Stanislaw Moskalewski

Microtubules and the organization of the Golgi complex

Electron microscopic and cytochemical studies indicate that microtubules play an important role in the organization of the Golgi complex in mammalian cells. During interphase microtubules form a radiating pattern in the cytoplasm, originating from the pericentriolar region (microtubule-organizing centre). The stacks of Golgi cisternae and the associated secretory vesicles and lysosomes are arranged in a circumscribed juxtanuclear area, usually centered around the centrioles, and show a defined orientation in relation to the rough endoplasmic reticulum. Exposure of cells to drugs such as colchicine, vinblastine and nocodazole leads to disassembly of microtubules and disorganization of the Golgi complex, most typically a dispersion of its stacks of cisternae throughout the cytoplasm. These alterations are accompanied by disturbances in the intracellular transport, processing and release of secretory products as well as inhibition of endocytosis. The observations suggest that microtubules are partly responsible for the maintenance and functioning of the Golgi complex, possibly by arranging its stacks of cisternae three-dimensionally within the cell and in relation to other organelles and ensuring a normal flow of material into and away from them. During mitosis, microtubules disassemble (prophase) and a mitotic spindle is built up (metaphase) to take care of the subsequent separation of the chromosomes (anaphase). The breaking up of the microtubular cytoskeleton is followed by vesiculation of the rough endoplasmic reticulum and partial atrophy, as well as dispersion of the stacks of Golgi cisternae. After completion of the nuclear division (telophase), the radiating microtubule pattern is re-established and the rough endoplasmic reticulum and the Golgi complex resume their normal interphase structure. This sequence of events is believed to fulfil the double function to provide tubulin units and space for construction of the mitotic spindle and to guarantee an approximately equal distribution of the rough endoplasmic reticulum and the Golgi complex on the two daughter cells.

Influence of colchicine and vinblastine on the Golgi complex and matrix deposition in chondrocyte aggregates. An ultrastructural study

Abstract 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 10 −5 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.

Influence of colchicine on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chondrocytes

Fetal guinea-pig epiphyseal chondrocytes were cultured in monolayers and as aggregates in the presence of antimicrotubular agents. Colchicine and vinblastine caused a dissociation of the Golgi complex, in addition to the disappearance of microtubules. Synthesis and secretion of proteoglycans and collagen were studied using radioactive precursors. Colchicine inhibited the synthesis of proteoglycans. The drug also inhibited secretion with an intracellular accumulation of these molecules. The proteoglycans secreted by the colchicine-treated cells had a smaller molecular size and contained a smaller proportion of aggregated molecules than proteoglycans in control cultures. However, there was no difference in the average size of the chondroitin sulfate side chains of the proteoglycan molecules. Nor was there any increase in the breakdown of proteoglycans in colchicine-treated cultures. Vinblastine was also found to inhibit synthesis and secretion of proteoglycans. Deuterium oxide also inhibited the synthesis of these molecules but stimulated their secretion into the medium. Colchicine caused an inhibition of both synthesis and secretion of collagen. It is suggested that the quantitative and qualitative effects of colchicine could be the result of disturbances in the Golgi complex, possibly in combination with a retarded translocation of secretory vacuoles. However, as the colchicine-treated chondrocytes were still able to continue a large part of their matrix biosynthesis with only moderate changes in the structure of the secreted molecules, it is probable that alternative pathways for the secretion of matrix molecules exist and/or the Golgi complex is able to retain a major part of its function despite the structural alterations.

In vitro influence of colchicine on the golgi complex in A- and B-cells of guinea pig pancreatic islets

Isolated guinea pig pancreatic islets were incubated (up to 4 hr) or cultured (up to 9 days) in the presence of colchicine. In control islets, the Golgi complex of the A-cells usually had a juxtanuclear location and was composed of a few dictyosomes. In the B-cells, the complex had a juxta- or perinuclear location and was considerably larger. Microtubules were present within and around the area occupied by the .Golgi complex. Exposure to colchicine caused, in addition to disappearance of the microtubules, marked changes in the Golgi complex. These occurred in a varying number of cells at 4 hr and in practically all cells at 24 hr. In both A- and B-cells, the dictyosomes became dispersed throughout the cytoplasm. The number and size of the cisternae were reduced in many of the cells. After transfer to a medium without colchicine, the Golgi complex regained its normal appearance. The above findings are interpreted as indicating that microtubules are responsible for the structural integrity of the Golgi complex. The changes in the dictyosomes are attributed to a postulated disruption of their functional relationship to the granular endoplasmic reticulum.

