Experimental
Cell Research 95 (1975) 440-454
INFLUENCE OF COLCHICINE AND VINBLASTINE ON THE GOLGI COMPLEX AND MATRIX DEPOSITION IN CHONDROCYTE AGGREGATES An Ultrastructural S. MOSKALEWSKI,
J. THYBERG,
Study
S. LOHMANDER
and U. FRlBERG
Department of Histology, Karolinska lnstitutet, S-10401 Stockholm 60, Sweden
SUMMARY Fetal guinea-pig epiphyseal chondrocytes were isolated enzymatically, aggregated, and the aggregates maintained in organ culture. As revealed by light and electron microscopy, the cultures produced a typical cartilaginous matrix, but no calcification occurred. Exposure of aggregating cells, or preformed aggregates, to colchicine or vinblastihe at IO+ M concentration led to disappearance of the microtubules, dissociation of the Golgi complex into single dictyosomes, and clustering of lysosomes. Thus, in treated cells the dictyosomes with accompanying vesicular structures were dispersed throughout the cytoplasm, whereas they were localized in a well-defined juxtanuclear region in control cells. The number and size of the cisternae forming a dictyosome were often reduced. Cells treated with vinblastine displayed macrotubules and an increased number of phagosomes. Both drugs reduced the deposition- of intercellular matrix. In cells first exposed to either of the drugs for 2 or 5 days and then transferred to fresh medium for 3 or 6 days, the microtubules reappeared, the Golgi complex regained its normal appearance, and the amount of matrix increased. These findings are discussed in view of present concepts of the role of microtubules in cell secretion.
A variety of antimicrotubular agents has been reported in the literature. Some of these, like colchicine, vinblastine and vincristine (for recent reviews see [ 1,2]), cause a disruption of the microtubules, whereas others such as ethanol, hexylene glycol [3, 41 and deuterium oxide [5,6] stabilize these organelles. Abundant evidence has accumulated indicating that antimicrotubular agents impair the secretion of many cell products, e.g. hormones such as insulin [7-91 and thyroid hormone [lo, 111. More recently, microtubules have also been implicated in the secretion of the macromolecules of connective tissue matrices. Thus, it has been found that colchicine and vinblastine, as well as other antimicrotubular Exptl
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agents, inhibit both secretion and synthesis of collagen in vitro [12-161. For example, it was reported that colchicine and vinblastine decreased the rate of secretion of collagen by cells isolated from chickembryo tendons by about 70 %, and that the amount of intracellular collagen concomitantly increased about two-fold [ 121. In a biochemical and ultrastructural study on bone cells, Ehrlich et al. [16] found that colchicine, in addition to the effect on secretion, also depressed collagen synthesis, whereas vinblastine inhibited synthesis of collagen as well as other proteins. These authors suggested that the reduction in collagen formation by colchicine is due to an accumulation of collagen in the Golgi
Znjluence
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vacuoles, with secondary inhibition of its synthesis. Likewise, colchicine and vinblastine have been found to depress both secretion and synthesis of glycosaminoglycans by isolated chick-embryo chondrocytes, but, contrarily, did not greatly reduce the synthesis of collagen by cartilaginous tibia anlagen [17]. On the basis of these findings it is usually assumed that microtubules are involved in the intracellular translocation of secretory vacuoles, and that, as regards matrix deposition, the primary effect of antimicrotubular agents in connective tissue cells is on secretion. Further experimental data are needed, however, to support this hypothesis and to elucidate the particulars of the functional role of microtubules in secretion. The present report is concerned with the influence of colchicine and vinblastine on chondrocyte fine structure and matrix deposition in vitro. Both short- and long-term effects were studied, as well as the ability of the cells to recover after prolonged exposure to these drugs. During the course of the experiments it was found that the antimicrotubular agents caused marked changes in the Golgi complex. As an attempt to determine whether these alterations were directly due to the absence of microtubules, or were secondary to inhibition of the synthesis of matrix components, the effects of puromycin, a potent protein synthesis inhibitor, on cellular fine structure was also examined. As experimental system we used chondrocytes isolated from cartilaginous epiphyses of guinea pig fetuses, aggregated, and thereafter maintained in organ culture. Because of the potential usefulness of this system for other studies on the function and metabolism of cartilage, the morphology of the chondrocyte aggregates was recorded for up to 25 days, i.e. beyond what
on chondrocyte
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was necessary for the experimental poses proper. MATERIAL
441 pur-
AND METHODS
Pregnant guinea pigs were obtained from a local deafer. The age of the fetuses was 40-50 days, as determined from the weight-age table presented by Draper [ 181.
Animais.
medium. Cells were grown in Ham’s F-12 medium supplemented by 10% fetal calf serum (BioCult), 0.3% tryptose phosphate broth (Bacto) and 50 pg of L-ascorbic acid, 150 pg of streptomycin sulfate (Glaxo) and 150 U/ml of benzylpenicillin (Glaxo). The same medium was used for aggregation of chondrocytes and for subsequent organ culture of aggregates. Culture
ofchondrocytes. The detailed procedure for chondrocyte isolation has been described previously [19, 201. Briefly, dissected fragments of cartilaginous epiphyses of long bones were pooled, digested with collagenase and DNase, and the liberated chondrocytes were then used for aggregation,
Zsolation
and organ culture of chondrocvtes. Chond&y& were aggregated in sihconized 25 ml Erlenmeyer flasks containing 3 x lo6 cells in 3 ml of medium. Each flask was spun at 75 rpm around its axis, using a modification of an apparatus described by Gaillard et al. [19]. After 48 h the aggregates were put on a piece of lens paper supported by a metal grid and maintained in organ culture dishes (Falcon). The medium was changed every 2-3 days. Samples for light and electron microscopy were taken at various time intervals covering I-25 days from the beginning of aggregation. Annregution
to antimicrotubular agens. Crystalline colchicine (Merck) and vinblastine sulfate (Sigma) were used at 10V5 M concentration. Three different experimental series were run. (a) For observations on shortterm effects, the cells were aggregated for 24 h in normal medium and the aggregates then exposed to colchicine or vinblastine for 40 min or 4 h. Alternatively, some of the aggregates were treated with puromvcin (Sigma), at a concentration of IO or 100 pgiml for4 h. (b) For observations on long-term effects, the cells were continuously exposed to either antimicrotubular agent, from the beginning of aggregation, for l-11 days. (c) For recovery studies, aggregates, after 2 or 5 days of exposure, were carefully rinsed, transferred to normal medium, and cultured for a further 3 or 6 days. Exposure
Light microscopy.Aggregates were fixed in the fluid
of Bouin-Hollande [21] or in glutaraldehyde as for electron microscopy. Sections were stained with hematoxylin and eosin, or with Alcian blue 8GS at pH 3.1 [22].
