Ferritin-Conjugated Antibodies Used for Labeling of Organelles Involved
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1 Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp , May 1974 Ferritin-Conjugated Antibodies Used for Labeling of Organelles Involved in the Cellular Synthesis and Transport of Procollagen (ferritin-antibody conjugates/golgi vacuoles/protein secretion) BJORN R. OLSEN AND DARWIN J. PROCKOP Department of Biochemistry, The Rutgers Medical School, College of Medicine and Dentistry of New Jersey, Piscataway, N.J Communicated by G. E. Palade, February 7, 1974 ABSTRACT An improved procedure s4as used to conjugate ferritin to antibodies specific for the NH2- terminal extensions on the precursor form of collagen known as procollagen. The ferritin-antibody conjugates were then used to determine the localization of procollagen in fibroblasts isolated from chick-embryo tendons. Procollagen was found in the cisternae of the endoplasmic reticulum, indicating that the protein passes into this compartment early in its biosynthesis. Specific labeling of large Golgi vacuoles was also observed, suggesting that this compartment is also involved in the secretory process. When cells were incubated with colchicine so that the secretion of procollagen was delayed, there was an increase of large, smooth-surfaced vacuoles in the cells and these vacuoles were labeled with the ferritin-antibody conjugate. Largely on the basis of classical studies on exocrine cells of the pancreas (1-3), it is generally accepted that proteins synthesized for "export" into the extracellular space pass into the cisternae of the endoplasmic reticulum, and that from there they are transported through Golgi vesicles to large Golgi vacuoles before secretion via secretory vesicles or granules. Some experimental evidence, largely morphological (4-9), has suggested that this general scheme also applies to the synthesis and secretion of the precursor form of collagen known as procollagen. However, it has been difficult to obtain definitive proof for this hypothesis. For example, autoradiographic studies with different types of cells synthesizing procollagen have raised the possibility that some or all of the protein passes into the cytoplasm and is secreted through the plasma membrane (10-12). Alternatively, it has been suggested that procollagen may pass directly from the cisternae of the endoplasmic reticulum into the extracellular space (5). We have recently prepared and purified specific antibodies (13) to the NH2-terminal extensions on the procollagen synthesized and secreted by chick-embryo fibroblasts. We here report on the use of these antibodies, together with an improved procedure for preparation of ferritin-antibody conjugates, to demonstrate the localization of procollagen in fibroblasts. MATERIALS AND METHODS Preparation and Purification of Antibodies to Procollagen. Procollagen was prepared from matrix-free fibroblasts (14) and was used to immunize a rabbit (13). Specific antibodies to the NH2-terminal extensions were then purified with an immunoadsorbent gel containing the isolated, disulfide-linked NH2-terminal extensions of procollagen. The gel was mixed with an equal amount of Sephadex G-25 (Pharmacia; fine 2033 grade) and poured into a column (0.9 X 5 cm) that was equilibrated at 40 with 0.1 M sodium phosphate buffer, ph 7.3. Three ml of antisera absorbed with 3 ml of fetal-calf serum were passed through the column. The column was washed with several volumes of 0.1 M sodium phosphate buffer and the antibody was eluted with 3 M KSCN. The eluted antibody was desalted on a Sephadex G-25 column equilibrated at 40 with 0.1 M sodium phosphate buffer, ph 7.3. Conjugation of the Purified Antibodies with Ferritin. Ferritin was activated with glutaraldehyde (15) by mixing 4.3 mg of the activated ferritin with 0.8 mg of the specific antibody and 0.3 mg of nonimmune IgG that had been previously labeled with ["25I]iodine (16). The mixture, in a final volume of 14.8 ml of 0.1 M sodium phosphate buffer, ph 7.3, was concentrated at 40 to 2.5 ml with an Amicon PM-30 filter. The concentrate was allowed to stand at 4 for 100 hr, and the conjugate was isolated by gel filtration on an 6% agarose column (Bio-Gel A-5m; mesh) (1.5 X 80 cm) that was equilibrated and eluted with 0.1 M Tris * HCl buffer, ph 7.5. Preparation of Fibroblasts for Electron Microscopy. Fibroblasts were isolated from tendons of 17-day-old chick embryos by digestion with collagenase and trypsin, and the cells were incubated for 3 hr at 370 in modified Krebs medium containing 10% fetal-calf serum (14). The cells were. fixed for E o wu m ir o 0.2 IDI 1000,_.n CY FRACTION NUMBER FIG. 1. Gel filtration of ferritin conjugated to antibodies specific for the NH2-terminal extensions on procollagen. Activated ferritin was reacted with antibodies to procollagen and gel filtration was carried out as described in text. The fraction size was 1.3 ml, and the void volume was about 50 ml. Symbols: Elution of ferritin as assayed by absorbance at 410 nm (O O); elution of IgG (@--*) as assayed by 125I in carrier IgG added to the specific antibody (see text). 2 a.
