Expression of Regulated Secretory Proteins Is Sufficient to Generate Granule-like Structures in Constitutively Secreting Cells
2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês
10.1074/jbc.m310613200
ISSN1083-351X
AutoresNicole Beuret, Hansruedi Stettler, Anja Renold, Jonas Rutishauser, Martin Spiess,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein α1-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30–70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas α1-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation. The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein α1-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30–70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas α1-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation. Endocrine and neuroendocrine cells possess a regulated secretory pathway in addition to the constitutive pathway present in all cells (1Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (737) Google Scholar). At the trans-Golgi network (TGN), 1The abbreviations used are: TGN, trans-Golgi network; POMC, pro-opiomelanocortin; A1Pi, α1-protease inhibitor; A1PiTS, tyrosine sulfation-tagged A1Pi; ACTH, adrenocorticotropic hormone; CgA and CgB, chromogranin A and B; EEA1, early endosome antigen 1; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; GFP, green fluorescent protein; PBS, phosphate-buffered saline; SgII, secretogranin II; Lamp-1, lysosome-associated membrane protein-1. 1The abbreviations used are: TGN, trans-Golgi network; POMC, pro-opiomelanocortin; A1Pi, α1-protease inhibitor; A1PiTS, tyrosine sulfation-tagged A1Pi; ACTH, adrenocorticotropic hormone; CgA and CgB, chromogranin A and B; EEA1, early endosome antigen 1; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; GFP, green fluorescent protein; PBS, phosphate-buffered saline; SgII, secretogranin II; Lamp-1, lysosome-associated membrane protein-1. regulated cargo proteins, such as peptide hormone precursors and granins, are sorted into secretory granules where they are stored in a densely packed form. By an external stimulus, the granules are triggered to fuse with the plasma membrane and to release their contents in a controlled manner. The regulated secretory pathway thus requires mechanisms to segregate regulated cargo from constitutively secreted proteins and to package them into specialized vesicles. These membrane-bounded organelles in addition recruit pro-hormone-processing enzymes as well as the components necessary for luminal acidification for transport of the granules to the cell periphery or along the axon and for the controlled fusion with the plasma membrane. So far, little is known regarding the machinery that is required to generate secretory granules. Two non-exclusive models have been proposed on how secretory granules are formed and how specific cargo selection is accomplished (2Arvan P. Castle D. Biochem. J. 1998; 332: 593-610Crossref PubMed Scopus (440) Google Scholar, 3Tooze S.A. Biochim. Biophys. Acta. 1998; 1404: 231-244Crossref PubMed Scopus (183) Google Scholar). The first model, termed "sorting-for-entry," is analogous to receptor-mediated endocytosis and mannose 6-phosphate receptor-dependent lysosomal transport (4Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (669) Google Scholar) where cargo binds to receptors, which in turn recruit a cytosolic coat. Similarly, regulated secretory proteins may be selected and other proteins excluded by interaction with receptors in the TGN membrane prior to granule formation. Consistent with this model, the propeptide of prosomatostatin (5Stoller T.J. Shields D. J. Cell Biol. 1989; 108: 1647-1655Crossref PubMed Scopus (102) Google Scholar) and a disulfide-bonded loop segment of chromogranin B (CgB) (6Krömer A. Glombik M.M. Huttner W.B. Gerdes H.H. J. Cell Biol. 1998; 140: 1331-1346Crossref PubMed Scopus (91) Google Scholar, 7Glombik M.M. Krömer A. Salm T. Huttner W.B. Gerdes H.H. EMBO J. 1999; 18: 1059-1070Crossref PubMed Scopus (101) Google Scholar) have been shown to be necessary and sufficient to mediate granule sorting, suggesting that they constitute sorting signals. An amphipathic loop of pro-opiomelanocortin (POMC) was also found to be necessary for sorting (8Cool D.R. Fenger M. Snell C.R. Loh Y.P. J. Biol. Chem. 1995; 270: 8723-8729Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Carboxypeptidase E was reported to bind to this loop and to be required for granule sorting (9Cool D.R. Normant