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• W2140172555 abstract "We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolateral endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin. This protein contains two PDZ domains (PSD-95, Dlg, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic domains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localized to both plasma membrane domains and to an intracellular vesicular compartment. Syntenin positive vesicles colocalized with internalized transferrin in the perinuclear region. In addition, syntenin colocalized in the apical supranuclear region with Rab5 and Rab11; the latter is a marker for the apical recycling endosomes in MDCK cells. We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolateral endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin. This protein contains two PDZ domains (PSD-95, Dlg, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic domains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localized to both plasma membrane domains and to an intracellular vesicular compartment. Syntenin positive vesicles colocalized with internalized transferrin in the perinuclear region. In addition, syntenin colocalized in the apical supranuclear region with Rab5 and Rab11; the latter is a marker for the apical recycling endosomes in MDCK cells. transferrin receptor 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene-sulfonate fluorescence-activated cell sorter fluorescence-activated organelle sorting Madin-Darby canine kidney baby hamster kidney horseradish peroxidase two-dimensional 2D gel electrophoresis PSD-95, Dlg, and ZO-1 postnuclear supernatant phosphate-buffered saline 1,4-piperazinediethanesulfonic acid Endocytosis includes “cellular drinking” (the unselective uptake of fluids) and the selective uptake of nutrients, hormones, and growth factors by receptors. Endocytosis occurs from both apical and basolateral surfaces in epthelial cells (1Simons K. Fuller S.E. Annu. Rev. Physiol. 1985; 1: 243-288Google Scholar, 2Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (551) Google Scholar). Clathrin-coated vesicles derived from the apical or basolateral plasma membrane lose their coat and fuse with early endosomes. Endocytosed cell surface receptors and other membrane proteins, as well as fluid phase markers, therefore appear first in early endosomes. Early endosomes comprise at least two functionally distinct compartments (3Ghosh R.N. Gelman D.L. Maxfield F.R. J. Cell Sci. 1994; 107: 2177-2189Crossref PubMed Google Scholar). Internalized receptors and ligands first enter the peripheral sorting endosomes. In these compartments, some membrane proteins are sorted away from those proteins destined for degradation. In particular, some receptor-ligand complexes and the remaining solutes are transported to late endosomes and lysosomes to be degraded, whereas the transferrin receptor (Tf-R)1 recycles back to the basolateral cell surface (4Ghosh R.N. Maxfield F.R. J. Cell Biol. 1995; 128: 549-561Crossref PubMed Scopus (95) Google Scholar). Recycling back to the plasma membrane can occur directly from the so-called sorting endosome (fast cycle) or indirectly via the recycling endosome (5Hopkins C.R. Gibson A. Shipman M. Strickland D.L. Trowbridge I.S. J. Cell Biol. 1994; 125: 1265-1274Crossref PubMed Scopus (217) Google Scholar). Work on endocytic compartments in epithelial MDCK cells has demonstrated that apical early endosomes are distributed below the apical microvilli above the ring of tight junctions and that basolateral early endosomes are found alongside the lateral as well as the basal membranes (6Parton R.G. Prydz K. Bomsel M. Simons K. Griffiths G. J. Cell Biol. 1989; 109: 3259-3272Crossref PubMed Scopus (151) Google Scholar, 7Bomsel M. Prydz K. Parton R.G. Gruenberg J. Simons K. J. Cell Biol. 1989; 109: 3243-3258Crossref PubMed Scopus (186) Google Scholar). Both compartments form three-dimensional networks of tubular cisternal and vesicular structures (2Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (551) Google