2 2034 Cell Biology: Olsen and Prockop Stt t v.ii 49 4 r 491z< < ; g X = > I r x. j-g, ;Xs _,b
3 Synthesis and Transport of Procollagen 2035 FIG. 2. Electron micrograph from control experiments in which cell fragments fixed for 3 hr with 1% formaldehyde were incubated with ferritin conjugated to nonimmune IgG. Washing of the cell fragments with phosphate buffer removed essentially all of the ferritin conjugates so that there was essentially no ferritin over the Golgi complex (G) or endoplasmic reticulum (ER). Magnification X 62,500. FIG. 3. Electron micrograph of cell fragments that were fixed for 3 hr with 1% formaldehyde and then incubated with ferritin conjugated to antibodies to procollagen. Heavy labeling of the endoplasmic reticulum is seen. Magnification X50,000. FIG. 4. Electron micrograph of cell fragments that were fixed for 15 hr with 1% formaldehyde and then incubated with ferritin conjugated to antibodies to procollagen. Because of the longer fixation time, the cell morphology was relatively well preserved, but the fragments were apparently less penetrable by the ferritin-conjugate. However, the specific labeling of the cisternae of the endoplasmic reticulum (ER) is still apparent. Inset: Labeling of perinuclear space (arrow) as well as endoplasmic reticulum (asterisk) in the same cell fragments. Magnification X62, hr or 15 hr at 40 with 1% formaldehyde in 60 mm sodium phosphate buffer, ph 7.3, containing 0.14 M sucrose. After centrifugation at 600 X g for 6 min, the cells were resuspended in 0.1 M sodium phosphate buffer and stored at 40 for hr. The fixed-cells were then homogenized with from 50 to 100 strokes in a Teflon and glass homogenizer (15). The cell fragments were sedimented by centrifugation at 20,000 X g for 10 min and the pellet was incubated with ferritin-conjugated antibodies to procollagen for hr at 4. Fragments from 50 to 100 million cells were incubated with 100,l of 0.1 M Tris- HCl, ph 7.4, containing the ferritin-labeled specific antibodies (1 mg of ferritin and 0.3 mg of antibody per ml). The cell fragments were washed twice with 5 ml of 0.1 M sodium phosphate bufferj ph 7.3; fixed at 40 for 1 hr with 3% glutaraldehyde in 60 mm sodium phosphate buffer, ph 7.3, containing 0.14 M sucrose; post-fixed at 4 for 1 hr with 1% osmium tetroxide in 60 mm sodium phosphate buffer, ph 7.3, containing 0.16 M sucrose; block-stained with 0.5% magnesium uranyl acetate for 30 min at room temperature; dehydrated in ethanol; and embedded in Araldite (Taab). Ultra-thin sections were examined in a JEM-1OOB electron microscope. RESULTS Properties of the Ferritin-Conjugated Antibodies. The ferritinconjugate of the purified antibodies was largely free of higher polymers of antibody, and gel filtration of the conjugate (Fig. 1) made it possible to select fractions (fractions 56-75) that were free both of unconjugated IgG and of polymeric ferritin. A large fraction of the ferritin was linked to the specific antibodies, since the molar ratio of ferritin to '25I-labeled IgG in the peak fractions (fractions in Fig. 1) was about 0.7. The amount of free ferritin was relatively small and it was readily removed from the cell fragments by washing with phosphate buffer (see below). Distribution of Ferritin-Conjugated Antibodies to Procollagen in Fibroblasts. A series of conditions was examined to establish an optimal method for preparing cell fragments. Fibroblasts were incubated with ['4C]proline for 3 hr so that the intracellular procollagen contained [14C]hydroxyproline (14). The medium was removed by centrifugation (14), and the cells were fixed in 1% formaldehyde for 1, 3, or 15 hr; Assays for nondialyzable ['4C]hydroxyproline indicated that about 80% of the initial [14C]procollagen was recovered in the cell fragments regardless of the fixation time. However, if the fixation was carried out for only 1 hr, there was a heavy background of the ferritin-conjugated antibody around the cell fragments as well as within them. This observation was probably explained by diffusion of the procollagen out of cell compartments because of insufficient crosslinking of the proteins in the fragments. If the fixation with 1% formaldehyde was extended to 3 or 15 hr, this background staining was almost completely eliminated. To test the effect of the fixation on antigenicity of procollagen, the cells were fixed with 1% formaldehyde for various