Kinetic studies of the intracellular transport of procollagen and fibronectin in human fibroblasts. Effects of the monovalent ionophore, monensin.

Rat embryo fibroblasts cultured in the presence of monensin exhibited an inhibited uptake of horseradish peroxidase . The inhibition was detected after 3 h, after which time the cells became increasingly vacuolated ; the concentration of monensin required to inhibit pinocytosis (0.3 ftM for half-maximum inhibition at 18 h) was similar to that found by others to inhibit secretion . Both the exchange of 5'-nucleotidase between the membranes of cytoplasmic organelles and the cell surface and the internalization of anti-5'-nucleotidase bound to the cell surface were inhibited by -90% in monensin-treated cells. The effects of monensin were reversible : cells cultured first with monensin, and then in fresh medium, exhibited control levels of horseradish peroxidase uptake, exchange of 5'-nucleotidase, and internalization of anti -5'-nucleotidase bound to the cell surface. After monensin treatment, the median density of both galactosyl transferace and 5'-nucleotidase increased from 1 .128 to 1 .148, and the median density of both N-acetyl-,8-glucosaminidase and horseradish peroxidase taken up by endocytosis decreased from 1 .194 to 1 .160 . The results indicate that monensin is a reversible inhibitor of pinocytosis and, presumably, therefore, of membrane recycling . They suggest that the inhibition of membrane recycling occurs at a step other than the fusion of pinocytic vesicles with lysosomes and is perhaps a consequence of an effect of the ionophore on the Golgi complex.The rate of membrane internalization by several different cells in culture as a result of fluid-phase pinocytosis exceeds the rate of membrane synthesis by at least an order of magnitude (1), suggesting that membrane is reutilized (recycled). More direct evidence for recycling has been obtained by studies analyzing the binding (and subsequent fate) of antibodies to the cell surface of rat fibroblasts (2, 3) or the flow of iodinated membrane from phagolysosomes to the cell surface in mouse macrophages (4, 5).At present, the intracellular pathway involved in the return of plasma membrane from lysosomes to the cell surface is unknown . As part of an attempt to identify the organelles involved, we have employed the ionophore monensin, which has been shown to inhibit secretion, probably as a result of an effect on the Golgi complex (6-8) . We show here that monensin treatment (a) inhibits the uptake of horseradish peroxidase by rat fibroblasts and (b) inhibits the exchange of 5'-nucleotidase between the cell surface and the interior. The ionophore has also been shown to alter the density of both lysosomes and membranes of the Golgi complex . MATERIALS AND METHODSRat embryo fibroblasts were cultured as described by Tulkens et at. (9) . Most experiments were performed with cells at the second subculture, grown to confluence on 35 mm dishes.THE IOURNAL OF CELL BIOLOGY " VOLUME 92 MARCH 1982 859-864

Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin.

Wilcox¹,

Kitson²,

Widnell³

1982

“…Moreover, secretion returns to normal upon removal of monensin. Subsequent studies with pulse labeling in conjunction with subcellular fractionations have shown that the passage oflabeled procollagen and fibronectin through the cell is hindered at specific locations (38). Also, our studies of the effect of monensin on chondrocytes THE IOURNAL OF CELL BioLOGy -VOLUME 87 December 1980 663-671 © The Rockefeller University Press -0021-9525/80/12/0663/09$1 .00 have demonstrated the disturbance of Golgi-related sulfation (34).…”

mentioning

confidence: 90%

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

Ledger¹,

Uchida²,

Tanzer³

1980

166

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.Experimental manipulation of the synthesis and secretion of procollagen has been achieved by the use of agents, such as inhibitors of proline hydroxylation (12), local anesthetics (9) and, for fibronectin as well, an inhibitor of protein glycosylation (18). Microtubule-disrupting drugs, such as colchicine, also inhibit secretion, apparently by impairing the transport of secretory vesicles to the cell surface (8). The use ofsuch agents is accompanied by dramatic changes in cell morphology (7) and, in some systems, the inhibition of procollagen synthesis (8). We have demonstrated (39) that the monovalent ionophore monensin reduces the rate of release ofprocollagen and fibronectin from human fibroblasts without seriously disturbing the synthesis of these macromolecules . Moreover, secretion returns to normal upon removal of monensin. Subsequent studies with pulse labeling in conjunction with subcellular fractionations have shown that the passage oflabeled procollagen and fibronectin through the cell is hindered at specific locations (38). Also, our studies of the effect of monensin on chondrocytes

“…2). Similarly, monensin has been observed to decrease the rate of intracellular movement of procollagen and fibronectin (33) and other proteins (17). It is unlikely that the longer time required for transport of vW protein in the presence of monensin is due to its accumulation in a post-Golgi compartment because there was no cellular accumulation of low molecular weight multimers in the presence of monensin.…”

Section: Effect Of Monensin On Vw Protein Production Storage and Secretionmentioning

confidence: 97%

Inhibition of disulfide bonding of von Willebrand protein by monensin results in small, functionally defective multimers.

Wagner¹,

Mayadas²,

Urban-Pickering³

et al. 1985

The biosynthesis of von Willebrand protein by human endothelial cells was impaired by the presence of the carboxylic ionophore monensin. Several processing steps that have been localized to the Golgi apparatus were affected in a dose-dependent manner, including carbohydrate processing, dimer multimerization, and precursor cleavage. Since multimerization was more susceptible to the ionophore than was precursor cleavage, it appears that these processing steps are separate events. As expected, dimer formation, which occurs in the rough endoplasmic reticulum, was unaffected by monensin. Thus, at high concentrations of monensin, only dimer molecules were produced and secreted. The observed inhibition of multimer formation and precursor cleavage were not likely the result of incomplete carbohydrate processing, since inhibition of complex carbohydrate formation by swainsonine did not interfere with the other processing steps. Monensin also affected the capacity of endothelial cells to store von Willebrand protein, as the ratio of secreted to cell-associated protein increased dramatically in the presence of monensin, and the processed forms could not be found in the treated cells. The low molecular weight multimers produced in the presence of monensin did not incorporate in the endothelial cells' extracellular matrix nor did they bind to the matrix of human foreskin fibroblasts. In summary, the presence of monensin in human endothelial cell culture produced experimental conditions that mimic Type IIA von Willebrand disease, in that the cells synthesized and secreted only low molecular weight von Willebrand protein multimers, which were functionally defective.