E. Shen F. Chen H.C. Pannell L. Zhang Y. Loh Y.P. Cell. 1997; 88: 73-83Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Its proposed role as a sorting receptor, however, is controversial (10Irminger J.C. Verchere C.B. Meyer K. Halban P.A. J. Biol. Chem. 1997; 272: 27532-27534Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The apparent substoichiometric amount of putative sorting receptors in secretory granules may be explained by the tendency of regulated secretory proteins to aggregate under the conditions of the trans-Golgi (low pH and high calcium concentrations) (e.g. Refs. 11Chanat E. Huttner W.B. J. Cell Biol. 1991; 115: 1505-1519Crossref PubMed Scopus (383) Google Scholar and 12Colomer V. Kicska G.A. Rindler M.J. J. Biol. Chem. 1996; 271: 48-55Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), which would allow each receptor to sort an entire polymer of cargo molecules. The alternative model, "sorting-by-retention," proposes that selective aggregation of regulated cargo in the TGN results in an immature granule. Captured non-granule molecules are subsequently removed in vesicles budding from maturing secretory granules by clathrin-coated vesicles and by so-called constitutive-like secretion, whereas specific granule cargo is retained (13Kuliawat R. Arvan P. J. Cell Biol. 1992; 118: 521-529Crossref PubMed Scopus (158) Google Scholar). This explains the presence of the mannose 6-phosphate receptor, clathrin, and AP-1 adaptors on immature secretory granules (14Dittie A.S. Hajibagheri N. Tooze S.A. J. Cell Biol. 1996; 132: 523-536Crossref PubMed Scopus (137) Google Scholar, 15Klumperman J. Kuliawat R. Griffith J.M. Geuze H.J. Arvan P. J. Cell Biol. 1998; 141: 359-371Crossref PubMed Scopus (235) Google Scholar). Recently, it has been proposed that a single protein of endocrine and neuronal cells, chromogranin A (CgA), controls secretory granule biogenesis (16Kim T. Tao-Cheng J.H. Eiden L.E. Loh Y.P. Cell. 2001; 106: 499-509Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Expression of CgA was even found to induce granular structures in transfected CV-1 fibroblast cells. The interpretation of these observations has been discussed controversially (17Day R. Gorr S.U. Trends Endocrinol. Metab. 2003; 14: 10-13Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 18Kim T. Tao-Cheng J.H. Eiden L.E. Loh Y.P. Trends Endocrinol. Metab. 2003; 14: 56-57Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Huh Y.H. Jeon S.H. Yoo S.H. J. Biol. Chem. 2003; 278: 40581-40589Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In the alternative view, condensing CgA might act as an aggregation or assembly factor similarly to the sorting-by-retention model (17Day R. Gorr S.U. Trends Endocrinol. Metab. 2003; 14: 10-13Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this study, we report that several other cargo proteins of the regulated secretory pathway of endocrine cells, peptide hormone precursors as well as granins, induce the formation of granule-like structures when expressed in cell lines normally lacking regulated secretion. Expression of cargo is sufficient to drive segregation of regulated and constitutive secretory proteins and the formation of dense membrane-bound accumulations with similar ultrastructural appearance as secretory granules, suggesting that initial granule formation requires no additional machinery specific to regulated secretory cells besides the regulated cargo itself. cDNA Constructs—cDNAs for regulated secretory proteins were gifts by G. Boileau (porcine POMC, University of Montreal), H. Gerdes (human secretogranin II (SgII) and CgB, University of Heidelberg), and M. Ito (human vasopressin, Northwestern University, Chicago, IL). The coding sequence of human oxytocin was assembled from the exons amplified by polymerase chain reaction from the gene (a gift by J. Amico, University of Pittsburgh). The cDNA of human α1-protease inhibitor (A1Pi) was from J. L. Brown (University of Colorado, Denver, CO). To C-terminally tag proteins with a c-Myc epitope, a KpnI restriction site was introduced by polymerase chain reaction in place of the stop codon for ligation to the Myc epitope sequence encoding EQKLISEEDLNstop. In the same way, the C1 epitope ETELDKASQEPPLLstop corresponding to the C-terminal sequence of the human asialoglycoprotein receptor H1, for which we have a rabbit anti-peptide antiserum (20Geffen I. Fuhrer C. Leitinger B. Weiss M. Huggel K. Griffiths G. Spiess M. J. Biol. Chem. 