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

Abstract

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

Role of retinoic acid in mouse neural crest cell development in vitro

“…RA was used at a final concentration of 10 nM-1 pM, which is within the physiological range (Thaller and Eichele, 1987). Where indicated, monensin (Sigma) was added to the culture medium to inhibit secretion of extracellular matrix proteins (Ledger et al, 1980). Monensin was used at 0.5-0.7 p M and the cells were exposed to it during the last 15 hr of the culture period before fixation for immunostainings (see Immunocytochemistry and the 2-p-(3,4-Dihydroxyphenyl)alanine (DOPA) Reaction).…”

Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187.

“…The monovalent carboxylic ionophore monensin has probably found the widest application. It is assumed that by blocking the intracellular transport, presumably somewhere within the Golgi apparatus, this com-© IRL Press Limited, Oxford, England. pound interferes with the biosynthesis of cellular membrane and secretory proteins (Ledger et al, 1980;Vassalli, 1977, 1978) and of the glycoproteins of several viruses, such as vesicular stomatitis virus, alphaviruses and coronaviruses (Johnson and Schlesinger, 1980;Straus and Lodish, 1980;Kaariainen et al, 1980;Niemann et al, 1982). However, with influenza virus, monensin did not inhibit glycoprotein processing and virus assembly (Alonso and Compans, 1981).…”

Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore.

“…As a result of viral inhibition of host protein synthesis, only one or a few viral membrane glycoproteins are synthesized in infected cells. These viruses possess limited genetic capacity, and the glycosylation and transport of their membrane glycoproteins to the cell surface are probably carried out by the same systems used by the host cell for biogenesis of its own membrane glycoproteins.Monovalent ionophores such as monensin, which is reported to interfere with the translocation of secretory as well as most membrane glycoproteins, have been used to characterize the pathways of intracellular glycoprotein transport in eucaryotic cells (18,(37)(38)(39)(40)(41)43). In addition, the effects of monensin on IgM and H2 glycosylation in lymphoid cells and fibronectin glycosylation in cultured human fibroblasts, as well as its influence on oligosaccharide maturation of…”