The role of microtubules in human natural killer cell-mediated cytotoxicity

The effect of four different microtubule (MT) inhibitors on the various stages of human natural killer (NK) cell-mediated cytotoxicity was studied. The MT-disrupting effect of the drugs was monitored by indirect immunofluorescence microscopy and transmission electron microscopy. All the drugs tested, vinblastine sulfate, demecolcine, nocodazole, and taxol, had only a slight inhibitory effect on NK activity. Cells with nonfunctional MT were capable of normal conjugate formation and polarization of actin-containing microfilaments. Natural killer cell cytotoxic factor (NKCF) activity produced by cells with nonfunctional MT was slightly diminished. MT disruption caused enlargement of Golgi cisternae, but did not, however, dissociate the overall structural organization of the Golgi complex. The results indicate that fresh human NK cells are capable of lytic activity without functional MT although MT play a small supportive role in production or secretion of NKCF and mediation of the lytic activity. Previous experiments by us and others have strongly suggested that NK cells mediate their cytolytic activity by directed secretion of toxic material. As NK cells with unfunctional microtubules are capable of close to normal secretion the results presented in this report are not inconsistent with the earlier suggested stimulus-secretion model.     

The arrangement of the Golgi complex beads is not controlled by the cytoskeleton

Exposure of insect fat body to treatments which disrupt microtubules (colchicine, vinblastine sulfate and cold treatment) blocks intracellular transport between the Golgi complex and the plasma membrane but does not affect Golgi complex bead rings or transport from rough endoplasmic reticulum to the Golgi complex. Drugs which disrupt microfilaments (cytochalasins B and D) do not affect the bead rings or intracellular transport of secretory proteins at any level. Thus, intracellular transport between the rough endoplasmic reticulum and the Golgi complex and the arrangement of the beads in rings are both independent of the cytoskeleton. The ring arrangement is presumably maintained by interconnection(s) with rough endoplasmic reticulum membrane.     

Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes

Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.     

Cold-stable microtubules in the cytoplasm of mouse embryo fibroblasts.

Treatment of cultured mouse embryo fibroblasts with Triton X-100 after prolonged incubation at 0 degrees C reveals a network of microtubules in the cytoplasm of cooled cells. This network of cold-stable microtubules was demonstrated by immunofluorescence microscopy, using a monospecific antibody against tubulin and by electron microscopy. The cold-stable microtubules, as well as the ordinary cytoplasmic microtubules, were sensitive to Ca ions and were not observed in the cells pre-treated with colchicine or vinblastine. The cold-stable microtubules do not seem to be in equilibrium with the pool of depolymerized tubulin at 0 degrees C.     

Vinblastine paracrystals from cultured cells are calcium-stable

Vinblastine has two distinct tubulin-related effects in 3T6 fibroblasts and J774.2 macrophages. It depolymerizes microtubules and it induces the formation of paracrystals in the cytoplasm. These paracrystals are retained in cytoskeletons prepared by Triton extraction and are stable to treatment with calcium. The direct addition of vinblastine to cytoskeletons does not alter the organization of microtubules. The two effects of vinblastine are concentration-dependent, as assayed by binding of [3H]taxol and tubulin immunofluorescence. At low concentrations, vinblastine depolymerizes cellular microtubules; at high concentrations the drug induces the formation of paracrystals.     

Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.     

Spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin

The integrity and intracellular distribution of the Golgi apparatus appear to depend upon microtubules. We have found that the microtubules rich in detyrosinated tubulin are located preferentially in the vicinity of the Golgi. Cells were double-stained with antibodies specific for either tyrosinated or detyrosinated tubulin and an antibody to prolactin or wheat germ agglutinin (Golgi markers). Microtubules rich in detyrosinated tubulin showed a close codistribution with the Golgi in three different cultured cell lines GH3, BS-C-1, and AtT20. Disruption of microtubules with nocodazole in GH3 cells resulted in fragmentation and dispersal of the Golgi apparatus as reported previously. During recovery of the microtubules and the Golgi complex after removal of the nocodazole, there was a spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin. Our results suggest that a functional relationship may exist between the structure and organization of the Golgi complex and the detyrosination of alpha- tubulin in microtubules.     

