The effects of vinblastine on the secretory ameloblastsAn ultrastructural cytochemical and immunocytochemical study in the rat incisor.код для вставкиСкачать
THE ANATOMICAL RECORD 219:113-126 (1987) The Effects of Vinblastine on the Secretory Ameloblasts: An Ultrastructural, Cytochemical, and lmmunocytochemical Study in the Rat Incisor A. NANCI, T.UCHIDA, AND H. WARSHAWSKY Dkpartements de Stomatologie et d‘Anatomie, UniuersitC de MontrCat, Montrbal, QuCbec H3C 357 (A.N.) and Department ofdnatomy, McGill University, Montreal (l!U., H. W), Quebec, H3A 2B2 Canada ABSTRACT Secretory ameloblasts synthesize the organic matrix of enamel and secrete it at two distinct “putative secretory sites” characterized by membrane infoldings (Nanci and Warshawsky, 1984a).The antimicrotubular agent vinblastine sulphate interferes with secretion. We have examined the effect of this drug on the ameloblast secretory sites and reevaluated the effect on the intracellular organization of the cell by using conditions that optimize fixation, cytochemistry (ZIO), and immunocytochemistry.Associated with the disappearance of secretory granules and Golgi-related structures from Tomes’ process was the loss of membrane infoldings at secretory sites. The Golgi apparatus appeared fragmented and numerous granule clusters were found throughout the cell body. These clusters were often seen in relation to extracellular patches of material in which no crystallites were seen. Immunocytochemistry revealed the presence of enamel proteins in the protein synthetic organelles, including various granule types, in lysosomes and in the extracellular patches. These data suggest that ameloblasts under the effect of vinblastine carry on secretory activities, but the product is not routed to the usual sites. It was confirmed that membrane infoldings characterize the sites where enamel proteins are normally secreted. The ameloblasts of inner enamel secretion in the rat incisor were postulated to possess two distinct putative secretory sites (Nanci and Warshawsky, 1984a). The interrod secretory site is located on the proximal portion of Tomes’ process and forms a cooperative front for the organization of interrod enamel (Warshawsky et al., 1981; Nanci and Warshawsky, 1984a).The rod secretory site is present on one surface of the interdigitating portion of Tomes’ process and organizes the individual rods that fill the interrod cavities (Warshawsky et al., 1981; Nanci and Warshawsky, 1984a). These putative secretory sites are characterized by three structural features: membrane infoldings at the growth front of enamel, accumulation of secretory granules in the proximity of the infoldings, and the association of granules with tubular and vesicular structures (Weinstock and Leblond, 1971;Smith, 1979;Warshawsky et al., 1981;Simmelink, 1982;Nanci and Warshawsky, 1984a). Vinblastine sulphate has been shown to interfere with the secretory process in various cell types (Ekholm et al., 1974; Ericson, 1980; Williams, 1981; Miake et al., 1982; Bennett et al., 1984) and specifically the secretory ameloblast (Moe and Mikkelsen, 1977a,b; Moe, 1979; Takuma et al., 1982; Takuma et al., 1984). The latter studies, as well as those using colcemid, an agent producing similar effects (Karim and Warshawsky, 19791, suggest that directional movement of secretory granules originating from the Golgi apparatus is affected. This results in accumulation of secretory granules close to 0 1987 ALAN R. LISS, INC. the Golgi saccules, which are now found throughout the cell body but not in Tomes’ process, and the possible release of these granules at abnormal sites along the ameloblast surface. In view of the usefulness of vinblastine in affecting the secretory process and the possibility of altering the distribution of secretory sites, inner and outer enamel secretory ameloblasts (Warshawsky and Smith, 1974) were examined after short-term administration of vinblastine, under conditions previously described to optimize fixation (Nanci and Warshawsky, 198413). The zinc iodide-osmium tetroxide method (ZIO) (Maillet, 1963; Kallenbach et al., 1963; Ozawa et al., 1983)was used to assess the redistribution of membranous profiles associated with the Golgi apparatus. Immunocytochemistry (Nanci et al., 1985) was used to verify whether enamel proteins persist in the cell at the various sites along the synthetic and secretory pathway, and whether the ectopically released material represents enamel proteins. Received September 19,1986; accepted March 2, 1987. Dr. Uchida’s present address is Department of Anatomy, Yamanashi Medical School, Tamaho, Yamanashi, 409-38, Japan. Address reprint requests to Dr. Antonio Nanci, Departement de Stomatologie, Facult6 de Medecine Dentaire. Universit6 de Montreal, C.P. 6128, Succ. A., Montreal, QuBbec, Canada H3C 357. 