Immunocytochemical Localization of Procollagen and Fibronectin in Human Fibroblasts : Effects of the Monovalent lonophore, 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. 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 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) . The simultaneous inhibition of secretion of both procollagen and fibronectin in fibroblasts evokes further interest because of the known affinity of these molecules in vitro (10) and their association together in the extracellular matrix (40) . Thus, the present investigation further characterizes the action of monensin on fibroblasts describing ultrastructural changes, the distribution of procollagen and fibronectin as determined by immunofluorescence microscopy, and considers the data in terms of the secretion of these proteins and the inhibition of their secretion by monensin . MATERIALS AND METHODS