the transmembrane chemokine. The fact that the tagged construct (Fig. 1E) was recognized by the antibody directed to the extracellular domain anti-CX3CL1 Ab (Fig. 1F) confirms that the expressed protein traverses the membrane. The presence of GFP and antibody fluorescence at the surface and in the juxtanuclear endomembrane vesicles implies that the full-length transmembrane protein is found in both locations (Fig. 1, G and H). The similarity of this distribution to that of the untagged protein indicates that linkage to GFP did not visibly alter the cellular targeting of CX3CL1. We also used confocal microscopy to verify that GFP-tagged CX3CL1 is indeed expressed in dual locations. As shown in Fig. 1I, cross-sectional analysis confirmed that CX3CL1-GFP is expressed not only in the plasma membrane but also within a juxtanuclear compartment. The endomembrane compartment appears to be maintained in its juxtanuclear location by microtubules, since treatment with colchicine caused the intracellular CX3CL1-GFP to disperse (Fig. 1J). Interestingly, treatment with colchicine also caused an appreciable decrease in plasma membrane CX3CL1-GFP signal. These data raise the possibility that cell surface CX3CL1 is in dynamic exchange with the pool of intracellular CX3CL1 and that this exchange relies on intact microtubules. Identification of the Juxtanuclear CX3CL1-associated Compartment—The identity and function of the intracellular CX3CL1 compartment were investigated next. We initially assessed whether CX3CL1 was located in lysosomes, recycling endosomes, or the Golgi apparatus, all of which are maintained in a juxtanuclear position by retrograde transport along microtubules. ECV-CX3CL1-GFP cells were labeled with a late endosomal/lysosomal marker, anti-CD63 Ab (Fig. 2A). As expected, some degree of co-localization was detected in the juxtanuclear region. To obviate the possibility that overlap was fortuitous, due to the presence of multiple organelles in the same general region, cells were treated with colchicine before staining (Fig. 2B). Under these conditions no significant degree of association between CD63 and the CX3CL1-containing vesicles was observed (Fig. 2, B and C; Pearson's colocalization coefficient r = 0.317 ± 0.041; p > 0.1), demonstrating that the latter compartment does not correspond to late endosomes/lysosomes. To investigate whether the intracellular CX3CL1 compartment is associated with recycling endosomes, ECV-CX3CL1-GFP cells were incubated with rhodamine-conjugated transferrin for 1 h at 37 °C with (Fig. 2E) or without (Fig. 2D) colchicine treatment. As shown in Fig. 2D, transferrin was indeed internalized to a juxtanuclear location resembling that of CX3CL1. However, when the organelles were dispersed by microtubule disruption, only partial overlap was observed (Fig. 2, E and F), yielding Pearson's correlation coefficient slightly greater than 0.5, which is statistically significant (r = 0.515 ± 0.071; p < 0.05) and indicative of partial colocalization. Thus, the intracellular CX3CL1-containing compartment overlaps with but is not entirely identical to the transferrin receptor-containing recycling endosomes. In similar experiments CX3CL1-containing vesicles were found to be distinct from the Golgi stacks, identified using anti-GM130 Ab (Fig. 2, G–I). A dissociation between the two compartments was also noted when the cells were pretreated with brefeldin (Fig. 2H; r = 0.322 ± 0.068; p > 0.1), which is known to disperse the Golgi cisternae while compacting both recycling endosomes and the trans-Golgi network. A sizable fraction of CX3CL1 remained compacted near the nucleus, consistent with location in recycling endosomes and/or trans-Golgi network. Collectively, these data demonstrate that CX3CL1 is expressed within a specialized juxtanuclear compartment that is distinct from late endosomes/lysosomes and Golgi cisternae but which partially co-localizes with transferrin receptor-associated recycling endosomes. SNARE Proteins in the Intracellular CX3CL1 Compartment—To further characterize the intracellular CX3CL1 compartment and to investigate its possible relationship with the plasmalemmal pool, we proceeded to study molecular entities that might mediate fusion of CX3CL1-bearing vesicles with the plasma membrane. The SNARE family of proteins is thought to mediate the docking and fusion of vesicles with their target membranes, thereby facilitating the delivery of cargo between compartments. In an attempt to identify SNARE proteins, ECV-CX3CL1 cells were transiently transfected with DNA constructs encoding VAMP-2-GFP, VAMP-3-GFP, or syntaxin-13-GFP, which are known components of the endocytic and recycling pathway. Cells were fixed, permeabilized, and immunostained with anti-CX3CL1 Ab to determine possible co-localization. As before, fortuitous overlap was minimized by pretreating the cells with colchicine. As illustrated in Fig. 3, the intracellular CX3CL1 compartment partially co-localized with syntaxin-13 (Fig. 3, A and B; r = 0.632 ± 0.032; p < 0.01) as well as with VAMP-3 (Fig. 3, C and D; r = 0.793 ± 0.009; p < 0.01). No significant co-localization with VAMP-2 was observed (Fig. 3, E and F; r = 0.395 ± 0.033; p > 0.1). These data support the notion that intracellular CX3CL1 represents a subcompartment of recycling endosomes and suggest that recycling may occur by SNARE-mediated fusion of endosomal and plasmalemmal membranes. Plasma Membrane CX3CL1 Undergoes Endocytosis in Diverse Cell Types—To ensure that the dual localization of CX3CL1 is not an idiosyncratic feature of ECV-304 cells, we transfected the tagged CX3CL1 in other cell types known to express the chemokine, namely primary porcine aortic endothelial cells and fibroblastic cells (COS-7). In both of these cell types, CX3CL1 was expressed with a distribution similar to that seen in ECV cells, namely, on the plasma membrane as well as in an intracellular location (see Fig. 4). The results from the preceding experiments suggested that intracellular CX3CL1 may represent a recycling endosomal pool. To verify this prediction, we incubated ECV-CX3CL1-GFP, COS-CX3CL1-GFP, and PAEC-CX3CL1-GFP cells with an Ab directed to the exofacial domain of CX3CL1 for 2 h at 37 °C. The cells were washed, fixed, and permeabilized before incubating with secondary Ab. In all three cell types tagged CX3CL1 was seen to accumulate in the juxtanuclear location in a time-dependent fashion (Fig. 4, C, D, G, H, K, and L), providing evidence of dynamic exchange between compartments. Labeling of the juxtanuclear pool was prevented when the experiment was performed at 4 °C. In this instance only the superficial CX3CL1 was labeled (Fig. 4, A, B, E, F, I, and J), implying that endocytosis is required to deliver the antibody to the intracellular pool. Recovery of Fluorescence afte