A novel 58-kDa protein associates with the Golgi apparatus and microtubules

With the aim of identifying proteins involved in linking microtubules to other cytoplasmic structures, microtubule-binding proteins were isolated from rat liver extracts by a taxol-dependent procedure. The major non-tubulin component, a 58-kDa protein (designated 58K), was purified to homogeneity by gel filtration chromatography. To aid further characterization of 58K, purified preparations of the protein were used as immunogen for the production of monoclonal antibodies. Five different monoclonals were obtained, and each of these reacted on immunoblots of liver homogenates with a single band that comigrated with 58K. Based on the results of immunochemical, peptide mapping, and microsequencing experiments, 58K was found to be unrelated structurally to similarly sized cytoskeleton-associated proteins, such as tubulin, tau, vimentin, or keratin, and to represent a new protein species. Several in vitro properties of 58K were found to be characteristic of microtubule-associated proteins. For instance, 58K cosedimented quantitatively with microtubules out of liver extracts, stimulated polymerization of tubulin, and bound to microtubules in a saturable manner. In contrast to traditional microtubule-associated proteins, however, 58K was not found to be distributed uniformly along microtubules in cells. Immunofluorescence microscopy of cultured hepatoma cells revealed, instead, that 58K is associated principally with the Golgi apparatus. Moreover, Golgi membranes isolated from rat liver were observed by immunoblotting to contain significant levels of 58K, which, upon subfractionation of the membranes, partitioned as if it were a peripheral membrane protein exposed to the cytoplasmic side of the Golgi. These collective results have been evaluated in terms of earlier evidence that the intracellular position and structural integrity of the Golgi relies on the presence and organization of microtubules. In that context, the observations reported here suggest that the in vivo function of 58K is to provide an anchorage site for microtubules on the outer surface of the Golgi.     

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 M 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 M 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.On leave of absence from the Department of Histology and Embryology, Institute of Biostructure, Medical School, Warsaw, Poland     

Demecolcine-induced enucleation of sheep meiotically maturing oocytes

The objective of this study was to investigate the possible effect of demecolcine, a microtubule-disrupting reagent, on induced enucleation (IE) of sheep meiotically maturing oocytes. Immunofluorescent staining with anti-tubulin antibodies was used to examine the spindle status of the oocytes. When the oocytes with intact germinal vesicles (GV) were cultured in the medium containing various concentrations of demecolcine (0.01 to 0.4 microg.mL-1) for 20 to 22 h, the spindle microtubule organization and first polar body (PB1) extrusion were inhibited by demecolcine in a dose-dependent manner. The highest IE rate (58.1%) was from the treatment with 0.04 microg.mL-1 demecolcine. Demecolcine treatment applied after germinal vesicle breakdown (GVBD) or at metaphase (M) yielded a PB1 extrusion rate and IE efficiency similar to the treatment applied at the onset of maturation. Analysis by immunofluorescence showed that both nonspindle microtubules and spindle microtubules were significantly disorganized by demecolcine. Combination treatment with demecolcine and cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) led to single pronuclear formation rather than PB1 extrusion. When demecolcine-treated oocytes were transferred into demecolcine-free medium, the ability to extrude PB1 was quickly restored and a 72.1% IE rate was obtained following such treatment. These results demonstrate that demecolcine can be used as a potential reagent for induced enucleation of sheep meiotically maturing oocytes and may greatly facilitate research in nuclear transfer.     

Distribution and content of microtubules in relation to the transport of lipid. An ultrastructural quantitative study of the absorptive cell of the small intestine

To determine whether microtubules are linked to intracellular transport in absorptive cells of the proximal intestine, quantitative ultrastructural studies were carried out in which microtubule distribution and content were determined in cells from fasting and fed animals. Rats were given a 1-h meal of standard chow, and tissue was taken from the mid-jejunum before, 1/2 h, and 6 h after the meal. The microtubule content of apical, Golgi, and basal regions of cells was quantitated by point-counting stereology. The results show) that microtubules are localized in intracellular regions of enterocytes (apical and Golgi areas) previously shown to be associated with lipid transport, and that the microtubule content within apical and Golgi regions is significantly (P less than 0.01) reduced during transport of foodstuffs. To determine the effect of inhibition of microtubule assembly on transport, colchicine or vinblastine sulfate was administered to postabsorptive rats, and the lipid and microtubule content of enterocytes determined 1 and 3 h later. After treatment with these agents, lipid was found to accumulate in apical regions of the cells; this event was associated with a significant reduction in microtubule content. In conclusion, the regional distribution of microtubules in enterocytes, the decrease in assembled microtubules after a fat-containing meal, and the accumulation of lipid after the administration of antimicrotubule agents suggest that microtubules are related to lipid transport in enterocytes.     

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

Embryonic chick frontal bones were cultured in the presence of colchicine or vinblastine and subsequently examined by tranmission 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 10 nm 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.     

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.     