times and the cells were ruptured by N2 cavitation (18) to make the procollagen available for reaction with antibody. Assay of the fragments by hemagglutination inhibition with erythrocytes coated with isolated disulfide-linked NH2-terminal extension from procollagen (13) indicated that fixation with 1% formaldehyde for up to 15 hr had no detectable effect on the antigenicity of the procollagen. Further control experiments were carried out with ferritin conjugated to nonimmune IgG from rabbits. When the cell fragments were incubated with nonimmune conjugate and not washed, some ferritin was seen in all cellular organelles such as nuclei, mitochondria, cisternae of the endoplasmic reticulum, and Golgi vacuoles. The results indicated, therefore, that the conjugate penetrated into these compartments. However, essentially all of this nonimmune conjugate was removed from the fragments by washing with phosphate buffer as described above (Fig. 2). In contrast, the ferritin conjugated to purified antibodies to procollagen specifically labeled the cisternae of the endoplasmic reticulum (Figs. 3 and 4) and some of the large Golgi vacuoles (Fig. 5). No significant labeling was seen in the cytoplasmic matrix except for a few sites at which there was discontinuity of the membrane of the endoplasmic reticulum. Also, no specific labeling was seen over the ribosomes free in the cytoplasm or over ribosomes attached to the membranes of the endoplasmic reticulum (Fgis. 3 and 4). The distribution of label in the cisternae of the endoplasmic reticulum was relatively uniform and was similar to that previously seen in similar cell fragments with ferritin-conjugates of antibodies to prolyl hydroxylase (15). Of special interest was the finding that ferritin-conjugates were present in the perinuclear space (Fig. 4, inset). Some large Golgi vacuoles were clearly labeled with the ferritin-conjugate (Fig. 5) but it was difficult to demonstrate specific labeling of the small vesicles within the Golgi complex. Incubating the cells with 1 AM colchicine for 3 hr greatly increased the number and size of the Golgi vacuoles (Figs. 6 and 7). The large vacuoles in the cells treated with colchicine were extensively labeled with the ferritinconjugated antiprocollagen (Fig. 6). DISCUSSION The purified antibodies used here are specific for the NH2- terminal extensions on procollagen containing interchain disulfide bonds or on individual pro-a chains in which the extensions contain intrachain disulfide bonds (13). The antibody does not react with pro-a chains if these are completely reduced and alkylated. The latter observation suggests that
4 2036 Cell Biology: Olsen and Prockop Proc. Nat. Acad. Sci. -USA ?1 - EUj wfa k.-..- (1974)
5 Synthesis and Transport of Procollagen 2037 FIG. 5. Electron micrograph of cell frag nts prepareas4,described in the legend of Fig. 4. Some labeling of large Golgi vacuoles (arrows) is apparent. Magnification X62,500. FIG. 6 Electron micrograph from an experiment in which cells were incubated with 1,uM1I colchicine for 3 hr at 370, fixed with 1% formaldehyde for 3 hr, and then incubated with ferritin-antibody conjugates as in Figs. 4 and 5. The treatment with colchicine has produced distended, smooth-surfaced vacuoles (see Fig. 7) which are heavily labeled with ferritin (arrows). Magnification X62,500. FIG. 7. Electron micrograph of tendon fibroblasts incubated with 1 1AM colchicine at 370 for 3 hr. The cells were fixed with 3% glutaraldehyde for 1 hr and post-fixed with 1% osmium tetroxide as described in text. The section was stained with 1% uranyl acetate in 70% methanol. Note the presence of large, smooth-surfaced vacuoles (V) similar to those seen in the cell fragments (see Fig. 6). Magnification X25,000. the antibody would not react with nascent forms of the polypeptides in which the intrachain disulfide bonds had not yet been synthesized, but they would react with single pro-a chains containing intrachain disulfide bonds before such chains are linked together with interchain bonds. The antibody also might react with NHrterminal extensions released from procollagen by proteolytic degradation of the protein if such proteolysis released the extensions in a largely intact form. However, previous studies with the cells used here indicate that this does not present a problem in interpreting the present data. In particular, pulse-label and chase