Electron
The material was fixed in 2% (Polaron) in 0.1 M cacodylate buffer,
microscopy.
glutaraldehyde
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Fine structure of cells. In l- and 2-dayold aggregates the cells were usually round or oval (fig. 3), but many of them had angular contours, or showed processes of varying size and shape. Nuclei were round or oval, with l-2 nucleoli. The granular endoplasmic reticulum was well-developed. In most cells its cisternae were narrow and filled with a material of high electron RESULTS density, while in others they were disStructure of normal aggregates tended, and their content was of lower Aggregates formed after 24 h of spinning density. Free ribosomes, frequently formof the isolated chondrocytes were flattened ing polysomes were also present. The Golgi and usually had an oval outline. The largest complex was prominent (figs 3,4), and conones reached a length of about 2 mm and a sisted of a few groups of flattened, parallel width of about 1 mm. Within the next 24 h cistemae, small vesicles and large vacuoles. the aggregates frequently formed long rib- In favorable sections up to 6 separate stacks bons by attaching to each other in the polar of cisternae (dictyosomes) with accomregions. panying vesicular structures could be disLight microscopy. In l-day-old aggre- tinguished; the dictyosomes were usually gates the intercellular substance, as visualcomposed of 3-5 cisternae. No direct conized by Alcian blue staining, was scanty nection between the different dictyosomes and consisted mainly of a narrow rim could be seen, but they all lay in a wellaround the cells (fig. 1). After 2-5 days the defined, juxtanuclear area. Microtubules matrix was better developed, particularly could be observed both at the periphery of in the central parts of the aggregates. Some this area and between the dictyosomes (fig. cells were degenerated and lay shrunken 4). Occasionally, l-2 centrioles were also in their lacunae. Cell nests could not be found within the same region. Some Golgi distinguished. During the continued cultivacuoles contained ruthenium red-stainable vation, up to 25 days, the amount of intermaterial. Mitochondria were small and had cellular substance increased but little (fig. few cristae. The majority of the cells con2). In older cultures, many cells were de- tained membrane-bounded dense bodies generated, and the intercellular matrix in (lysosomes), frequently with incorporated their vicinity remained unstained or stained cell remnants and/or myelinlike figures. only weakly. In 5-day-old and older aggregates the diversity of cell shapes was more pronounced than before. The number of disFig. 1. Fragment of l-day-old, non-exposed chondrocells successively increased, cyte-aggregate. Cells in close contact, small amount of integrated intercellular substance. H.E. x300. particularly in the central parts of the aggreFig. 2. Fragment of a 25day-old, non-exposed aggregates. Non-degenerated chondrocytes regate. Increased amount of intercellular substance as mained, however, in considerable numbers compared with l-day-old aggregates. H.E. x300. Fig. 3. 2-day-old, non-exposed aggregate. Chondrountil the end of the cultivation period. The cytes separated by intercellular substance. Cell on the majority of them progressively accumuright contains a prominent Golgi complex (G). X8000. lated dense bodies, which frequently conpH 7.3, for at least 2 h, posttixed in 1% osmium tetroxide in Veronal acetate buffer with added salts [23], dehydrated in ethanol, and embedded in Spun low viscosity medium [24]. To demonstrate proteoglycans some aggregates were stained by addition of 0.05% of ruthenium red to the osmium fixative [25]. Thin sections were cut with an LKB Ultrotome I. and double-stained with uranyl acetate followed by ‘lead citrate [26]. The specimens were examined in a Philips EM 300 electron microscope.
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tamed inclusions of the types mentioned above. Fine structure of intercellular substance. In l-day-old aggregates intercellular matrix was mainly seen in the central parts. In 2day-old aggregates the amount of matrix was increased, but the difference between central and peripheral parts was still evident. There were also considerable variations between different areas within the aggregates. The intercellular substance contained dispersed collagen fibrils and matrix granules stainable with ruthenium red (fig. 5). The fibrils had an appearance typical of cartilage collagen. They had a diameter of 150-250 A and exhibited but a faint crossstriation. With time the intercellular substance became more prominent. Even after 25 days of culture, however, the matrix was less dense than in fetal, cartilaginous epiphyses (unpublished observations). In older aggregates many cells were surrounded by a clear zone, containing single matrix granules and practically no fibrils. No signs of calcification were noted. A finding of incidental interest was that the outer surfaces of peripheral cells were covered by a ruthenium red-stainable material. This material often had a patchy distribution and could originate from the
Fig. 4. Chondrocyte from 2-day-old, non-exposed aggregate. Well developed Golgi complex with numerous microtubules (arrows) within, and at the periphery of, the complex. x26000. Fig. 5. Intercellular substance from l-day-old, non-. exposed aggregate. Collagen fibres with faint crossstriation and matrix granules stained with ruthenium red. x70000. Fig. 6. 2-day-old aggregate formed in the presence of colchicine. The amount of intercellular substance is smaller than in the corresponding control aggregate (cf fin. 3). None of the cells contains an intact Golgi complex: x8000.
on chondrocyte
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445 (cf
r271). Effects of antimicrotubular agents Neither colchicine nor vinblastine interfered with the aggregation of the chondrocytes. No microtubules could be seen in exposed cells at any of the intervals covering the period of observation from 40 min to 11 days. Both agents also evoked striking changes in the Golgi complex, primarily a dissociation of the complex into single dictyosomes and their spreading throughout the cytoplasm. The first signs of this dissociation could be recognized already after 40 min, particularly in vinblastine-treated cultures. Thus, in some cells the distances between individual dictyosomes were somewhat increased, with interposition of ground cytoplasm with endoplasmic reticulum. The spreading was essentially complete after 4 h of exposure, and the dictyosomes, with accompanying vesicular structures, were now distinctly separated by other organelles, and frequently located on opposite sides of the nucleus (figs 6-10). The dissociation of the dictyosomes persisted throughout the experimental period (11 days). Furthermore, most of the dictyosomes showed an altered structure after dispersion. Many of them had cisternae that were fewer and shorter than in the controls. Such dictyosomes were accompanied by a group of a few vacuoles, while small vesicles were scanty. Other dictyosomes displayed a more normal complement of cisternae, but lacked vacuoles, and were thus associated mainly with small vesicles (figs 8-10). Intermediate forms were also present. No ruthenium red-stainable material was demonstrable within the vacuoles of dispersed dictyosomes. Exptl
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The dissociation of the Golgi complex was accompanied by the formation of clusters of lysosomes in the close vicinity of some of the dispersed dictyosomes (figs 11, 12). In addition, a considerable increase in the number of microfilaments was apparent in the chondrocytes after a few days of treatment with either drug. Exposure to vinblastine lead to an enhanced autophagocytosis in the chondrocytes; early signs thereof could be observed already after 40 min (fig. 13). After 4 h many cells contained lysosomes into which e.g. fragments of granular endoplasmic reticulum had been incorporated. At later intervals the chondrocytes showed a markedly increased number of phagosomes and phagolysosomes. In older cultures residual bodies with myelin-like figures were numerous. A second effect exclusively seen after exposure to vinblastine was the appearance of distinct, parallel arrays of macrotubules (figs 11, 12) of the type described previously and supposed to contain the microtubular protein, tubulin [28-3 11. The macrotubules were first seen after 1-2 days of treatment; their numbers increased with time. They had an average diameter of about 350-400 A and were accompanied by numerous polysomes. Crystals of the kind which have been reported to occur, in other types of cells, under the influence of vinblastine [28] were not encountered.