Laminin and fibronectin in normal and malignant neuroectodermal cells

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical localization of glucocorticoid receptor in human gingival fibroblasts and evidence for a colocalization of glucocorticoid receptor with cytoplasmic microtubules

The cellular distribution of the glucocorticoid receptor (GR) in relation to various intracellular and plasma membrane structures in human fibroblasts was studied using indirect immunofluorescence techniques with monoclonal and polyclonal antibodies. During interphase, GR was located predominantly in the cytoplasm, showing a similar pattern as tubulin. In mitotic cells, GR and tubulin were localized in mitotic spindles and in telophase midbodies. Colchicine and vinblastine induced a similar redistribution of GR and tubulin to the cell periphery. This redistribution was reversible for colchicine but not for vinblastine. Vinblastine also induced paracrystals containing GR and tubulin. These results support the hypothesis that GR interacts in vivo with cytoplasmic microtubules.     

Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network

Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.     

Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells

Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, Fl-amnion (transformed amnion), WISH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (WI-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 micrograms/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.     

A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. II. Effects on cell movement, organization of microtubules, and intermediate filaments, and arrangement of Golgi elements

A rat monoclonal antibody against yeast alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) that reacts specifically with the tyrosylated form of alpha- tubulin and readily binds to tubulin in microtubules when injected into cultured cells (see Wehland, J., M. C. Willingham, and I. V. Sandoval, 1983, J. Cell Biol., 97:1467-1475) was used to study microtubule organization and function in living cells. Depending on the concentration of YL 1/2 that was injected the following striking effects were observed: (a) When injected at a low concentration (2 mg IgG/ml in the injection solution), where microtubules were decorated without changing their distribution, intracellular movement of cell organelles (saltatory movement) and cell translocation were not affected. Intermediate concentrations (6 mg IgG/ml) that induced bundling but no perinuclear aggregation of microtubules abolished saltatory movement and cell translocation, and high concentrations (greater than 12 mg IgG/ml) that induced perinuclear aggregation of microtubules showed the same effect. (b) YL 1/2, when injected at intermediate and high concentrations, arrested cells in mitosis. Such cells showed no normal spindle structures. (c) Injection of an intermediate concentration of YL 1/2 that stopped saltatory movement caused little or no aggregation of intermediate filaments and no dispersion of the Golgi complex. After injection of high concentrations, resulting in perinuclear aggregation of microtubules, intermediate filaments formed perinuclear bundles and the Golgi complex became dispersed analogous to results obtained after treatment of cells with colcemid. (d) When rhodamine-conjugated YL 1/2 was injected at concentrations that stopped saltatory movement and arrested cells in mitosis, microtubule structures could be visualized and followed for several hours in living cells by video image intensification microscopy. They showed little or no change in distribution and organization during observation, even though these microtubule structures appeared not to be stabilized by injected YL 1/2 since they were readily depolymerized by colcemid or cold treatment and repolymerized upon drug removal or rewarming to 37 degrees C, respectively. These results are discussed in terms of the participation of microtubules in cellular activities such as cell movement and cytoplasmic organization and in terms of the specificity of YL 1/2 for the tyrosylated form of alpha-tubulin.     

Intracellular localization of factor VIII-related antigen and fibronectin in cultured human endothelial cells: evidence for divergent routes of intracellular translocation

Cultured human endothelial cells synthesize and secrete both fibronectin and factor VIII-related antigen (VIIIR:Ag). In immunofluorescence microscopy, intracellular fibronectin was seen diffusely perinuclearly whereas VIIIR:Ag was located both diffusely in the perinuclear cytoplasm and in distinct rod-shaped granules. These granules could, moreover, be visualized with fluorochrome-coupled Ricinus communis agglutinin I (RCA), which also stained the Golgi apparatus as a reticular juxtanuclear structure, and they were identified as Weibel-Palade bodies by immunoelectron microscopy. Puromycin treatment depleted intracellular fibronectin but did not affect the granular localization of VIIIR:Ag. A short exposure of the cells to monensin caused a juxtanuclear accumulation of fibronectin at the Golgi region whereas VIIIR:Ag only was seen in rounded cytoplasmic granules. A prolonged monensin treatment brought about a cytoplasmic accumulation of fibronectin-containing vesicles whereas VIIIR:Ag showed no accumulation and there was no codistribution between granules containing fibronectin or VIIIR:Ag. Type IV procollagen, on the other hand, was distinctly co-localized with fibronectin. In monensin-treated cells RCA mainly stained the VIIIR:Ag-containing vesicles whereas Concanavalin A (Con A) appeared to label the fibronectin-containing vesicles. Immunoelectron microscopy of these cells revealed VIIIR:Ag in some vacuolar structures and typical Weibel-Palade bodies could not be identified. Exposure of the cells to tunicamycin, on the other hand, caused a prominent cytoplasmic accumulation of VIIIR:Ag and, within 96 h, led to the disappearance of most of the VIIIR:Ag-positive granules but did not affect the intracellular distribution of fibronectin. These results, which show that metabolical inhibitors affect differently the intracellular compartmentalization of fibronectin and VIIIR:Ag, indicate, that the two glycoproteins have divergent intracellular pathways in cultured human endothelial cells.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.