1993; 268: 20772-20777Abstract Full Text PDF PubMed Google Scholar), was fused to CgB and SgII. The cDNAs were subcloned into the SV40-based expression plasmids pECE or pCB6. For expression in COS-1 cells without plasmid amplification, SgII-Myc was also cloned into pcDNA5 (Invitrogen), which lacks SV40 sequences. Cell Culture and Transfection—Madin-Darby canine kidney strain II and HepG2 hepatoma cells were grown in minimal essential medium, COS-1, Chinese hamster ovary (CHO)-K1, human embryo kidney (HEK)293, and NIH-3T3 cells in Dulbecco's minimal essential medium supplemented with 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum at 37 °C with 7.5% CO2. Cells were transfected using Lipofectin (Invitrogen) with 2 μg of plasmid DNA/35-mm dish at ∼40% confluency and processed after 48 h. To reduce expression levels of SgII-Myc in COS cells, the SgII-Myc expression plasmid was gradually diluted with pEGFP-N1 (Clontech), whereby the total amount of plasmid was kept constant at 2 μg/35-mm plate. Expression levels were assayed by immunoblot analysis using rabbit anti-secretoneurin antiserum (21Kirchmair R. Hogue-Angeletti R. Gutierrez J. Fischer-Colbrie R. Winkler H. Neuroscience. 1993; 53: 359-365Crossref PubMed Scopus (239) Google Scholar), a gift from R. Fischer-Colbrie (University of Innsbruck), at a dilution of 1:1000. To generate stable cell lines, HEK293 cells were transfected with pCB6 containing the vasopressin precursor cDNA and subjected to selection with 1 mg/ml G418-sulfate (Invitrogen). Resistant lines were cloned and analyzed by immunoblotting and immunofluorescence microscopy. Immunofluorescence and Antibodies—We used rabbit anti-neurophysin II antibodies against pro-vasopressin, which also recognizes neurophysin I of pro-oxytocin (ICN); rabbit anti-human adrenocorticotropic hormone (ACTH) to detect POMC (Sigma); sheep anti-human TGN46 (Serotech); rabbit anti-A1Pi antiserum from Jerry L. Brown (University of Colorado Health Sciences Center, Denver, CO); rabbit anti-human EEA1 (early endosome antigen 1) antiserum from H. Stenmark (Norwegian Radium Hospital, Oslo, Norway); rabbit anti-protein-disulfide isomerase from H. P. Hauri (Biozentrum, Basel, Switzerland); monoclonal antibodies against the c-Myc epitope (9E10) (22Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2151) Google Scholar) against giantin from H. P. Hauri, against lysosome-associated membrane protein-1 (Lamp-1) from J. Rohrer (FMI, Basel, Switzerland), and against Rab5 from R. Jahn (MPI, Göttingen, The Netherlands); and a rabbit antipeptide antiserum recognizing the C1-epitope (20Geffen I. Fuhrer C. Leitinger B. Weiss M. Huggel K. Griffiths G. Spiess M. J. Biol. Chem. 1993; 268: 20772-20777Abstract Full Text PDF PubMed Google Scholar). Antibodies were used at a dilution of 1:100 with the exception of anti-ACTH (1:200) and anti-Rab5 (1:80). As secondary antibodies, non-cross-reacting Cy3-labeled goat anti-mouse, Cy2-labeled goat anti-rabbit, Cy3-labeled donkey anti-sheep, and Cy2-labeled donkey anti-rabbit immunoglobulin antibodies (from Jackson Immunoresearch and Amersham Biosciences) were used as appropriate according to the manufacturers' recommendations. Cells were grown on 14-mm glass coverslips, fixed with 3% paraformaldehyde for 15 min at room temperature, washed in phosphate-buffered saline (PBS), quenched with 50 mm NH4Cl in PBS, and permeabilized with 0.1% Triton X-100 for 10 min. Nonspecific antibody binding was blocked with PBS containing 1% bovine serum albumin. The fixed cells were incubated at room temperature with primary antibodies for 1 h, washed with PBS with albumin, and stained with fluorescent secondary antibodies in PBS with albumin for 30 min. After several washes with PBS with albumin, PBS, and water, the coverslips were mounted in Mowiol 4-88 (Hoechst). Staining patterns were analyzed using a Zeiss Axioplan 2 microscope with a KX Series imaging system (Apogee Instruments) or a Zeiss Axiovert 200 M confocal LSM 510 Meta microscope. Electron Microscopy—To enrich transfected cells for ultrastructural analysis, COS-1 cells were cotransfected with an expression plasmid encoding pro-vasopressin or SgII-Myc and a plasmid pEGFP-N1 encoding green fluorescent protein (GFP). The cells were brought into suspension by trypsinization and were sorted for GFP fluorescence using a MoFlo cell sorter (Cytomation, Fort Collins, CO). They were then fixed with 3% paraformaldehyde, 0.5% glutaraldehyde in PBS, pH 7.4, for 1 h