Fig. 7. Chondrocyte from 2-day-old aggregate formed in the nresence of colchicine. The Go& comnlex is dispersed into single, inconspicuous -dictydsomes (arrows). Otherwise cell structure appears normal. x 16000. Fin. 8. Two dictvosomes from the cell shown in tie. 7. Short, inconspicuous cistemae accompanied by &all and medium-sized vesicles. x36 000. Fig. 9. Dictyosome with a reduced number of cisternae accompanied by a few large vacuoles and small vesicles; 2 days of colchicine treatment. x36000. 30-751814
on chondrocyte
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Both colchicine and vinblastine caused a reduction in the amount of intercellular matrix deposited in the aggregates of the long-term series. Already in l-day-old aggregates the intercellular substance was clearly less developed than in the controls (fig. 6), but as in the latter contained both collagen fibrils and matrix granules. The amount of matrix increased with time of culture but to a lesser extent than in nonexposed aggregates. Effects ofpuromycin. Puromycin did not, at either concentration, affect the number and structure of the microtubules. Nor did it cause any dissociation of the Golgi complex (fig. 14); the number of dictyosomes, and the number of cisternae within these, were unaltered. The Golgi vacuoles were, however, reduced in number and lacked the typical content seen in control cells. Conversely, the small Golgi vesicles were more numerous. In some cells autophagocytosis of, inter alia, granular endoplasmic reticulum was noted. Moreover, some cells displayed clumping of chromatin. Recovery. Chondrocytes, exposed for 2 or 5 days to either of the antimicrotubular agents, and then cultured for 3 or 6 days in drug-free medium, showed a normal complement of microtubules. In cells recovering from vinblastime treatment macrotubules were no longer present. Moreover, in most cells the dictyosomes were again localized in one well-defined area (fig. 15). The cisternae were well-developed and the number of large vacuoles and small vesicles was similar to that in the control cells. In a few chondrocytes, however, single dictyosomes remained separated from the main Golgi area even after 6 days. Clustering of lysosomes was no longer observed. This recovery in cellular fine structure was accompanied by an increase in the amount of deposited matrix, which did not, however, Exptl
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attain the level existing in non-exposed cultures of comparable age. DISCUSSION Experimental system Chondrocytes were chosen for the present study since they are readily isolated and produce large amounts of intercellular matrix in vitro. The experimental system was based on the methods for isolation and aggregation of chondrocytes proposed by Gaillard et al. [19]. Both in the experiments of these authors, in which fetal, mouse epiphyseal chondrocytes were aggregated and cultured for up to 10 days, and in our experiments, with fetal guinea pig material, the aggregated cells elaborated a typical intercellular matrix. The accumulation thereof was most conspicuous during the first few days of culture. In both studies, however, no further maturation of the cartilage was observed. On the other hand, guinea-pig fetal chondrocytes, isolated as in the present study and transplanted intramuscularly into young guinea pigs, are capable of reforming cartilage in which both hypertrophy of cells and calcification of the matrix are evident [20]. Moreover, primordia of long bones consisting of small-
IO. Chondrocyte from l-day-old aggregate formed in the presence of vinblastine. Dispersion of Golgi complex into single dictyosomes (arrows). Scanty intercellular substance. X 13 000. (Inset). Separated dictyosome from the same cell (crossed arrow). Inconspicuous cisternae, a few large vacuoles and small vesicles. x40000. Fig. II. Chondrocyte from an S-day-old aggregate treated continuously with vinblastine. A bundle of macrotubules with accompanying polysomes. Abundant microtilaments. Clusters of lysosomes occur in the vicinity of single dictyosdmes (arrows). x 14000. Fig. Z2. Chondrocyte from an &day-old aggregate, exposed continuously to vinblastine. Bundle of macrotubules in cross-section and a group of lysosomes. x38000. Fig.
on chondrocyte
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449
celled precartilages are capable of developing into bone in culture [32, 333. This points to some inadequacy of the in vitro conditions to support maturation of cartilage formed by isolated chondrocytes. Znfluence of colchicine and vinblastine on cell structure The presence of microtubules within the Golgi complex has previously been established both in plant [34] and animal cells, e.g. chondrocytes [35]. In the present study microtubules were regularly observed within and in the vicinity of the Golgi complex in the control cells. Both colchicine and vinblastine caused a rapid disappearance of the microtubules in the cytoplasm of the chondrocytes. Microtubules could not be seen at any of the intervals studied, which indicates that the concentration of the drugs remained effective between changes of medium. The disappearance of the microtubules was followed by dissociation of the Golgi complex and spreading of the dictyosomes throughout the cytoplasm. This would seem to imply that microtubules are necessary to maintain the structural integrity of the Golgi complex. As far as we can ascertain the first observations on the influence of colchicine and vinblastine on the Golgi complex were described by Robbins & Gonatas [36]. They found a fragmentation and circumferential distribution of the Golgi apparatus in HeLa cells exposed to these agents for 10 h. More recently, in a study on a mouse embryonic cell line, treatment with colchicine, vinblastine and vincristine was reported to result in ‘explosion’ of the Golgi field and distribution of individual ‘Golgi organelles’ over the entire cytoplasm [37]. In the main, these observations seem to be in agreement with those of the present study. Our explanation of the mechanism of Exprl
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Fig.
IS. Chondrocyte from an 8day-old aggregate treated with vinblastine for 5 days and then cultured in normal medium for 3 days. Reformed Golgi complex
with structure corresponding to that in untreated cells, cf fig. 4. C, centriole; DZf, dense body with myelinlikefigures. x26000.
dictyosome dispersion as due to destruction of microtubules could be challenged on the basis of the feedback theory of Ehrlich et al. [16], according to which depression of secretion would lead to secondary inhibition of synthesis and, presumably, functional and morphological alterations of the Golgi apparatus. Such an argument
would, however, be difficult to accept for cartilage cells. Our observations did not suggest any accumulation of secretory products in the Golgi complex. Further, if secondary inhibition of the formation of matrix components occurred it could possibly evoke changes in the Golgi complex in situ, but should not be expected to cause dispersion of the dictyosomes throughout the cytoplasm. This reasoning is supported by the observations in the control experiment with puromycin, a general inhibitor of protein synthesis [38]. Thus, this agent did not cause any dissociation of the Golgi complex although a clear reduction in the number of secretory vacuoles was observed. These effects on the Golgi complex
Fig. 13. Chondrocyte from l-day-old aggregate, exposed to vinblastine for 40 min. Beainnina of disnersion of Golgi complex and format& ofautophagosomes encompassing fragments of granular endoplasmic reticulum (arrows). x 17 000. (Zraser). Forming autophagosome from the same cell (crossed arrow). X60000. Fig. 14. Chondrocyte from l-day-old aggregate exposed to puromycin (100 w/ml) for 4 h. Dictyosomes are located in a well-defined, juxtanuclear area. x39000.
