Retarded fibronexus formation and cell attachment on type V collagen.

Abstract

We previously found that type V collagen repressed the attachment and spread of aortic smooth muscle cells. The present study was carried out to investigate the effects of type V collagen on the formation of fibronectin and F-actin filaments of human dermal fibroblasts in relation to cell attachment and spread, using an immunofluorescent technique and morphometry. The number and area of the cells attached to type V collagen at 1 and 3 hours after seeding were significantly lower than those of cells on other substrates, including collagen types I, III and IV, and bovine serum albumin. However, there was no significant difference in the attachment and spread among the cells on these substrates after 24 hours. Cultured fibroblasts exhibited two patterns of fibronectin; one was a clear, linear fibronectin localized mainly in the cellular margins, and the other was a granular or flocculent fibronectin found in the perinuclear areas. The former was stained in non-permeabilized cells, but not in trypsin-treated cells (cell surface fibronectin). In contrast, the latter was not detected in nonpermeabilized cells, but was found in trypsin-treated cells (perinuclear fibronectin). Most of the cells cultured on type V collagen did not form either linear cell surface fibronectin or F-actin filaments at 3 hours. In contrast, many cells on collagen types I, III, and IV developed both cell surface fibronectin and F-actin filaments, the distributions of which were partially coincident. Colocalization of linear cell surface fibronectin and F-actin filaments was found in cells on all of the substrates after 24 hours. Perinuclear fibronectin showed similar patterns, and was not colocalized with F-actin filaments on different substrates at 3 and 24 hours of culture. Solid-phase substrates induced a better cellular attachment at 3 hours than serum adhesive factors. The administration of monensin, which inhibits the secretion of protein products, decreased the intensity of the fluorescence of cell surface fibronectin in fibroblasts, which was observed in a clear line. These results suggest that the retardation of the initial attachment and spread of fibroblasts on type V collagen is related to an inhibition in the formation of the fibronexus, a close transmembranous association of individual fibronectin fibers and F-actin filaments.