Scholar). In vitro fusion assays with endosomes derived from the MDCK cell line have confirmed that there is a specific set of early endosomes associated with each plasma membrane domain. These endosomes show homotypic fusion but no heterotypic fusion activities with each other (7Bomsel M. Prydz K. Parton R.G. Gruenberg J. Simons K. J. Cell Biol. 1989; 109: 3243-3258Crossref PubMed Scopus (186) Google Scholar, 8Bomsel M. Parton R. Kuznetsov S.A. Schroer T.A. Gruenberg J. Cell. 1990; 62: 719-731Abstract Full Text PDF PubMed Scopus (228) Google Scholar). Recent evidence suggests that in epithelia, apical and basolateral endocytic pathways converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. Up to now, marker proteins for early endosomes, such as Rab5, were found on both apical and basolateral early endosomes (9Harder T. Gerke V. J. Cell Biol. 1993; 123: 1119-1132Crossref PubMed Scopus (149) Google Scholar). Other proteins, such as annexin II, show, in addition to miscellaneous subcellular localizations, an association with peripheral transferrin-labeled endosomes in epithelial cells (10Bucci C. Wandinger-Ness A. Lütcke A. Chiariello M. Bruni C.B. Zerial M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5061-5065Crossref PubMed Scopus (111) Google Scholar), whereas Rab25 and Rab11a were found to be associated with the apical recycling system of polarized MDCK cells (11Casanova J.E. Wang X. Kumar R. Bhartur S.G. Navarre J. Woodrum J.E. Altschuler Y. Ray G.S. Goldenring J.R. Mol. Biol. Cell. 1999; 10: 47-61Crossref PubMed Scopus (342) Google Scholar). In addition, it was shown that recycling of endocytosed transferrin and transferrin receptor through this pericentriolar endosomal compartment requires hydrolysis of GTP on Rab11 (12Ren M. Xu G. Zeng J. De Lemos-Chiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6187-6192Crossref PubMed Scopus (390) Google Scholar, 13Ullrich O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1075) Google Scholar). The complexity of endocytic recycling and the limited number of marker proteins identified to date suggests that there are many yet unidentified molecules involved in regulating endocytic trafficking in polarized cells. One approach to gain more knowledge about the apical and basolateral specifities for endocytosis is to analyze the overall molecular composition and organization of apical and basolateral early endocytic vesicles. For this purpose, the lipophilic fluorescent probe trimethylamino-diphenylhexatriene (TMA-DPH) that intercalates in membrane structures has been used for studying endocytosis (14Illinger D. Poindron P. Fonteneau P. Modollel M. Kuhry J.-G. Biochim. Biophys. Acta. 1990; 1030: 73-81Crossref PubMed Scopus (39) Google Scholar, 15Cupers P. Veithen A. Kiss A. Baudhuin P. Courtoy P.J. J. Cell Biol. 1994; 127: 725-735Crossref PubMed Scopus (100) Google Scholar, 16Illinger D. Kuhry J.G. J. Cell Biol. 1994; 125: 783-794Crossref PubMed Scopus (31) Google Scholar, 17Masumoto N. Tasaka K. Mizuki J. Tahara M. Miyake A. Tanizawa O. Biochem. Biophys. Res. Commun. 1993; 197: 207-213Crossref PubMed Scopus (18) Google Scholar, 18Illinger D. Duportail G. Mely Y. Poirelmorales N. Gerard D. Kuhry J.G. Biochim. Biophys. Acta. 1995; 1239: 58-66Crossref PubMed Scopus (61) Google Scholar, 19Wang Z.X. Moran M.F. Science. 1996; 272: 1935-1939Crossref PubMed Scopus (146) Google Scholar, 20Illinger D. Poindron P. Kuhry J.G. Biol. Cell. 1991; 73: 131-138Crossref PubMed Scopus (27) Google Scholar). TMA-DPH is a cationic analogue of DPH that has also proven useful as a probe for studying bulk-phase endocytosis (14Illinger D. Poindron P. Fonteneau P. Modollel M. Kuhry J.-G. Biochim. Biophys. Acta. 1990; 1030: 73-81Crossref PubMed Scopus (39) Google Scholar, 15Cupers P. Veithen A. Kiss A. Baudhuin P. Courtoy P.J. J. Cell Biol. 1994; 127: 725-735Crossref PubMed Scopus (100) Google Scholar, 16Illinger D. Kuhry J.G. J. Cell Biol. 1994; 125: 783-794Crossref PubMed Scopus (31) Google Scholar, 17Masumoto N. Tasaka K. Mizuki J. Tahara M. Miyake A. Tanizawa O. Biochem. Biophys. Res. Commun. 