114 A. NANCI, T. UCHIDA, AND H. WARSHAWSKY chondria (m). Numerous cisternae of rough endoplasmic reticulum (rER) and various types of granules (g) appear de nouo in the cytoplasm. The proximal cell web (pew) of the ameloblast and the desmosomes (d) associated with the cells of the stratum intermedium (SI) have been retained. Some nuclei (N) seem to have been displaced distally. fg, Fig. 1. The infranuclear compartment of inner enamel secretory granule with flocculent content, 17,500, ameloblasts appears to contain fewer than normal numbers of mito- Figs. 1-7. Electron micrographs of inner and outer enamel secretory ameloblasts two hours after vinblastine injection and fixed with a mixture of acrolein, formaldehyde, and glutaraldehyde. The sections were stained with uranyl acetate and lead citrate. EFFECTS OF VINBLASTINE ON SECRETORY AMELOBLASTS MATERIALS AND METHODS Male Sherman or Wistar rats, 100 g weight, were injected through the jugular vein with 5 mg/100 g body weight of vinblastine sulphate (Sigma Chemical Company) in physiological saline Woe and Mikkelsen, 1977a). One or 2 hours after the injection, the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital and perfused through the left ventricle with lactated Ringer’s solution (Abbott)for about 30-45 seconds followed by the fixative for 10 minutes. Tissue Preparation for Ultrastructural Analysis Three rats were used to study the ultrastructural effects of vinblastine sulphate 2 hours after injection. The fixative used was a mixture of 2% acrolein, 2.5% glutaraldehyde, and 3%formaldehyde in 0.06 M sodium cacodylate buffer containing 0.05% CaC12, pH 7.3 (Nanci and Warshawsky, 198413). After perfusion, the mandibles were dissected and immersed in the same fixative for 3 hours at 4°C. The incisors were then dissected from the alveolar bone and washed in 0.1M sodium cacodylate buffer containing 5% sucrose, pH 7.3. All teeth were postfixed in osmium tetroxide reduced with potassium ferrocyanide (Karnovsky, 1971) for 2 hours at 4°C. Tissues were then left overnight at 4°C in 0.1 M sodium cacodylate washing buffer, dehydrated in acetone and embedded in Epon. Thin sections in the cross and tangential plane of the incisor were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with a Philips 400 electron microscope at 80 kV. Zinc Iodide-Osmium Tetroxide Method (ZIO) Four vinblastine sulphate and 2 normal saline injected rats were used for ZIO staining. One hour after the injection, the animals were perfused with a mixture of 2% acrolein, 2.5% glutaraldehyde, and 3% formaldehyde in 0.05 M sodium phosphate buffer, pH 7.3. The mandibles were dissected and immersed in the same fixative for 2 hours at room temperature. The incisors were then dissected from the alveolar bone and washed in 0.1 M sodium phosphate buffer. The ZIO staining was performed according to Reinecke and Walther (1978). Abbreviations d dg f fg g G GS k ir iT N m PCW ph Pf? Pm PT R r rER Tomes SI desmosome dark granule filament granule with flocculent material granule Golgi apparatus stacks of Golgi saccules lysosome infranuclear compartment interrod growth site interdigitating portion of Tomes’ process nucleus mitochondria proximal cell web phagosome pale granule particulate material proximal portion of Tomes’ process rod rod growth site rough endoplasmic reticulum Tomes’ process stratum intermedium 115 Briefly, the incisors were washed in a tris-HC1 buffer solution, pH 3.8 at 4°C. They were then incubated in the ZIO reagent for 18 hours at 4°C in the dark, washed in tris-HC1buffer, dehydrated in acetone, and embedded in Epon. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with a Philips 400 electron microscope at 80 kV. lmmunocytochemicalMethod Using the Protein A-Gold Technique Two vinblastine injected rats were fixed with 2% glutaraldehyde in 0.08 M cacodylate buffer containing 0.05% CaC12, pH 7.3. The mandibles were dissected and further fixed by immersion in the same fixative for 2 hours at 4°C. They were then decalcified in 4.13%EDTA for 14 days at 4°C (Warshawsky and Moore, 1967). Longitudinal segments of the incisor were cut, washed in 0.1 M cacodylate buffer containing 5% sucrose, and postfixed in potassium ferrocyanide reduced osmium tetroxide (Karnovsky, 1971). They were dehydrated in graded acetone and flat embedded in Epon. Thin sections were cut with a diamond knife and mounted on 200-mesh nickel grids having a carbon-coated formvar film. The sections were processed for immunocytochemistry using the modified protein A-gold immunocytochemical technique for the detection of antigenic sites on osmium postfixed tissues (Bendayan and Zollinger, 1983; Bendayan, 1984; Nanci et al., 1985) Briefly, the sections were pretreated with sodium metaperiodate for 1 hour and then incubated with a rabbit polyclonal antibody against SDS-denatured mouse amelogenins (Slavkin et al., 1982)at a 1/30 dilution in 0.01 M phosphate-buffered saline containing 1%ovalbumin for 3 hours at room temperature followed by protein A-gold complex for 30 minutes. These antibodies have been found t o crossreact with several mammalian species; however, they do not distinguish between amelogenins and enamelins (Slavkin et al., 1982). As a control, the antibody was absorbed with excess antigen. Control experiments with protein A-gold alone and preimmune serum were reported previously (Nanci et al., 1985). After the immunocytochemical procedure, the sections were stained with uranyl acetate and lead citrate, and examined with a Philips 410 electron microscope at 80kV. RESULTS Morphological Alterations Induced by Vinblastine Although the external conformation of ameloblasts exposed to high doses of vinblastine sulphate was not significantly affected after 1 or 2 hours, their internal organization was disrupted. Ameloblasts from both regions of inner (Figs. 1,2,3,4,6,7)and outer (Figs. 5,111 enamel secretion seemed to have been affected in a similar manner. Microtubules were rarely observed, but the filaments associated with junctional complexes (particularly the proximal one) in many cases still appeared to persist (Figs. 1,14). Occasional accumulations of filaments were observed throughout the cytoplasm. The infranuclear mitochondria1 compartment contained fewer mitochondria than normal, but abundant profiles of rough endoplasmic reticulum and numerous granules of varying size and density were now present (Fig. 1). Nuclei appeared to have been displaced distally (Figs. 1, 141, resulting in an elongated infranuclear comDartment. The normal tubula; configuration of the Golgi 116 A. NANCI, T. UCHIDA, AND H. WARSHAWSKY Fig. 2. Cross-sectioned inner enamel secretory ameloblasts. In some cells the Golgi apparatus (G)is no longer a cylindrical tube but is fragmented into separate stacks of saccules that have been displaced to the periphery of the cell. Clusters of granules (arrows) are also seen near the cell membrane and are often associated with extracellular patches of granular material (*). ly, lysosome; N, nucleus. x 14,850. EFFECTS OF VINBLASTINE ON SECRETORY AMELOBLASTS 117 Fig. 3. Distal portion of inner enamel secretory ameloblasts. The rough endoplasmic reticulum (rER) extends into the proximal portion of Tomes’ process ( p a , but the interdigitating portion (iT) appears devoid of organelles. A fine particulate material (pm) accumulates in some processes. The membrane associated with both the interrod (ir) and rod (r) growth sites is not infolded. g, granule; ly, lysosome. x 13,750. apparatus was broken into stacks of saccules distributed out the entire cell and extended into Tomes’ process throughout the supranuclear compartment (Figs. 2,11, (Figs. 33). Clusters of secretory-like, dense-content 16). Occasionally, membrane profiles resembling Golgi granules were found throughout the cell pigs. 2,3,7, saccules appeared in the infranuclear compartment. 11). These granules were either dark or pale-staining The rough endoplasmic reticulum was dispersed through- (Figs. 2,7) similar to those found in normal ameloblasts Fig. 4. Tomes’ processes (Tomes) from inner enamel secretory amelo- points (arrows). Some granules with flocculent material (fg) and phablasts appear devoid of organelles and the membrane associated with gosomes (ph) are also seen in the proximal portion. The membrane interrod (ir) and rod (r) growth sites lacks significant infolding. x 14,000. associated with the growing interrod (ir) and rod (r) enamel is not significantly infolded. x 13,750. Fig. 5. Distal portion of outer enamel secretory ameloblasts. Rough Fig. 6.Cross section of the interdigitating portions of Tomes’ proendoplasmic reticulum (rER) extends into the proximal portion of Tomes’ process (pT) and occasionally some cisternae reach the interdi- cesses in inner enamel secretion showing “fissures” (arrows). The rod gitating portion (iT). This latter portion appears fissured at several (R) associated with these processes seem to be subdivided. x 13,750. Fig. 7. Electron micrograph showing the various types of granules Fig. 8 . Thin section of inner enamel secretory ameloblasts fixed with observed in ameloblasts treated with vinblastine. The cluster of dense- glutaraldehyde only and incubated with antiamelogenins antibodies content granules in cell 1contains both pale (pg) and dark (dg) gran- revealed by the protein A-gold complex. The section was stained with ules. In cell 2, two clusters of granules with flocculent material (fg), uranyl acetate and lead citrate. Both pale (pg) and dark (dg) granules are seen. Between the cells there is a patch of granular material (*I m, in cell 1 and granules with a core of flocculent material (fg) in cell 2 mitochondria; N, nucleus. X29,250. are labeled by gold particles. Only the core portion of these granules is labeled. The rough endoplasmic reticulum (rER), the patch of extracellular material (*) and a larger lysosome-like granule (ly) also show labeling by gold particles. x37,200. Fig. 9. Immunocytochemical preparation as Figure 8. Profiles of Tomes’ process of inner enamel secretion, cut in different planes of section, are devoid of organelles and show no significant labeling. An accumulation of filaments (f) is seen in one process. The cell surfaces associated with rod growth sites are smooth (r). The enamel shows an intense labeling by gold particles. x 16,350. Fig. 10. Immunocytochemical preparation as Figure 8. This electron micrograph shows three patches of granular material (*I found laterally between cells of inner enamel secretion and labeled by gold particles. Clusters of dense-content granules (g), labeled by gold particles, are associated with these patches. X42,250. Fig. 11. Supranuclear compartment of outer enamel secretory ameloblasts from vinblastine injected animals fixed with the aldehyde mixture. Several phagosomes (ph) and lysosomes (ly) are seen in proximity to the Golgi apparatus (GI. Some of these phagosomes appear to have engulfed intact granules (arrows). ~22,500. Fig. 12.Immunocytochemical preparation as Figure 8. The granules (arrows) present in phagosomes (ph) are labeled by gold particles. The Golgi apparatus (G) shows some labeling. In some instances the saccules (curved arrows) contain accumulations of labeled material. ~42,250. 122 A. NANCI, T.UCHIDA, AND H. WARSHAWSKY Figs. 13-18.Inner enamel secretory ameloblasts from normal untreated (Figs. 13,15,17) and vinblastine injected (Figs. 14,16,18) rats stained for Golgi-related structures with the zinc iodide-osmium tetroxide method (210).Sections were stained with uranyl acetate and lead citrate. Fig. 13. Few stained structures (arrows) are seen in the infranuclear (in) compartment of ameloblasts from untreated normal rats. N, nucleus. x 11,600. Fig. 14. The infranuclear compartment (in) of vinblastine-treated ameloblasts contains many stained tubular and vesicular structures (arrows). These structures are clustered similar to the granules with flocculent content seen in figure 1. N, nucleus. ~ 1 1 , 6 0 0 . EFFECTS OF VINBLASTINE ON SECRETORY AMELOBLASTS Fig. 15. The Golgi apparatus of normal untreated ameloblasts is a large, centrally located, cylindrical structure consisting of heavily stained interconnected stacks (GS)of saccules. X24,300. 123 Fig. 16.After vinblastine treatment, the Golgi apparatus appears fragmented into separate stacks of saccules fGS) clearly visualized by the ZIO staining. x 11,600. 124 A. NANCI, T.UCHIDA, AND H. WARSHAWSKY (Nanci and Warshawsky, 1984a). Another granule-type was also observed; these were irregular in shape and had a core of flocculent material (Figs. 1,7). Such granules were often seen in close association with Golgi saccules and were occasionally interconnected by empty membranous channels. The dense-content granule clusters were often in proximity to extracellular patches of granular material found laterally between cells (Figs. 2,7). No crystal-like structures were seen in this material. Large lysosome-like granules (Figs. 2,3,11)and phagosomes (Figs. 5,11) containing cellular debris or granules were also seen, particularly in outer enamel secretory ameloblasts. The most dramatic alterations were observed in Tomes’ process. The area corresponding to the proximal portion of Tomes’ process (Figs. 3,5) and occasionally the interdigitating portion (Fig. 5) contained rough endoplasmic reticulum