at room temperature, washed with PBS, incubated with 1% osmium tetroxide for 1 h, washed with water, and dehydrated by successive 15-min incubations with 50, 70, 90, and 100% ethanol followed by 1 h with Epon/acetone 1:1, 1 h with Epon/acetone 2:1, and twice for 2 h with Epon and 24–48 h at 60 °C. Thin sections were stained with 6% uranyl acetate for 1 h and with lead acetate for 2 min. For immunogold labeling, GFP-positive cells expressing SgII-Myc were fixed with 3% paraformaldehyde, 0.5% glutaraldehyde in PBS for 1 h, washed with PBS, fixed again with 0.5% osmium tetroxide for 1 h, washed with water, and incubated 15 min each with 50 and 70% ethanol, 1 h with ethanol/LR White 2:1, 1 h with LR White (from Polysciences, Warrington, PA), and 24–48 h at 60 °C. Thin sections were then blocked twice for 5 min with 2% bovine serum albumin in PBS, incubated with anti-Myc antibody 9E10 in 2% bovine serum albumin for 3 h, washed three times for 10 min with PBS, and again twice for 5 min with 2% bovine serum albumin, incubated for 1 h with goat anti-mouse immunoglobulin (from British Biocell) coupled to 10-nm gold, washed with PBS and with water, and contrasted with 6% uranyl acetate for 1 h and with lead acetate for 2 min. Storage and Stimulation Assays—To analyze the secretion behavior, COS-1 cells transfected with A1PiTS (23Leitinger B. Brown J.L. Spiess M. J. Biol. Chem. 1994; 269: 8115-8121Abstract Full Text PDF PubMed Google Scholar), CgB-C1, or SgII-C1 were labeled with [35S]sulfate (0.5 mCi/ml, from Amersham Biosciences) in sulfate-free medium (from Invitrogen) for 90 min at 19 °C followed by a chase at 37 °C for up to 6 h in medium containing excess unlabeled sulfate. The medium was removed and replaced by fresh chase medium at different times. The labeled protein secreted into the medium as well as that retained in the cells at the end of the experiment was immunoprecipitated and analyzed by gel electrophoresis and autoradiography. Signals were quantified by PhosphorImager. To analyze stimulated secretion, COS-1 cells expressing SgII-C1 were labeled with [35S]sulfate for 90 min at 19 °C followed by a chase of 3 h, after which the medium was replaced by medium with or without 1 μm A23187 (Sigma; prepared by adding a 100-fold concentrated stock solution in Me2SO or just Me2SO to the medium). Labeled protein secreted into the medium during this time was analyzed by immunoprecipitation, gel electrophoresis, and autoradiography. The total amount of labeled SgII-C1 was determined by immunoprecipitation from the medium and the cell lysate of parallel aliquots of transfected cells after the labeling period. Cell integrity as judged by trypan blue exclusion was not affected after 30 min with 1 μm A23187. To estimate the effect of stimulation on the density of granule-like structures, transfected COS-1 cells expressing SgII-Myc were incubated for 30 min with fresh medium with or without 1 μm A23187 and then fixed and processed for immunofluorescence. 50 random cells of each condition were photographed, and the granule-like structures were counted, excluding the Golgi/TGN area of the cells where structures could not be separated from each other by a person unaware of the sample identity. The numbers were normalized for the size of the cells as estimated by measuring the area from tracings of the cell outlines excluding the Golgi area using Adobe Photoshop. Pro-vasopressin Expressed in COS-1 Cells Accumulates in Granular Structures—The vasopressin precursor protein is normally expressed in specific neurons of the hypothalamus. Upon translocation into the endoplasmic reticulum (ER), it is transported through the secretory pathway to the TGN where it is packaged into immature secretory granules. The precursor is processed by pro-hormone convertases to the nonapeptide hormone, the carrier protein neurophysin II, and a C-terminal glycopeptide. Granules are transported to the nerve terminals in the neurohypophysis, and its contents are released as the granule membrane is triggered to fuse with the plasma membrane. Upon expression in transfected COS-1 cells, which lack a regulated secretory pathway, pro-vasopressin is not proteolytically cleaved and is in its majority constitutively secreted (∼85% in 2 h) (see Ref. 24Beuret N. Rutishauser J. Bider M.D. Spiess M. J. Biol. Chem. 1999; 274: 18965-18972Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). When subjected to indirect immunofluorescence analysis using an antibody