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as well as the effect on other cell components in the chondrocytes are similar to those described in a study on exocrine cells in the pancreas of puromycin-treated rats r391. The dissociation of the Golgi complex was accompanied, in some dictyosomes, by a reduction in the number of the cisternae. This could be due to their splitting off during or after the process of dispersion. Taking into consideration present concepts of the formation and function of the Golgi complex, another and more likely explanation is that the dispersion of the dictyosomes is followed by a disturbance of the formation of new cisternae. Thus, electron microscopic observations suggest that new cisternae are formed at the outer or forming (cis) face of the dictyosome by fusion of vesicles arriving from the endoplasmic reticulum. On the inner, maturing (tram) face of the dictyosome the cisternae seem to transform into vacuoles containing secretory products (for reviews, see [40421). In the goblet cells of the intestine new Golgi cisternae were estimated to form every 2-4 min [43]. From the above it follows that, if for some reason the inflow of vesicles from the endoplasmic reticulum to the dictyosome would be interrupted, its morphology and function would be seriously affected. No formation of new cisternae at the outer surface of the dictyosome would occur. On the inner surface transformation of the cisternae into secretory vacuoles could probably continue for a short time, i.e. as long as the materials needed for their formation had not been exhausted. Such an inactive dictyosome could be expected to have a reduced number of cisternae and to be devoid of secretory products. It seems reasonable to assume that the dispersion of the dictyosomes will, at least temporarily, Exptl CellRes
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affect their normal relationship to the endoplasmic reticulum. After dispersion the dictyosomes may possibly reestablish a functional contact with this organelle. Conceivably, differences in the efficiency of such contact could explain the variations in number of cisternae of the dictyosomes and associated vacuoles and vesicles within single cells. The short-term effects of antimicrotubular agents on the Golgi complex have also been studied in connective tissue cells other than chondrocytes. Thus, in tendon fibroblasts incubated with colchicine for 3 h, the Golgi vacuoles were found to be increased both in number and size [44]. In cells of cranial bones, exposed to colchicine for about 1 h, there were increased numbers of Golgi-associated vacuoles and vesicles [16]. After incubation with vinblastine, the Golgi complex was small, contained few large vacuoles and had a larger than usual number of associated small vesicles. Moreover, the cisternae of the granular endoplasmic reticulum were vesicular in appearance as compared with the long, interconnecting channels in the control cells. These observations contrast with those reported here. Thus, dissociation of the Golgi complex was not noted in the above studies, although discernible already after 40 min in our material. Furthermore, in the chondrocytes the alterations in the number of Golgi vacuoles and vesicles comprised, at all intervals, a decrease rather than an increase; any clear changes in the granular endoplasmic reticulum were not observed even after prolonged exposure. Obviously, more experimental data are needed to explain these differences in the response of various connective tissue cell types to antimicrotubular agents. In addition to the changes, induced by colchicine and vinblastine, in the Golgi
Influence
of colchicine
and vinbiustine
complex of the chondrocytes there was a clustering of lysosomes and, particularly with the latter agent, an enhanced autophagocytosis. Similar effects of antimicrotubular drugs have been observed in studies on other cell types both in vivo and in vitro 136, 45,461. The changes in cellular fine structure caused by colchicine and vinblastine were reversible. Thus, after withdrawal of the drugs the Golgi complex of the chondrocytes regained its normal appearance. The Golgi complex could be restored from the dissociated dictyosomes, or formed de novo with concomitant or subsequent destruction of the dispersed components. The restoration of cellular morphology was accompanied by an increased deposition of matrix components, indicating that also a functional recovery of the chondrocytes took place. Influence of colchicine and vinblastine on the secretion of matrix components It is generally accepted that the Golgi complex is involved in the synthesis and secretion of matrix components in connective tissue cells. Thus, there is considerable morphological and biochemical evidence indicating that the formation of both collagen and proteoglycans is initiated in the granular endoplasmic reticulum, and then completed during transport through the cisternae of the reticulum to the Golgi complex, as well as within this latter system, where the molecules finally are accumulated in vacuoles before being released to the extracellular space by exocytosis. However, as far as collagen is concerned it has been suggested that other secretory pathways may also exist (for recent reviews see [47-49]). During recent years antimicrotubular
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agents have been found to interfere with both secretion and synthesis of collagen in cultured bones and fibroblasts [12-161, and of glycosaminoglycans in isolated chondrocytes [17]. On the basis of these findings it has been suggested that disruption of microtubules interferes with the movement of Golgi vacuoles to, the cell surface and that this results in accumulation of secretory products in the Golgi complex, which then secondarily leads to an inhibition of synthesis [IL171. In the present study, aggregates formed and cultured in the presence of colchicine and vinblastine showed a reduced content of intercellular matrix. This reduction comprised both collagen fibrils and matrix granules, the latter demonstrated to contain proteoglycans by ruthenium red staining [50, 511. Considerable amounts of intercellular matrix werk, nevertheless, laid down in the treated aggregates. Hence, the chondrocytes retained part of their capacity for synthesizing and secreting extracellular macromolecules in spite of the disappearance of the microtubules and the structural alterations of the Golgi complex. It is evident from our results that the reduction of matrix deposition in aggregates treated with colchicine and vinblastine could be due to a primary disfunction of the Golgi complex as well as to a disturbance in the transport of secretory vacuoles. Since separation of these two effects is not feasible at present, no definite conclusions as to the role of microtubules in the translocation of secretory vacuoles in chondrocytes would seem justified. Financial support, including personal fellowshios for Dr S. Mosk&wski, was obtained from the Swedish Medical Research Council (oroi. no 12X-33553, the Swedish Cancer Society (proj: nd. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Swedish Committee on International Health Relations, the Swedish Institute, the A. 0. SW&d Foundation, the M. Bergvail Foundation. and from the Funds of Karolinska Exptl
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Institutet. Antibiotics were generously supplied by Glaxo Laboratories Ltd (Sweden). The authors are also indebted to Mrs Eva Lundberg and Miss Karin Askfors for technical assistance, and to Mrs Ingrid Wtilma for preparation of the manuscript. S. M. is on leave from the Department of Histology and Embryology, Medical Academy, Warsaw, Poland.
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43. 44. 45. 46. 47. 48. 49. 50. 51.
Spurr, A R, J ultrastruct res 26 (1969) 3 I. Luft, J H, Anat ret 171 (1971) 347. Reynolds, E S, J cell biol I7 (1963) 20X. Temmink, J H M & Spiele, H, J cell biol 61 (1974) 548. Bensch, K G & Malawista, S E. J cell biol 40 (l%9) 95. Krishan, A, J ultrastruct res 31 (1970) 272. Tyson, G E & Bulger, R E, Am j anat I35 (1972) 319. - Z Zellforsch 141 (1973) 443. Biggers, J D, Cells and tissues in culture (ed E N Willmer) vol. 2, p. 198. Academic Press, New York (l%5). Fell, H B & Robinson, R, Biochem j 23 (1929) 767. Mollenhauer, H, H, J cell sci 15 (1974) 89. Thyberg, J, Nilsson, S & Friberg, U. Z Zellforsch 146 (1973) 83. Robbins, E & Gonatas, N K, J histochem cytothem 12 (1964) 704. De Brabander, M, Aerts, F, Van De Veire, R & Borgers, M, Nature 253 (1975) 119. Stewart, P R, The ribonucleic acids (ed P R Stewart & D S Letham) p. 151. Springer-Vet-lag, Berlin (1973). Longndcker: D S, Shinozuka, H & Farber, E, Am j pathol 52 (l%8) 891. Beams, H W & Kessel, R G, Int rev cytol23 (1968) 209. Favard, P, Handbook of molecular cytology (ed A Lima-de-Faria) D. 1130. North-Holland, Amsterdam-London (1969). Morre, D J, Mollenhauer, H H & Bracker, C E, Results and problems in cell differentiation (ed J Reinert & H Ursprung) vol. 2. Origin and continuity of cell organell&, p. 82. Springer-Verlag, Berlin (1971). Neutra, M & Leblond, C P, J cell biol 30 (1966) 119. Olsen, B & Prockop, D J, Proc natl acad sci US 71 (1974) 2033. Ekholm. R. Ericsson. L E. Josefsson. J 0 & Melander, A, Endocrinology 94 (1974) 64l. Arstila, A U, Nuuja, I J M & Trump, B F, Exp cell res 87(1974)249. Miller, E J & Matukas, V J, Fed proc 33 (1974) 1197. Rod&, L, Metabolic conjugation and metabolic hydrolysis (ed W H Fishman) p. 345. Academic Press. New York (19701. Weinstock, M & Leblond, C P, Fed proc 33 (1974) 1205. Luft, J H, Anat ret 171 (1971) 369. Thyberg, J, Lohmander, S & Friberg, U, J ultrastruct res 45 (1973) 407.