Relationship between the Golgi complex and microtubules enriched in detyrosinated or acetylated α-tubulin: studies on cells recovering from nocodazole and cells in the terminal phase of cytokinesis

Double immunofluorescence microscopy was used to study the relationship between the Golgi complex and microtubules enriched in posttranslationally modified tubulins in cultured mouse L929 fibroblasts. In interphase cells, the elements of the Golgi complex were grouped around the microtubule-organizing center. From here, tyrosinated microtubules extended to the periphery of the cells, whereas the distribution of detyrosinated and acetylated microtubules largely overlapped with that of the Golgi complex. Treatment of cells with 10 μM nocodazole led to the disruption of all microtubules and dispersion of the Golgi elements. Following withdrawal of the drug, tyrosinated microtubules reformed first, followed by acetylated and then detyrosinated microtubules. In parallel, the Golgi elements moved back toward the juxtanuclear region and reestablished a close spatial relationship first with the acetylated and later also with the detyrosinated microtubules. Long-term recovery in the presence of 0.15 or 0.3 μM nocodazole allowed partial reformation of tyrosinated and acetylated microtubules, whereas no or only a few detyrosinated microtubules were detected. At the same time, the Golgi elements were grouped closer together around or on one side of the nucleus in close relation to acetylated microtubules. In synchronized cells released from a mitotic block, a radiating array of tyrosinated microtubules was first formed, followed by acetylated and detyrosinated microtubules. The Golgi elements initially came together in a few groups and thereafter took an overall morphology similar to that in interphase cells. During this reunification, they showed a close spatial relationship to acetylated microtubules, whereas detyrosinated microtubules appeared only later. Microtubules enriched in acetylated and/or detyrosinated tubulin thus appear to take part in establishing and maintaining the organization of the Golgi elements within an interconnected supraorganellar system. Whether the acetylation and detyrosination of tubulin are directly involved in this process or merely represent two modifications within this subpopulation of microtubules remains unknown.

Cold and Metabolic Inhibitor Effects on Cytoplasmic Microtubules and the Golgi Complex in Cultured Rat Epiphyseal Chondrocytes

SummaryPrevious work has shown that exposure of cultured chondrocytes to colchicine leads to disappearance of microtubules and dispersion of the dictyosomes of the Golgi complex throughout the cytoplasm. Here, the effects of cold and metabolic inhibitors on cultured chondrocytes have been investigated in order to characterize further the relationship between these organelle systems. After incubation of cells for 24 h at 4° C most, but not all microtubules disappeared, indicating the existence of cold-resistant microtubules. Dictyosomes remained united in one area, until transfer of cultures to 37° C, when they dispersed throughout the cytoplasm in about one-third of the cells. In cells exposed simultaneously to cold and colchicine, microtubules disappeared completely, but spreading of dictyosomes occurred only in some cells and became generalized first upon warming. Application of the metabolic inhibitors sodium azide or sodium fluoride (10-2 M) or 2-deoxyglucose (5×10-2 M) together with sodium cyanide (10-2 M) inhibited microtubule removal by colchicine. Consequently, spreading of the Golgi complex was prevented. These findings support the concept of an important role of microtubules in the organization of the Golgi complex. Moreover, depolymerization of microtubules by colchicine appears to be an energy dependent process.