directed against neurophysin II, many transfected cells showed the expected weak staining of the ER typical of a secretory protein whose rate-limiting step in secretion is protein folding and ER exit. In ∼50% of the expressing cells, however, intense additional staining was found in punctate patterns, often near the nucleus but frequently also in the periphery of the cells (Fig. 1, A–C). The fluorescent structures varied in apparent size between 2 μm often observed in transient transfections, suggesting that they might be toxic. Although the formation of vasopressin accumulations does not depend strongly on expression level, it is unknown why it is triggered in only a fraction of the cells. A Constitutive Cargo Protein Is Segregated from Granule-like Structures—When pro-vasopressin was coexpressed with SgII-Myc in COS-1 cells, the two proteins colocalized to the same structures (Fig. 4, A and A′). Similarly, colocalization was observed for coexpressed CgB-Myc and SgII-C1 (SgII tagged with the 10-amino acid epitope C1) (Fig. 4, B and B′). In contrast, when a constitutively secreted protein, A1Pi, was expressed in COS-1 cells, the cells showed reticular ER and perinuclear Golgi staining. Even when expressed in cells producing SgII-Myc, A1Pi (Fig. 4, C and D) was not detectable in the SgII-Myc accumulations (C′ and D′). These results suggest that the formation of granule-like structures is specific for regulated cargo and that there is segregation of constitutive and regulated secretory proteins. Granule-like Structures Are Post-Golgi Organelles—In Fig. 5, the colocalization of organelle markers with SgII accumulations was analyzed to test whether the protein might form undegradable accumulations in the ER (e.g. due to misfolding), in lysosomes, or compartments en route. Antibodies against the ER chaperone protein-disulfide isomerase did not colocalize with the punctate structures containing SgII (Fig. 5A). Outside the compact Golgi area, SgII structures were devoid of giantin (Fig. 5B), a marker of the Golgi stacks, and no colocalization was observed with the early endosome markers EEA1 (Fig. 5C) or Rab5 (Fig. 5D) or with Lamp-1 (Fig. 5E), a marker for late endosomes and lysosomes. Immunolocalization of the TGN marker TGN46 in COS-1 cells expressing SgII-Myc showed the expected strong colocalization in the TGN and some staining of the granule-like structures near the center of the cell but less or no TGN46 staining in peripheral SgII accumulations (Fig. 5, F—F″). This finding is consistent with the model that these structures are derived from the TGN but are losing TGN46 progressively by the budding of vesicles for constitutive-like secretion. The same conclusions were obtained from double-labeling experiments for pro-vasopressin structures and the ER marker p63, giantin, Rab5, transferrin receptor, Lamp-1, or TGN46 (data not shown). Ultrastructural Morphology of Granule-like Structures—To facilitate the analysis by electron microscopy, COS-1 cells were transfected simultaneously with expression plasmids for the vasopressin precursor or SgII-Myc and for GFP. The cells were then trypsinized and subjected to fluorescence-activated cell sorting to isolate the transfected GFP-producing cells. Upon processing of cells expressing pro-vasopressin for electron microscopy, structures of 0.4–1-μm diameter were observed that were quite homogeneously filled with dense material and surrounded by a membrane (Fig. 6, A and B) and were not detectable in untransfected COS-1 cells. Granule-like structures formed by SgII-Myc had a very similar ultrastructural appearance and an average size of 0.64 μm (±0.16 μm; n = 65). Unlike our anti-neurophysin antibodies, the antibody against the Myc epitope was suitable for immunogold electron microscopy. Anti-Myc antibody in combination with a secondary antibody coupled to 10-nm-gold particles clearly decorated the dense material of the granular structures, demonstrating the presence of SgII-Myc (Fig. 6, C and D). The granule-like structures produced in COS-1 cells are similar in ultrastructural appearance to secretory granules observed in different endocrine tissues but larger because natural granules are typically only 100–400 nm in diameter (27Cross P.C. Mercer K.L. Cell and Tissue Ultrastructure. A Functional Perspective. W. H. Freeman and Co., New York1993Google Scholar). Storage and Stimulated Secretion in COS-1 Cells—In pulse-chase experi
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