1993; 197: 207-213Crossref PubMed Scopus (18) Google Scholar, 20Illinger D. Poindron P. Kuhry J.G. Biol. Cell. 1991; 73: 131-138Crossref PubMed Scopus (27) Google Scholar, 21Illinger D. Poindron P. Kuhry J.G. Biol. Cell. 1991; 71: 293-296Crossref PubMed Scopus (8) Google Scholar, 22Böck G. Steinlein P. Haberfellner M. Gruenberg J. Huber L.A. Celis J.E. Cell Biology: A Laboratory Handbook. 2. Academic Press, San Diego, CA1998: 63-69Google Scholar). In addition, the dye does not diffuse through tight junctions, and for this reason, it has been used successfully in anisotropy experiments to estimate in situ the lipid order of the plasma membrane of polarized MDCK II cells (23Le Grimellec C. Friedlander G. Giocondi M.C. Am. J. Physiol. 1988; 255: F22-F32PubMed Google Scholar). Here we combined the use of TMA-DPH with conventional density gradient centrifugation (24Gruenberg J. Gorvel J.-P. Magee A.I. Wileman T. Protein Targeting: A Practical Approach. Oxford University Press, Oxford1992: 187-216Google Scholar) with high speed organelle sorting in a flow cytometer (fluorescence-activated organelle sorting (FAOS)) to analyze the apical and basolateral endosomal proteins in MDCK cells (22Böck G. Steinlein P. Haberfellner M. Gruenberg J. Huber L.A. Celis J.E. Cell Biology: A Laboratory Handbook. 2. Academic Press, San Diego, CA1998: 63-69Google Scholar, 25Böck I. Steinlein P. Huber L.A. Tr. Cell Biol. 1997; 7: 499-503Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Using high resolution 2D gel electrophoresis (2DE) and protein microsequencing, we identified the adaptor protein syntenin in apical endocytic vesicles. Syntenin contains a tandem repeat of two PDZ domains. PDZ domains mediate protein-protein interactions and typically bind to short amino acid motifs at the carboxyl terminus of interacting proteins, including certain ion channels and transmembrane receptors. As such, syntenin reacts with the FYA carboxyl-terminal amino acid sequence of syndecans (26Grootjans J.J. Zimmermann P. Reekmans G. Smets A. Degeest G. Durr J. David G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13683-13688Crossref PubMed Scopus (340) Google Scholar) and with the carboxyl terminus of B-type ephrins (27Lin D. Gish G.D. Songyang Z. Pawson T. J. Biol. Chem. 1999; 274: 3726-3733Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). By combining a novel strategy for subcellular fractionation with biochemical analysis and transient overexpression of syntenin, we could for the first time demonstrate an association of this recently identified adaptor molecule with the apical recycling compartment of MDCK cells. MDCK I and II cells were cultured and seeded on filters (Costar; pore size, 0.4 μm) as described previously (10Bucci C. Wandinger-Ness A. Lütcke A. Chiariello M. Bruni C.B. Zerial M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5061-5065Crossref PubMed Scopus (111) Google Scholar, 28Huber L.A. Pimplikar S. Parton R.G. Virta H. Zerial M. Simons K. J. Cell Biol. 1993; 123: 35-45Crossref PubMed Scopus (379) Google Scholar). Before each experiment, the confluency of the monolayer was confirmed by measuring transepithelial electrical resistance. In vivo staining with TMA-DPH was carried out with a baby hamster kidney cell line (BHK-21) grown on coverslips (29Gruenberg J. Griffiths G. Howell K. J. Cell Biol. 1989; 108: 1301-1317Crossref PubMed Scopus (454) Google Scholar). Monoclonal anti-human Tf-R antibodies were obtained from Roche Molecular Biochemicals, and the monoclonal antibodies against E-cadherin and annexin II were from Transduction Laboratories. Polyclonal antibodies against Rab4, Rab5, and Rab7 were raised against synthetic peptides derived from the carboxyl terminus and prepared in our laboratory as outlined (30Zerial M. Parton R. Chavrier P. Frank R. Methods Enzymol. 