Received February 28, 1975 Revised version received April 17, 1975
Cell Research 95 (1975) 440-454
INFLUENCE OF COLCHICINE AND VINBLASTINE ON THE GOLGI COMPLEX AND MATRIX DEPOSITION IN CHONDROCYTE AGGREGATES An Ultrastructural S. MOSKALEWSKI,
J. THYBERG,
Study
S. LOHMANDER
and U. FRlBERG
Department of Histology, Karolinska lnstitutet, S-10401 Stockholm 60, Sweden
SUMMARY Fetal guinea-pig epiphyseal chondrocytes were isolated enzymatically, aggregated, and the aggregates maintained in organ culture. As revealed by light and electron microscopy, the cultures produced a typical cartilaginous matrix, but no calcification occurred. Exposure of aggregating cells, or preformed aggregates, to colchicine or vinblastihe at IO+ M concentration led to disappearance of the microtubules, dissociation of the Golgi complex into single dictyosomes, and clustering of lysosomes. Thus, in treated cells the dictyosomes with accompanying vesicular structures were dispersed throughout the cytoplasm, whereas they were localized in a well-defined juxtanuclear region in control cells. The number and size of the cisternae forming a dictyosome were often reduced. Cells treated with vinblastine displayed macrotubules and an increased number of phagosomes. Both drugs reduced the deposition- of intercellular matrix. In cells first exposed to either of the drugs for 2 or 5 days and then transferred to fresh medium for 3 or 6 days, the microtubules reappeared, the Golgi complex regained its normal appearance, and the amount of matrix increased. These findings are discussed in view of present concepts of the role of microtubules in cell secretion.
A variety of antimicrotubular agents has been reported in the literature. Some of these, like colchicine, vinblastine and vincristine (for recent reviews see [ 1,2]), cause a disruption of the microtubules, whereas others such as ethanol, hexylene glycol [3, 41 and deuterium oxide [5,6] stabilize these organelles. Abundant evidence has accumulated indicating that antimicrotubular agents impair the secretion of many cell products, e.g. hormones such as insulin [7-91 and thyroid hormone [lo, 111. More recently, microtubules have also been implicated in the secretion of the macromolecules of connective tissue matrices. Thus, it has been found that colchicine and vinblastine, as well as other antimicrotubular Exptl
Cell Res 95 (1975)
agents, inhibit both secretion and synthesis of collagen in vitro [12-161. For example, it was reported that colchicine and vinblastine decreased the rate of secretion of collagen by cells isolated from chickembryo tendons by about 70 %, and that the amount of intracellular collagen concomitantly increased about two-fold [ 121. In a biochemical and ultrastructural study on bone cells, Ehrlich et al. [16] found that colchicine, in addition to the effect on secretion, also depressed collagen synthesis, whereas vinblastine inhibited synthesis of collagen as well as other proteins. These authors suggested that the reduction in collagen formation by colchicine is due to an accumulation of collagen in the Golgi
Znjluence
of colchicine
and vinblastine
vacuoles, with secondary inhibition of its synthesis. Likewise, colchicine and vinblastine have been found to depress both secretion and synthesis of glycosaminoglycans by isolated chick-embryo chondrocytes, but, contrarily, did not greatly reduce the synthesis of collagen by cartilaginous tibia anlagen [17]. On the basis of these findings it is usually assumed that microtubules are involved in the intracellular translocation of secretory vacuoles, and that, as regards matrix deposition, the primary effect of antimicrotubular agents in connective tissue cells is on secretion. Further experimental data are needed, however, to support this hypothesis and to elucidate the particulars of the functional role of microtubules in secretion. The present report is concerned with the influence of colchicine and vinblastine on chondrocyte fine structure and matrix deposition in vitro. Both short- and long-term effects were studied, as well as the ability of the cells to recover after prolonged exposure to these drugs. During the course of the experiments it was found that the antimicrotubular agents caused marked changes in the Golgi complex. As an attempt to determine whether these alterations were directly due to the absence of microtubules, or were secondary to inhibition of the synthesis of matrix components, the effects of puromycin, a potent protein synthesis inhibitor, on cellular fine structure was also examined. As experimental system we used chondrocytes isolated from cartilaginous epiphyses of guinea pig fetuses, aggregated, and thereafter maintained in organ culture. Because of the potential usefulness of this system for other studies on the function and metabolism of cartilage, the morphology of the chondrocyte aggregates was recorded for up to 25 days, i.e. beyond what
on chondrocyte
aggregates
was necessary for the experimental poses proper. MATERIAL
441 pur-
AND METHODS
Pregnant guinea pigs were obtained from a local deafer. The age of the fetuses was 40-50 days, as determined from the weight-age table presented by Draper [ 181.
Animais.
medium. Cells were grown in Ham’s F-12 medium supplemented by 10% fetal calf serum (BioCult), 0.3% tryptose phosphate broth (Bacto) and 50 pg of L-ascorbic acid, 150 pg of streptomycin sulfate (Glaxo) and 150 U/ml of benzylpenicillin (Glaxo). The same medium was used for aggregation of chondrocytes and for subsequent organ culture of aggregates. Culture
ofchondrocytes. The detailed procedure for chondrocyte isolation has been described previously [19, 201. Briefly, dissected fragments of cartilaginous epiphyses of long bones were pooled, digested with collagenase and DNase, and the liberated chondrocytes were then used for aggregation,
Zsolation
and organ culture of chondrocvtes. Chond&y& were aggregated in sihconized 25 ml Erlenmeyer flasks containing 3 x lo6 cells in 3 ml of medium. Each flask was spun at 75 rpm around its axis, using a modification of an apparatus described by Gaillard et al. [19]. After 48 h the aggregates were put on a piece of lens paper supported by a metal grid and maintained in organ culture dishes (Falcon). The medium was changed every 2-3 days. Samples for light and electron microscopy were taken at various time intervals covering I-25 days from the beginning of aggregation. Annregution
to antimicrotubular agens. Crystalline colchicine (Merck) and vinblastine sulfate (Sigma) were used at 10V5 M concentration. Three different experimental series were run. (a) For observations on shortterm effects, the cells were aggregated for 24 h in normal medium and the aggregates then exposed to colchicine or vinblastine for 40 min or 4 h. Alternatively, some of the aggregates were treated with puromvcin (Sigma), at a concentration of IO or 100 pgiml for4 h. (b) For observations on long-term effects, the cells were continuously exposed to either antimicrotubular agent, from the beginning of aggregation, for l-11 days. (c) For recovery studies, aggregates, after 2 or 5 days of exposure, were carefully rinsed, transferred to normal medium, and cultured for a further 3 or 6 days. Exposure
Light microscopy.Aggregates were fixed in the fluid
of Bouin-Hollande [21] or in glutaraldehyde as for electron microscopy. Sections were stained with hematoxylin and eosin, or with Alcian blue 8GS at pH 3.1 [22].