and occasional granules. However, the interdigitating portion of Tomes’ process was completely devoid of other organelles (Figs. 3,4,5,). Rod and interrod crystallites abutted on the membrane of Tomes’ process; however, no characteristic membrane infoldings (Nanci and Warshawsky, 198413)were associated with either the rod or interrod growth sites (Figs. 3,4,5). In many instances, Tomes’ process and the associated enamel rod appeared fissured (Fig. 6). Occasionally, fine particulate material (Fig. 3) or filaments (Fig. 9) accumulated in Tomes’ process. ZIO Staining of Golgi-RelatedStructures In untreated animals, ZIO stained the Golgi apparatus, which appeared as a large interconnected and centrally located cylindrical structure (Fig. 15). In Tomes’ process tubular and vesicular elements in the central core of organelles were also reactive. Some secretorylike granules stained; others did not (Fig. 17). Few stained structures were present in the infranuclear compartment (Fig. 13.) In vinblastine injected rats the Golgi apparatus was fragmented into several ZIO stained bodies dispersed throughout the supranuclear cytoplasm (Fig. 16). Whereas no reactive elements were seen in Tomes’ process (Fig. 18),stained vesicular elements were now present in the infranuclear compartment (Fig. 14). lmmunocytochemical Labeling Sections of tissue from vinblastine treated animals incubated with the antiamelogenins antibody were specifically labeled (Figs. 8,9,10,12).The enamel (Fig. 9) and extracellular patches of granular material (Figs. 8,101 were labeled with numerous gold particles. Intracellularly, the rough endoplasmic reticulum (Fig. 8) and the Golgi apparatus showed some labeling (Fig. 12). Gold particles were also present over the various granule types (Figs. 8,101 and over larger lysosome-likegranules (Fig. 8). Occasionally, lysosomes containing small secretory-like granules were encountered. The small granules were labeled, but the matrix of the lysosome was not (Fig. 12). Only few, randomly dispersed gold particles were observed over the tissue section when the antibody was absorbed with excess antigen. DISCUSSION Morphological Alterations Induced by Vinblastine The present study has shown that vinblastine sulphate, which disrupts microtubules, produces severe ef- fects on secretory ameloblasts as early as 1 to 2 hours after injection. Both inner and outer enamel secretory ameloblasts appeared similarly affected, emphasizing the functional similarity between these regions of amelogenesis. In accordance with previous reports (Moe and Mikkelsen, 1977a; Takuma et al., 1982), and similar to the effects of colcemid (Karim and Warshawsky, 19791, the major ultrastructural alterations produced by this drug are the disorganization of organelles within ameloblasts and the loss of directional movement resulting in the accumulation of secretory granules at Golgi sites throughout the cell. The build-up of these granules eventually results in their ectopic release. ZIO, although nonspecific, stains Golgi-related structures (Ozawa et al., 1983). It is thus particularly suited to visualize the alterations of the Golgi network induced by vinblastine. The present study suggests that in normal ameloblasts there are fine, membrane-lined tubular channels that are associated with the Golgi apparatus and extend into Tomes’ process, possibly related to one type of secretory granule. After vinblastine administration, the Golgi apparatus fragments into separate stacks of saccules, which disperse throughout the supranuclear region of the cell. The tubular channels related to the Golgi apparatus, which normally extend into Tomes’ process, are lost. This implies that the network of channels turns over at the same rate as the secretory granules in Tomes’ process. The appearance of ZIO structures in the infranuclear compartment reflects the appearance of Golgi related structures, presumably de nouo, since intracellular migration is arrested. We postulate that this is an attempt by the cell to revert to its embryonic orientation where the Golgi apparatus occupied the infranuclear cytoplasm. Radioautographic studies using 3H-fucose(Bennett et al., 1984)have shown that glycoprotein synthesis continues after vinblastine administration, but there is a temporary inhibition of migration and directional exocytosis of secretory granules. Colcemid, a drug that produces similar ultrastructural changes in secretory ameloblasts, was also shown not to abolish protein synthesis but to delay protein migration and secretion (Karim and Warshawsky, 1979).It has been suggested that secretory ameloblasts possess two secretory sites and that these are characterized by membrane infoldings (Nanci and Warshawsky, 1984a). We have found that vinblastine produces a dramatic loss of these infoldings. Membrane infoldings may reflect a general attribute of all secretory sites, or they may be specifically involved with events leading to the organization of enamel. In cases of rapid secretory activity, excess membrane could result and become infolded as a mechanism for its removal. The loss of infolded membrane at the ameloblast secretory sites after vinblastine could be the result of secretory granules no longer reaching and fusing with the membrane at these sites. The fact that infoldings were not found at ectopic sites may reflect insaicient exocytosis or it may argue against this being a general feature of secretory sites. On the other hand, the loss of membrane infoldings concomitant with the loss of secretory granules and other organelles from Tomes’ process suggests that infoldings reflect cellular activity related to secretion and extracellular organization of enamel. Regardless of the origin of these membrane infoldings, they are always associated with the growing end of rod and inter- EFFECTS OF VINBLASTINE ON SECRETORY AMELOBLASTS 125 Fig. 17. In normal animals, Tomes’ process (Tomes) contains a subFig. 18. Vinblastine treatment abolishes most stained structures stantial network of stained tubular and vesicular elements. Whereas from Tomes’ process Vomes). x 11,600. some secretory-like granules stain, others do not. X 15,900. 126 A. NANCI, T. UCHIDA, AND H. WARSHAWSKY rod enamel (Weinstock and Leblond, 1971; Warshawsky et al., 1981; Nanci and Warshawsky, 1984a) and they disappear when secretion is arrested. It is thus confirmed that membrane infoldings are a characteristic feature of the natural sites where enamel proteins are released, and we favor the view that membrane infoldings may represent intense cellular motility related in some way to the structuring of enamel, either by orienting the matrix or the crystallites. lmmunocytochernical Localization of fnarnel Proteins The immunocytochemical labeling obtained indicates that the small granules forming clusters, irrespective of their shape and density, contain enamel proteins. The labeling of large lysosome-like granules raises the possibility that secretory ameloblasts degrade a portion of their secretory product (posttranslational degradation, crinophagy) or have a resorptive activity (Nanci et al., 1985). The presence of secretory granules in lysosomes (Figs. 11,121represents classical crinophagy (Smith and Farquhar, 1966). Such a presence has never been reported in normal ameloblasts and seems to be induced or accentuated by vinblastine treatment. Likewise, the extracellular patches of material found laterally between cells consist of enamel proteins. This material is generally not found, at least with such a frequency, between secretory ameloblasts. The patches may thus represent ectopic secretion (Moe and Mikkelsen, 1977a; Takuma et al., 1982, 1984; Karim and Warshawsky, 1979).As reported by Takuma et al. (19841, we also have seen no crystal-like structures within these patches. Assuming that both classes of enamel protein (amelogenins and enamelins) are secreted in tandem, the absence of mineralization within the patches suggests that the enamel layer, as opposed to the patches, must represent a microenvironment that favors crystal initiation and growth and that de nouo crystal formation requires more than the simple presence of enamel proteins. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Ms. Annie Belanger. We are grateful to Dr. H.C. Slavkin of the University of Southern California for having generously supplied the antibodies. This work was supported by grants from the Medical Research Council of Canada. LITERATURE CITED Bendayan, M. (1984)Protein A-gold electron microscopic immunocytochemistry: Methods, applications and limitations. J. Elect. Microsc. Tech., 1243-270. Bendayan, M., and M. Zollinger (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J. Histochem. Cytochem., 31:lOl-109. Bennett, G., S. Parsons, and E. Carlet (1984) Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins. I. Inhibition of glycoprotein migration in various rat cell types as shown by light microscope radioautography after injection of 3H-fucose. Am. J. Anat., 170.521-530. Ekholm, R., L.E. 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