1992; 219: 398-407Crossref PubMed Scopus (33) Google Scholar). Polyclonal antibodies against Rab11 (31Urbé S. Huber L.A. Zerial M. Tooze S.A. Parton R.G. FEBS Lett. 1993; 334: 175-182Crossref PubMed Scopus (186) Google Scholar) and monoclonal antibodies against Rab5 (9Harder T. Gerke V. J. Cell Biol. 1993; 123: 1119-1132Crossref PubMed Scopus (149) Google Scholar) were used as described previously. Myc-tagged syntenin was detected with an anti 9E10 monoclonal antibody or a polyclonal affinity-purified antibody generated in our laboratory against a Myc peptide (32Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2165) Google Scholar). All secondary antibodies were obtained from Dianova. Lyophilized TMA-DPH was from Molecular Probes and reconstituted as a 20 mmstock solution in Me2SO (Fluka). BHK-21 cells were washed 2–3 times with prechilled PBS2+(PBS, 1 mm CaCl2, 1 mmMgCl2) at 4 °C. Drops of TMA-DPH diluted to 100 μm in NaOAc transport buffer (250 mm HEPES, pH 7.4, 1.15 m NaOAc, 25 mm MgCl2) were added on top of the monolayer. After incubation for 3–4 min on an ice-cold metal plate, the dye was removed by briefly washing with prechilled PBS2+. Internalization was carried out in an incubator at 37 °C for various time points. Double stainings with acridine orange were peformed as described (33Matteoni R. Kreis T.E. J. Cell Biol. 1987; 105: 1253-1265Crossref PubMed Scopus (372) Google Scholar). Filter-grown MDCK II cells were washed twice in ice-cold PBS2+ for 5 min at 4 °C and once for 2 min in prechilled NaOAc buffer. TMA-DPH diluted to 50 μm in NaOAc transport buffer was added to the apical or basolateral cell surface and the cells incubating for 2 min at 4 °C. Immediately after TMA-DPH binding, the filters were incubated at 37 °C in a water bath for 2 min with prewarmed medium. Following internalization, MDCK II cells were washed with ice-cold PBS and scraped, and a postnuclear supernatant (PNS) was prepared as described previously (22Böck G. Steinlein P. Haberfellner M. Gruenberg J. Huber L.A. Celis J.E. Cell Biology: A Laboratory Handbook. 2. Academic Press, San Diego, CA1998: 63-69Google Scholar, 24Gruenberg J. Gorvel J.-P. Magee A.I. Wileman T. Protein Targeting: A Practical Approach. Oxford University Press, Oxford1992: 187-216Google Scholar, 34Fialka I. Pasquali C. Lottspeich F. Ahorn H. Huber L.A. Electrophoresis. 1997; 18: 2582-2590Crossref PubMed Scopus (70) Google Scholar). The PNS from one and a half filter inserts was loaded on top of one SW60 tube (Beckman) containing a continuous sucrose gradient (10–40% sucrose). After centrifugation (100,000 × g at 4 °C for 16 h), 20 fractions were collected with an Auto Densi-Flow fraction collector (Labconco Corp.) and analyzed. To generate amounts suitable for preparative FACS sorting, up to 10 filter inserts were used. Horseradish peroxidase (HRP) (5–10 mg/ml; Sigma) was internalized from fetal calf serum-free medium into early endosomes and further chased into late endosomes as described previously (24Gruenberg J. Gorvel J.-P. Magee A.I. Wileman T. Protein Targeting: A Practical Approach. Oxford University Press, Oxford1992: 187-216Google Scholar). HRP activity was assayed using o-dianisidine and peroxide as substrates as described (24Gruenberg J. Gorvel J.-P. Magee A.I. Wileman T. Protein Targeting: A Practical Approach. Oxford University Press, Oxford1992: 187-216Google Scholar). Specific activity of the fractions is given as ng of HRP/mg of protein. Protein concentrations were measured using the Micro BCA protein assay reagent kit (Pierce) following the manufacturer's specifications. All protein precipitations were carried out with the CHCl3/methanol method (35Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3142) Google Scholar). Precipitated proteins were dissolved in SDS-sample buffer and separated by 10% SDS-polyacrylamide gel electrophoresis, followed by semidry electrophoretic transfer onto nitrocellulose membranes. Proteins were detected using specific antibodies as outlined previously (34Fialka I. Pasquali C. Lottspeich F. Ahorn H. Huber L.A. Electrophoresis. 