Electron
The material was fixed in 2% (Polaron) in 0.1 M cacodylate buffer,
microscopy.
glutaraldehyde
Exptl
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et ul.
1
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2
influence
of colchicine
and vinblastine
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Fine structure of cells. In l- and 2-dayold aggregates the cells were usually round or oval (fig. 3), but many of them had angular contours, or showed processes of varying size and shape. Nuclei were round or oval, with l-2 nucleoli. The granular endoplasmic reticulum was well-developed. In most cells its cisternae were narrow and filled with a material of high electron RESULTS density, while in others they were disStructure of normal aggregates tended, and their content was of lower Aggregates formed after 24 h of spinning density. Free ribosomes, frequently formof the isolated chondrocytes were flattened ing polysomes were also present. The Golgi and usually had an oval outline. The largest complex was prominent (figs 3,4), and conones reached a length of about 2 mm and a sisted of a few groups of flattened, parallel width of about 1 mm. Within the next 24 h cistemae, small vesicles and large vacuoles. the aggregates frequently formed long rib- In favorable sections up to 6 separate stacks bons by attaching to each other in the polar of cisternae (dictyosomes) with accomregions. panying vesicular structures could be disLight microscopy. In l-day-old aggre- tinguished; the dictyosomes were usually gates the intercellular substance, as visualcomposed of 3-5 cisternae. No direct conized by Alcian blue staining, was scanty nection between the different dictyosomes and consisted mainly of a narrow rim could be seen, but they all lay in a wellaround the cells (fig. 1). After 2-5 days the defined, juxtanuclear area. Microtubules matrix was better developed, particularly could be observed both at the periphery of in the central parts of the aggregates. Some this area and between the dictyosomes (fig. cells were degenerated and lay shrunken 4). Occasionally, l-2 centrioles were also in their lacunae. Cell nests could not be found within the same region. Some Golgi distinguished. During the continued cultivacuoles contained ruthenium red-stainable vation, up to 25 days, the amount of intermaterial. Mitochondria were small and had cellular substance increased but little (fig. few cristae. The majority of the cells con2). In older cultures, many cells were de- tained membrane-bounded dense bodies generated, and the intercellular matrix in (lysosomes), frequently with incorporated their vicinity remained unstained or stained cell remnants and/or myelinlike figures. only weakly. In 5-day-old and older aggregates the diversity of cell shapes was more pronounced than before. The number of disFig. 1. Fragment of l-day-old, non-exposed chondrocells successively increased, cyte-aggregate. Cells in close contact, small amount of integrated intercellular substance. H.E. x300. particularly in the central parts of the aggreFig. 2. Fragment of a 25day-old, non-exposed aggregates. Non-degenerated chondrocytes regate. Increased amount of intercellular substance as mained, however, in considerable numbers compared with l-day-old aggregates. H.E. x300. Fig. 3. 2-day-old, non-exposed aggregate. Chondrountil the end of the cultivation period. The cytes separated by intercellular substance. Cell on the majority of them progressively accumuright contains a prominent Golgi complex (G). X8000. lated dense bodies, which frequently conpH 7.3, for at least 2 h, posttixed in 1% osmium tetroxide in Veronal acetate buffer with added salts [23], dehydrated in ethanol, and embedded in Spun low viscosity medium [24]. To demonstrate proteoglycans some aggregates were stained by addition of 0.05% of ruthenium red to the osmium fixative [25]. Thin sections were cut with an LKB Ultrotome I. and double-stained with uranyl acetate followed by ‘lead citrate [26]. The specimens were examined in a Philips EM 300 electron microscope.
Expel Cell Res 95 (1975)
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Exptl
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Res 95 (1975)
et al.
Influence
of colchicine
and vinblastine
tamed inclusions of the types mentioned above. Fine structure of intercellular substance. In l-day-old aggregates intercellular matrix was mainly seen in the central parts. In 2day-old aggregates the amount of matrix was increased, but the difference between central and peripheral parts was still evident. There were also considerable variations between different areas within the aggregates. The intercellular substance contained dispersed collagen fibrils and matrix granules stainable with ruthenium red (fig. 5). The fibrils had an appearance typical of cartilage collagen. They had a diameter of 150-250 A and exhibited but a faint crossstriation. With time the intercellular substance became more prominent. Even after 25 days of culture, however, the matrix was less dense than in fetal, cartilaginous epiphyses (unpublished observations). In older aggregates many cells were surrounded by a clear zone, containing single matrix granules and practically no fibrils. No signs of calcification were noted. A finding of incidental interest was that the outer surfaces of peripheral cells were covered by a ruthenium red-stainable material. This material often had a patchy distribution and could originate from the
Fig. 4. Chondrocyte from 2-day-old, non-exposed aggregate. Well developed Golgi complex with numerous microtubules (arrows) within, and at the periphery of, the complex. x26000. Fig. 5. Intercellular substance from l-day-old, non-. exposed aggregate. Collagen fibres with faint crossstriation and matrix granules stained with ruthenium red. x70000. Fig. 6. 2-day-old aggregate formed in the presence of colchicine. The amount of intercellular substance is smaller than in the corresponding control aggregate (cf fin. 3). None of the cells contains an intact Golgi complex: x8000.
on chondrocyte
aggregates
serum present in the culture
medium
445 (cf
r271). Effects of antimicrotubular agents Neither colchicine nor vinblastine interfered with the aggregation of the chondrocytes. No microtubules could be seen in exposed cells at any of the intervals covering the period of observation from 40 min to 11 days. Both agents also evoked striking changes in the Golgi complex, primarily a dissociation of the complex into single dictyosomes and their spreading throughout the cytoplasm. The first signs of this dissociation could be recognized already after 40 min, particularly in vinblastine-treated cultures. Thus, in some cells the distances between individual dictyosomes were somewhat increased, with interposition of ground cytoplasm with endoplasmic reticulum. The spreading was essentially complete after 4 h of exposure, and the dictyosomes, with accompanying vesicular structures, were now distinctly separated by other organelles, and frequently located on opposite sides of the nucleus (figs 6-10). The dissociation of the dictyosomes persisted throughout the experimental period (11 days). Furthermore, most of the dictyosomes showed an altered structure after dispersion. Many of them had cisternae that were fewer and shorter than in the controls. Such dictyosomes were accompanied by a group of a few vacuoles, while small vesicles were scanty. Other dictyosomes displayed a more normal complement of cisternae, but lacked vacuoles, and were thus associated mainly with small vesicles (figs 8-10). Intermediate forms were also present. No ruthenium red-stainable material was demonstrable within the vacuoles of dispersed dictyosomes. Exptl
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rt al.