Electron microscopic studies on the uptake of colloidal thorium dioxide particles by isolated fetal guinea-pig chondrocytes and the distribution of labeled lysosomes in cartilage formed by transplanted chondrocytes

SummaryChondrocytes isolated from fetal, guinea-pig epiphyses were grown in monolayer culture, exposed to thorium dioxide particles, and studied ultrastructurally after varying intervals. The exogenous marker was ingested by endocytosis and subsequently accumulated in lysosomes. After intramuscular injection into young guinea pigs, the thorium dioxidelabeled chondrocytes formed a typical hyaline cartilage. This consisted mainly of rounded or polygonal cells with large, eccentrically located nuclei. The cytoplasm showed an extensive granular endoplasmic reticulum and a well-developed Golgi complex, suggesting active synthesis and secretion of matrix components. Among the other cytoplasmic organelles, lysosomes containing variable amounts of marker particles were observed. After 2–3 weeks the transplants showed signs of cellular degeneration and disintegration. During these processes, lysosomes remained structurally intact and, furthermore, retained the incorporated marker. Thus, thorium dioxide-labeled bodies were found in former chondrocyte lacunae and in the intercellular substance proper. In the latter location labeled bodies could be observed in close proximity to early mineral deposits. These results are discussed with special reference to the cellular origin and lysosomal nature of matrix vesicles in calcifying cartilages.The skilled technical assistance of Mrs. Eva Lundberg and Miss Karin Askfors and the secretarial assistance of Mrs. Ingrid Wäälma are gratefully acknowledged.

Bone formation in cartilage produced by transplanted epiphyseal chondrocytes

SummaryChondrocytes were isolated from rat epiphyseal cartilage, cultured in vitro, and exposed to exogenous tracers which accumulated in their lysosomes. The cells were then injected into the posterior tibial muscle of animals from the same outbred strain, where they reconstructed calcifying hyaline cartilage. The mineralization of the tissue was followed by ingrowth of blood capillaries from the host bed. Macrophage-like cells surrounding the vessels phagocytized degenerated chondrocytes and unmineralized matrix, whereas multinucleated chondroclasts removed some of the mineralized cartilage matrix. Mesenchyme-like cells accompanying the invading vessels attached to the remaining septa of calcified cartilage matrix and developed into osteoblasts depositing bone matrix on the surface of these septa. The apparent lack of inherent tracer labeling of the lysosomes in the different bone cells indicate that they were derived from the host. No signs of transformation of chondrocytes into bone cells were observed.When isolated rat epiphyseal chondrocytes were injected into the wall of the hamster cheek pouch, calcifying cartilage was reconstructed without signs of subsequent ossification. Transplantation of cartilage reconstructed in the hamster into the dorsal muscles of rats was, however, followed by formation of bone by a sequence analogous to that described above. Such an osteogenetic response was also obtained when the cartilage had been devitalized before transplantation.These experiments show that calcified cartilage, developing in or grafted into an intramuscular site, is able to induce and serve as a substrate for endochondral bone formation, similar to that occurring during normal development. They further indicate that bone induction by calcified cartilage does not require the presence of living chondrocytes.

The Elastogenetic Process in Transplants and Cultures of Isolated Auricular Chondrocytes

Chondrocytes isolated from auricular cartilage of rabbits in the early postnatal period were transplanted intramuscularly into closely related animals and the reconstruction of cartilage and formation of elastic fibers monitored. Elastogenesis was most produced in transplants from younger donors. Auricular chondrocytes from 7 day old rabbits also produced elastic fibers in tissue culture, undergoing morphological changes similar to those occurring in vivo. The ability to form elastic fibers declined after prolonged cultivation.

Effects of antimicrotubular agents on the fine structure of the Golgi complex in embryonic chick osteoblasts

SummaryEmbryonic chick frontal bones were cultured in the presence of colchicine or vinblastine and subsequently examined by transmission electron microscopy. In control cultures the osteoblasts showed a large Golgi complex consisting of dictyosomes arranged in a well-defined juxtanuclear area. Microtubules were particularly numerous within this Golgi area although they could be observed throughout the cytoplasm. Colchicine and vinblastine caused the disappearance of cytoplasmic microtubules, while bundles of 10nm diameter filaments appeared more frequently. In addition, cell polarity was lost and the Golgi complex became disorganized, with the dictyosomes randomly dispersed in the cytoplasm and showing a decreased number of cisternae and an increased number of vacuoles, the latter generally lacking stainable material. Increased number of autophagosomes were also noted.These findings indicate that microtubules function in the organization of the Golgi complex in osteoblasts. In view of the well documented role of this organelle system in collagen secretion it is suggested that previously observed secretory disturbances produced by antimicrotubular drugs may be due to a defective transfer of material to the dictyosomes and/or a defect in the packaging and transport of such material away from them.