1997; 18: 2582-2590Crossref PubMed Scopus (70) Google Scholar). TMA-DPH was titrated by flow cytometry using labeled PNS to avoid complex quenching effects from the Förster-type fluorescence resonance autotransfer (18Illinger D. Duportail G. Mely Y. Poirelmorales N. Gerard D. Kuhry J.G. Biochim. Biophys. Acta. 1995; 1239: 58-66Crossref PubMed Scopus (61) Google Scholar). The best compromise between the highest amount of sortable material and the minimum of false positive structures was obtained at 50 μm TMA-DPH (data not shown). Fractions enriched in TMA-DPH-labeled endosomes were diluted 1:100 in PBS and analyzed using a FACS Vantage Turbo Sort Option (Becton Dickinson, San Jose, CA) equipped with an Argon laser tuned to 40 MW multiline UV and 210 mW 488 nm output using a 4xx/44-nm bandpass filter and a 50-μm nozzle. System threshold was set on forward light scatter, and photomultiplier tube voltage was adjusted using unlabeled PNS. Fractions containing more than 10% of labeled vesicles were gated for preparative sorts. The concentration of these fractions was adjusted to give event rates of approximately 15,000 events/s at lowest possible sample differential pressure at a sheath pressure of 45 psi. Droplet formation frequency was adjusted to approximately 70 kHz. The large volumes (typically more than 20 ml) of sorted material was concentrated by several steps of sec-butanol extraction and finally CHCl3/methanol precipitated (35Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3142) Google Scholar). 2DE was performed following in-gel sample reswelling as described (34Fialka I. Pasquali C. Lottspeich F. Ahorn H. Huber L.A. Electrophoresis. 1997; 18: 2582-2590Crossref PubMed Scopus (70) Google Scholar, 36Pasquali C. Fialka I. Huber L.A. Electrophoresis. 1997; 18: 2573-2581Crossref PubMed Scopus (120) Google Scholar). The gels were stained with ammoniacal silver, scanned, and analyzed as described (34Fialka I. Pasquali C. Lottspeich F. Ahorn H. Huber L.A. Electrophoresis. 1997; 18: 2582-2590Crossref PubMed Scopus (70) Google Scholar, 36Pasquali C. Fialka I. Huber L.A. Electrophoresis. 1997; 18: 2573-2581Crossref PubMed Scopus (120) Google Scholar, 37Appel R.D. Palagi P.M. Walther D. Vargas J.R. Sanchez J.C. Ravier F. Pasquali C. Hochstrasser D.F. Electrophoresis. 1997; 18: 2724-2734Crossref PubMed Scopus (138) Google Scholar). Preparative amounts of total membrane fractions from MDCK II cells (34Fialka I. Pasquali C. Lottspeich F. Ahorn H. Huber L.A. Electrophoresis. 1997; 18: 2582-2590Crossref PubMed Scopus (70) Google Scholar,36Pasquali C. Fialka I. Huber L.A. Electrophoresis. 1997; 18: 2573-2581Crossref PubMed Scopus (120) Google Scholar) were precipitated, dissolved, and separated as outlined for analytical gels and stained with Coomassie (34Fialka I. Pasquali C. Lottspeich F. Ahorn H. Huber L.A. Electrophoresis. 1997; 18: 2582-2590Crossref PubMed Scopus (70) Google Scholar, 36Pasquali C. Fialka I. Huber L.A. Electrophoresis. 1997; 18: 2573-2581Crossref PubMed Scopus (120) Google Scholar). Candidate proteins were identified in several gels and spots collected for microsequencing. Microsequencing was performed as described (34Fialka I. Pasquali C. Lottspeich F. Ahorn H. Huber L.A. Electrophoresis. 1997; 18: 2582-2590Crossref PubMed Scopus (70) Google Scholar). A polymerase chain reaction fragment amplified with Bam HI-Xho I linkers encoding the cDNA of syntenin (amino acids residues 2–298) was generated from a human syntenin cDNA clone obtained from the IMAGE Consortium (unique IMAGE Consortium identifier 33203 and GenBankTM accession number R19118). This Bam HI-Xho I fragment was subcloned in frame, into a Bam HI-Xho I downstream of a Myc epitope in a pcDNA-3 expression vector (Invitrogen) containing a Kozak consensus sequence. Thus, an amino-terminally Myc-tagged human syntenin cDNA was created that could be recognized by the 9E10 anti-Myc antibody (32Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2165) Google Scholar). DNA sequencing was performed to confirm the integrity of this pcDNAIII-Myc-syntenin construct. Filter-grown MDCK I Rab5 cells were transfected with pcDNAIII-Myc-syntenin using a PEI/Ad5 system as described previously (38Bischof J. Vietor I. Cotten M. Huber L.A. Biol. Chem. Hoppe-Seyler. 