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and vinblastine
The dissociation of the Golgi complex was accompanied by the formation of clusters of lysosomes in the close vicinity of some of the dispersed dictyosomes (figs 11, 12). In addition, a considerable increase in the number of microfilaments was apparent in the chondrocytes after a few days of treatment with either drug. Exposure to vinblastine lead to an enhanced autophagocytosis in the chondrocytes; early signs thereof could be observed already after 40 min (fig. 13). After 4 h many cells contained lysosomes into which e.g. fragments of granular endoplasmic reticulum had been incorporated. At later intervals the chondrocytes showed a markedly increased number of phagosomes and phagolysosomes. In older cultures residual bodies with myelin-like figures were numerous. A second effect exclusively seen after exposure to vinblastine was the appearance of distinct, parallel arrays of macrotubules (figs 11, 12) of the type described previously and supposed to contain the microtubular protein, tubulin [28-3 11. The macrotubules were first seen after 1-2 days of treatment; their numbers increased with time. They had an average diameter of about 350-400 A and were accompanied by numerous polysomes. Crystals of the kind which have been reported to occur, in other types of cells, under the influence of vinblastine [28] were not encountered.
Fig. 7. Chondrocyte from 2-day-old aggregate formed in the nresence of colchicine. The Go& comnlex is dispersed into single, inconspicuous -dictydsomes (arrows). Otherwise cell structure appears normal. x 16000. Fin. 8. Two dictvosomes from the cell shown in tie. 7. Short, inconspicuous cistemae accompanied by &all and medium-sized vesicles. x36 000. Fig. 9. Dictyosome with a reduced number of cisternae accompanied by a few large vacuoles and small vesicles; 2 days of colchicine treatment. x36000. 30-751814
on chondrocyte
aggregates
447
Both colchicine and vinblastine caused a reduction in the amount of intercellular matrix deposited in the aggregates of the long-term series. Already in l-day-old aggregates the intercellular substance was clearly less developed than in the controls (fig. 6), but as in the latter contained both collagen fibrils and matrix granules. The amount of matrix increased with time of culture but to a lesser extent than in nonexposed aggregates. Effects ofpuromycin. Puromycin did not, at either concentration, affect the number and structure of the microtubules. Nor did it cause any dissociation of the Golgi complex (fig. 14); the number of dictyosomes, and the number of cisternae within these, were unaltered. The Golgi vacuoles were, however, reduced in number and lacked the typical content seen in control cells. Conversely, the small Golgi vesicles were more numerous. In some cells autophagocytosis of, inter alia, granular endoplasmic reticulum was noted. Moreover, some cells displayed clumping of chromatin. Recovery. Chondrocytes, exposed for 2 or 5 days to either of the antimicrotubular agents, and then cultured for 3 or 6 days in drug-free medium, showed a normal complement of microtubules. In cells recovering from vinblastime treatment macrotubules were no longer present. Moreover, in most cells the dictyosomes were again localized in one well-defined area (fig. 15). The cisternae were well-developed and the number of large vacuoles and small vesicles was similar to that in the control cells. In a few chondrocytes, however, single dictyosomes remained separated from the main Golgi area even after 6 days. Clustering of lysosomes was no longer observed. This recovery in cellular fine structure was accompanied by an increase in the amount of deposited matrix, which did not, however, Exptl
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Res 95 (1975)
448
Exprl
Moskalewski
Cell Res 95 (1975)
et al.
Influence
of colchicine
and vinblastine
attain the level existing in non-exposed cultures of comparable age. DISCUSSION Experimental system Chondrocytes were chosen for the present study since they are readily isolated and produce large amounts of intercellular matrix in vitro. The experimental system was based on the methods for isolation and aggregation of chondrocytes proposed by Gaillard et al. [19]. Both in the experiments of these authors, in which fetal, mouse epiphyseal chondrocytes were aggregated and cultured for up to 10 days, and in our experiments, with fetal guinea pig material, the aggregated cells elaborated a typical intercellular matrix. The accumulation thereof was most conspicuous during the first few days of culture. In both studies, however, no further maturation of the cartilage was observed. On the other hand, guinea-pig fetal chondrocytes, isolated as in the present study and transplanted intramuscularly into young guinea pigs, are capable of reforming cartilage in which both hypertrophy of cells and calcification of the matrix are evident [20]. Moreover, primordia of long bones consisting of small-
IO. Chondrocyte from l-day-old aggregate formed in the presence of vinblastine. Dispersion of Golgi complex into single dictyosomes (arrows). Scanty intercellular substance. X 13 000. (Inset). Separated dictyosome from the same cell (crossed arrow). Inconspicuous cisternae, a few large vacuoles and small vesicles. x40000. Fig. II. Chondrocyte from an S-day-old aggregate treated continuously with vinblastine. A bundle of macrotubules with accompanying polysomes. Abundant microtilaments. Clusters of lysosomes occur in the vicinity of single dictyosdmes (arrows). x 14000. Fig. Z2. Chondrocyte from an &day-old aggregate, exposed continuously to vinblastine. Bundle of macrotubules in cross-section and a group of lysosomes. x38000. Fig.
on chondrocyte
aggregates
449
celled precartilages are capable of developing into bone in culture [32, 333. This points to some inadequacy of the in vitro conditions to support maturation of cartilage formed by isolated chondrocytes. Znfluence of colchicine and vinblastine on cell structure The presence of microtubules within the Golgi complex has previously been established both in plant [34] and animal cells, e.g. chondrocytes [35]. In the present study microtubules were regularly observed within and in the vicinity of the Golgi complex in the control cells. Both colchicine and vinblastine caused a rapid disappearance of the microtubules in the cytoplasm of the chondrocytes. Microtubules could not be seen at any of the intervals studied, which indicates that the concentration of the drugs remained effective between changes of medium. The disappearance of the microtubules was followed by dissociation of the Golgi complex and spreading of the dictyosomes throughout the cytoplasm. This would seem to imply that microtubules are necessary to maintain the structural integrity of the Golgi complex. As far as we can ascertain the first observations on the influence of colchicine and vinblastine on the Golgi complex were described by Robbins & Gonatas [36]. They found a fragmentation and circumferential distribution of the Golgi apparatus in HeLa cells exposed to these agents for 10 h. More recently, in a study on a mouse embryonic cell line, treatment with colchicine, vinblastine and vincristine was reported to result in ‘explosion’ of the Golgi field and distribution of individual ‘Golgi organelles’ over the entire cytoplasm [37]. In the main, these observations seem to be in agreement with those of the present study. Our explanation of the mechanism of Exprl
Cd
Res 95 (1975)
450
Erptl
Moskalewski
Cell Res 95 (1975)
et al.
Inji’uence
of colchicine
and vinblastine
on chondrocyte
aggregates
45 1
Fig.