experiments with the cells have shown that there is little if any intracellular degradation of the procollagen which would lead to release of peptides containing intact antigenic determinants (14, 20). Therefore, most of the antigen in the cells reacting with the ferritin-antibody conjugates must consist of either procollagen or of pro-a chains in which the NHrterminal extensions are intact and contain intrachain disulfide bonds. The results demonstrated specific labeling of the cisternae of the endoplasmic reticulum and no significant labeling of the cytoplasmic matrix or any other cellular compartment. These observations conclusively establish that the newly synthesized polypeptide chains of procollagen pass into the cisternae during or after their assembly on ribosomes. Ferritin-antibody conjugates also labeled the perinuclear space, indicating that some procollagen passes into this compartment early in its biosynthesis. The presence of procollagen in the cisternae of the endoplasmic reticulum agrees with the recent demonstration (15) that prolyl hydroxylase, the enzyme that synthesizes the hydrox-yproline in collagen by the hydroxylation of peptide-bound proline, is also found in the cisternae of the endoplasmic reticulum. Labeling with ferritin-antibody conjugates was also seen over the large Golgi vacuoles, suggesting that these cellular organelles participate in the secretory process. Specific labeling was not seen over the small vesicles in the Golgi complex, an observation that might be explained by incomplete penetration of these vesicles whose diameter (about 500 A) is not much larger than the anticipated size of the ferritin-antibody conjugates ( A). It is also possible, however, that these vesicles, which are smaller than the largest dimension of the triple helix of procollagen (about 3000 A), do not participate in the secretion of this protein in these cells. The data presented here did not conclusively establish that all the procollagen in the cells was secreted through the Golgi vacuoles since it was not possible to carry out kinetic studies on the transfer of protein from one compartment to another. However, the observations suggesting that secretion involved the large Golgi vacuoles were further supported by the experiments with colchicine. Previous studies demonstrated that treatment of the cells with colchicine delaved the secretion of procollagen without affecting its rate of synthesis and therefore led to an intracellular accumulation of the protein (14, 21, 22). The results presented here showed that this intracellular accumulation was accompanied by a distension of the Golgi vacuoles and by an increase in the amount of procollagen in such vacuoles. This work was supported in part by N.I.H. Grant AM-16,516 from the U.S. Public Health Service. We gratefully acknowledge the expert technical assistance of Mrs. Nancy Doerr. 1. Palade, G. E., Siekevitz, P. & Caro, L. G. (1961) in Ciba Foundation Symposium on the Exocrine Pancreas, eds. de Reuck, A. V. S. & Cameron, M. P. (J. & A. Churchill Ltd., London), pp Jamieson, J. D. & Palade, G. E. (1967) J. Cell Biol. 34, Jamieson, J. D. & Palade, G. E. (1971) J. Cell Biol. 50, Revel, J. P. & Hay, E. D. (1963) Z. Zellforsch. Mikroskop. Anat. 61, Ross, R. & Renditt, E. P. (1965) J. Cell Biol. 27, Trelstad, R. L. (1971) J. Cell Biol. 48, Weinstock, M. (1972) Z. Zellforsch. Mikroskop. Anat. 129, Coulombre, A. J. & Coulombre, J. L. (1972) Develop. Biol. 28, Hay, E. D. & Dodson, J. W. (1973) J. Cell Biol. 57, Salpeter, M. M. (1968) J. AMorphol. 124, Cooper, G. W. & Prockop, D. J. (1968) J. Cell Biol. 38, Reith, E. J. (1968) J. Ultrastruct. Res. 21, Dehm, P., Olsen, B. R. & Prockop, D. J. (1974) Eur. J. Biochem., in press. 14. Dehm, P. & Prockop, D. J. (1972) Biochim. Biophys. Acta 264, Olsen, B. R., Berg, R. A., Kishida, Y. & Prockop, D. J. (1973) Science 182, Marchalonis, J. J. (1969) Biochem. J. 113, Kraehenbuhl, J. P. & Jamieson, J. D. (1972) Proc. Nat. Acad. Sci. USA 69, Hunter, M. J. & Commerford, S. L. (1961) Biochim. Biophys. Acta 47, Schofield, J. D. & Prockop, D. J. (1973) Clin. Orthop. Relat. Res. 97, Schofield, J. D., Uitto, J. & Prockop, D. J. (1974) Biochemistry, in press. 21. Ehrlich, H. P. & Bornstein, P. (1972) Nature New Biol. 238, Diegelmann, R. F. & Peterkofsky, B. (1972) Proc. Nat. Acad. Sci. USA 69,
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