1999; 380: 269-273Google Scholar). For internalization of transferrin, the cells were washed with Hanks' balanced salts (Life Technologies, Inc.) and incubated in serum-free medium containing 50 μmdesferoxamine mesylate (Sigma) for 3 h at 37 °C to deplete the cells from Fe2+ and enrich ligand-free transferrin receptor on the plasma membrane. Thereafter, the cells were washed with ice-cold PBS2+. The filters were then placed on a drop of prewarmed medium (on a piece of parafilm) containing 100 μg/ml transferrin-Alexa 488™ (Molecular Probes), overlaid with prewarmed medium, and incubated at 37 °C for 15 min. Internalization was stopped by placing the filters in ice-cold PBS2+, followed by three washes to remove free transferrin. Subsequently, cells were extracted with 0.05% saponin in cytoskeleton buffer (10 mmPIPES, pH 6.8, 150 mm NaCl, 5 mm EGTA, 5 mm glucose, 5 mm MgCl2) and processed for immunofluorescence as described (28Huber L.A. Pimplikar S. Parton R.G. Virta H. Zerial M. Simons K. J. Cell Biol. 1993; 123: 35-45Crossref PubMed Scopus (379) Google Scholar). The filter pieces were mounted in 50% glycerol in cytoskeleton buffer containing 4%n-propyl gallate (Sigma), and confocal microscopy images were obtained using a Leica TCS NT confocal microscope (Leica, Heidelberg, Germany). Images were processed using the Imaris and Colocalization software packages (Bitplane AG, Zürich, Switzerland) after deconvolution using measured point-spread functions with the Huygens software (Scientific Volume Imaging, Hilversum, Netherlands). TMA-DPH has been shown to interact with living BHK-21 cells by instantaneous incorporation into the plasma membrane. To confirm the predicted properties of TMA-DPH as endocytic membrane tracer (14Illinger D. Poindron P. Fonteneau P. Modollel M. Kuhry J.-G. Biochim. Biophys. Acta. 1990; 1030: 73-81Crossref PubMed Scopus (39) Google Scholar, 20Illinger D. Poindron P. Kuhry J.G. Biol. Cell. 1991; 73: 131-138Crossref PubMed Scopus (27) Google Scholar, 21Illinger D. Poindron P. Kuhry J.G. Biol. Cell. 1991; 71: 293-296Crossref PubMed Scopus (8) Google Scholar), we established an internalization protocol in BHK-21 cells, in which filling kinetics of endosomes are well characterized. For this purpose, we prebound TMA-DPH to the cell surface at 4 °C and internalized for 2 and 30 min by warming up the sample to 37 °C. The distribution of TMA-DPH in living cells was analyzed by direct fluorescence microscopy (Fig.1). TMA-DPH bound to the plasma membrane at 4 °C (Fig. 1 A) and could be internalized into an early endocytic compartment 2 min after raising the temperature to 37 °C (Fig. 1 B). In addition, the perinuclear compartment (Fig.1 C) that was labeled after 30 min of TMA-DPH internalization was colocalized with acridine orange, a marker for acidic late endocytic/lysosomal compartments (Fig. 1 D). This confirms that TMA-DPH is rapidly incorporated into the plasma membrane and follows the normal intracellular traffic of internalization, thus behaving as a suitable marker for membrane endocytosis under the applied conditions. We first compared the distribution of fluid phase internalized HRP with internalized TMA-DPH after subcellular fractionation of cells on sucrose gradients. We internalized HRP in filter-grown MDCK II cells through the apical or basolateral medium. Cells were homogenized, and the PNS was then loaded on top of a continuous sucrose gradient (10–40%) and centrifuged to equilibrium. A total of 20 fractions per gradient were collected and analyzed for their HRP activity and total amounts of protein (Fig.2 A). The apical and basolateral internalization profiles showed a peak area that spanned fractions 10–16, corresponding to 25–35% of sucrose concentration (1.104–1.151 g/cm3). We next examined the gradient distribution for TMA-DPH-labeled early endocytic structures. After binding TMA-DPH to the basolateral or apical plasma membrane for 2 min at 4 °C, the fluorescent dye was internalized for 2 min at 37 °C (Fig. 2 B), and cells were fractionated as in Fig.2 A. When compared with the distribution profiles obtained by HRP labeling of apical and basolateral endocytic vesicles of MDCK II cells, TMA-DPH labeling revealed a largely overlapping peak area between fractions 14 and 16, corresponding to 30–35% concentration of sucrose. Immunoblotting of these fractions revealed the presence of Tf-R, Rab5, and Rab4 as early endosomal markers (10Bucci C. Wandinger-Ness A. Lütcke A. Chiariello M. Bruni C.B. Zerial M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5061-5065Crossref PubMed Scopus (111) Google Scholar, 39Van der Sluijs P. Hull M. Zahraoui A. Tavitian A. Goud B. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6313-6317Crossref PubMed Scopus (220) Google Scholar, 40Van der Sluijs P. Hull M. Webster P. Mâle P. Goud B. Mellman I. Cell. 1992; 70: 729-740Abstract Full Text PDF PubMed Scopus (509) Google Scholar) and Rab7 as a marker protein for late endosomes (41Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Cell. 1990; 62: 317-329Abstract Full Text PDF PubMed Scopus (882) Google Scholar) in the overlapping peak areas (outlined in Fig. 2 C). However, this sucrose concentration corresponded to the density of early endocytic organelles rather than to the reported density of late endocytic vesicles (42Aniento F. Gruenberg J. Cold. Spring Harbor Symp. Quant. Biol. 1995; 60: 205-209Crossref PubMed Scopus (13) Google Scholar), emphasizing that conventional gradient fractionation is not sufficient to separate (a) early from late endocytic structures, or (b) apical from basolateral endosomes in MDCK II cells. Gradient fractions containing >10% TMA-DPH-labeled apical or basolateral vesicles, as referred to the PNS fraction, were pooled and subsequently sorted in a flow cytometer as described under “Experimental Procedures.” Proteins of sorted fractions were concentrated and precipitated, and their protein content was determined. 5 μg of protein (equivalent to about 40 million sorted fluorescent events) derived from apical or basolateral sorts and 5 μg of protein precipitated from the starting fraction were separated by 10% Tris-glycine SDS-polyacrylamide gel electrophoresis. The gel was transferred onto a nitrocellulose membrane, stained with Ponceau-S to confirm equal loading of samples (Fig.3 A), and prepared for immunodetection. Apical as well as basolateral fractions revealed an enrichment of early endocytic markers after FACS sorting (Fig.3 B). Rab5a is a common component of the apical and basolateral endocytic machinery in polarized MDCK cells (9Harder T. Gerke V. J. Cell Biol. 1993; 123: 1119-1132Crossref PubMed Scopus (149) Google Scholar) and was strongly enriched in both fractions. However, Tf-R, a basolaterally endocytosed and recycling transmembrane protein (43Nunez M.T. Tapia V. Arredondo M. J. Nutr. 1996; 126: 2151-2158Crossref PubMed Scopus (20) Google Scholar, 44Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Crossref PubMed Scopus (128) Google Scholar), was only enriched in basolateral fractions but was clearly present in the apical compartment. A marker protein for the plasma membrane, E-cadherin, was significantly decreased in the sorted fractions, as well as the late endosomal GTPase Rab7. Annexin II, a Ca2+-, phospholipid-, and actin-binding protein implicated in the regulation of vesicular traffic (10Bucci C. Wandinger-Ness A. Lütcke A. Chiariello M. Bruni C.B. Zerial M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5061-5065Crossref PubMed Scopus (111) Google Scholar, 45Emans N. Gorvel J.-P. Walter C. Gerke V. Kellner R. Griffiths G. Gruenberg J. J. Cell Biol. 1993; 120: 1357-1369Crossref PubMed Scopus (229) Google Scholar, 46Harder T. Kellner R. Parton R.G. Gruenberg J. Mol. Biol. Cell." @default.
• W2140172555 created "2016-06-24" @default.
• W2140172555 creator A5034693291 @default.
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• W2140172555 date "1999-09-01" @default.
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• W2140172555 title "Identification of Syntenin as a Protein of the Apical Early Endocytic Compartment in Madin-Darby Canine Kidney Cells" @default.
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