IS. Chondrocyte from an 8day-old aggregate treated with vinblastine for 5 days and then cultured in normal medium for 3 days. Reformed Golgi complex
with structure corresponding to that in untreated cells, cf fig. 4. C, centriole; DZf, dense body with myelinlikefigures. x26000.
dictyosome dispersion as due to destruction of microtubules could be challenged on the basis of the feedback theory of Ehrlich et al. [16], according to which depression of secretion would lead to secondary inhibition of synthesis and, presumably, functional and morphological alterations of the Golgi apparatus. Such an argument
would, however, be difficult to accept for cartilage cells. Our observations did not suggest any accumulation of secretory products in the Golgi complex. Further, if secondary inhibition of the formation of matrix components occurred it could possibly evoke changes in the Golgi complex in situ, but should not be expected to cause dispersion of the dictyosomes throughout the cytoplasm. This reasoning is supported by the observations in the control experiment with puromycin, a general inhibitor of protein synthesis [38]. Thus, this agent did not cause any dissociation of the Golgi complex although a clear reduction in the number of secretory vacuoles was observed. These effects on the Golgi complex
Fig. 13. Chondrocyte from l-day-old aggregate, exposed to vinblastine for 40 min. Beainnina of disnersion of Golgi complex and format& ofautophagosomes encompassing fragments of granular endoplasmic reticulum (arrows). x 17 000. (Zraser). Forming autophagosome from the same cell (crossed arrow). X60000. Fig. 14. Chondrocyte from l-day-old aggregate exposed to puromycin (100 w/ml) for 4 h. Dictyosomes are located in a well-defined, juxtanuclear area. x39000.
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Moskalewski
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as well as the effect on other cell components in the chondrocytes are similar to those described in a study on exocrine cells in the pancreas of puromycin-treated rats r391. The dissociation of the Golgi complex was accompanied, in some dictyosomes, by a reduction in the number of the cisternae. This could be due to their splitting off during or after the process of dispersion. Taking into consideration present concepts of the formation and function of the Golgi complex, another and more likely explanation is that the dispersion of the dictyosomes is followed by a disturbance of the formation of new cisternae. Thus, electron microscopic observations suggest that new cisternae are formed at the outer or forming (cis) face of the dictyosome by fusion of vesicles arriving from the endoplasmic reticulum. On the inner, maturing (tram) face of the dictyosome the cisternae seem to transform into vacuoles containing secretory products (for reviews, see [40421). In the goblet cells of the intestine new Golgi cisternae were estimated to form every 2-4 min [43]. From the above it follows that, if for some reason the inflow of vesicles from the endoplasmic reticulum to the dictyosome would be interrupted, its morphology and function would be seriously affected. No formation of new cisternae at the outer surface of the dictyosome would occur. On the inner surface transformation of the cisternae into secretory vacuoles could probably continue for a short time, i.e. as long as the materials needed for their formation had not been exhausted. Such an inactive dictyosome could be expected to have a reduced number of cisternae and to be devoid of secretory products. It seems reasonable to assume that the dispersion of the dictyosomes will, at least temporarily, Exptl CellRes
95 (1975)
affect their normal relationship to the endoplasmic reticulum. After dispersion the dictyosomes may possibly reestablish a functional contact with this organelle. Conceivably, differences in the efficiency of such contact could explain the variations in number of cisternae of the dictyosomes and associated vacuoles and vesicles within single cells. The short-term effects of antimicrotubular agents on the Golgi complex have also been studied in connective tissue cells other than chondrocytes. Thus, in tendon fibroblasts incubated with colchicine for 3 h, the Golgi vacuoles were found to be increased both in number and size [44]. In cells of cranial bones, exposed to colchicine for about 1 h, there were increased numbers of Golgi-associated vacuoles and vesicles [16]. After incubation with vinblastine, the Golgi complex was small, contained few large vacuoles and had a larger than usual number of associated small vesicles. Moreover, the cisternae of the granular endoplasmic reticulum were vesicular in appearance as compared with the long, interconnecting channels in the control cells. These observations contrast with those reported here. Thus, dissociation of the Golgi complex was not noted in the above studies, although discernible already after 40 min in our material. Furthermore, in the chondrocytes the alterations in the number of Golgi vacuoles and vesicles comprised, at all intervals, a decrease rather than an increase; any clear changes in the granular endoplasmic reticulum were not observed even after prolonged exposure. Obviously, more experimental data are needed to explain these differences in the response of various connective tissue cell types to antimicrotubular agents. In addition to the changes, induced by colchicine and vinblastine, in the Golgi
Influence
of colchicine
and vinbiustine
complex of the chondrocytes there was a clustering of lysosomes and, particularly with the latter agent, an enhanced autophagocytosis. Similar effects of antimicrotubular drugs have been observed in studies on other cell types both in vivo and in vitro 136, 45,461. The changes in cellular fine structure caused by colchicine and vinblastine were reversible. Thus, after withdrawal of the drugs the Golgi complex of the chondrocytes regained its normal appearance. The Golgi complex could be restored from the dissociated dictyosomes, or formed de novo with concomitant or subsequent destruction of the dispersed components. The restoration of cellular morphology was accompanied by an increased deposition of matrix components, indicating that also a functional recovery of the chondrocytes took place. Influence of colchicine and vinblastine on the secretion of matrix components It is generally accepted that the Golgi complex is involved in the synthesis and secretion of matrix components in connective tissue cells. Thus, there is considerable morphological and biochemical evidence indicating that the formation of both collagen and proteoglycans is initiated in the granular endoplasmic reticulum, and then completed during transport through the cisternae of the reticulum to the Golgi complex, as well as within this latter system, where the molecules finally are accumulated in vacuoles before being released to the extracellular space by exocytosis. However, as far as collagen is concerned it has been suggested that other secretory pathways may also exist (for recent reviews see [47-49]). During recent years antimicrotubular
on chondrocyte
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453
agents have been found to interfere with both secretion and synthesis of collagen in cultured bones and fibroblasts [12-161, and of glycosaminoglycans in isolated chondrocytes [17]. On the basis of these findings it has been suggested that disruption of microtubules interferes with the movement of Golgi vacuoles to, the cell surface and that this results in accumulation of secretory products in the Golgi complex, which then secondarily leads to an inhibition of synthesis [IL171. In the present study, aggregates formed and cultured in the presence of colchicine and vinblastine showed a reduced content of intercellular matrix. This reduction comprised both collagen fibrils and matrix granules, the latter demonstrated to contain proteoglycans by ruthenium red staining [50, 511. Considerable amounts of intercellular matrix werk, nevertheless, laid down in the treated aggregates. Hence, the chondrocytes retained part of their capacity for synthesizing and secreting extracellular macromolecules in spite of the disappearance of the microtubules and the structural alterations of the Golgi complex. It is evident from our results that the reduction of matrix deposition in aggregates treated with colchicine and vinblastine could be due to a primary disfunction of the Golgi complex as well as to a disturbance in the transport of secretory vacuoles. Since separation of these two effects is not feasible at present, no definite conclusions as to the role of microtubules in the translocation of secretory vacuoles in chondrocytes would seem justified. Financial support, including personal fellowshios for Dr S. Mosk&wski, was obtained from the Swedish Medical Research Council (oroi. no 12X-33553, the Swedish Cancer Society (proj: nd. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Swedish Committee on International Health Relations, the Swedish Institute, the A. 0. SW&d Foundation, the M. Bergvail Foundation. and from the Funds of Karolinska Exptl
Cell Res 95 (1975)
Institutet. Antibiotics were generously supplied by Glaxo Laboratories Ltd (Sweden). The authors are also indebted to Mrs Eva Lundberg and Miss Karin Askfors for technical assistance, and to Mrs Ingrid Wtilma for preparation of the manuscript. S. M. is on leave from the Department of Histology and Embryology, Medical Academy, Warsaw, Poland.
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Received February 28, 1975 Revised version received April 17, 1975
