Our object was to obtain information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured fibroblasts. We have carried out double irnmunoelectron-microscopic labeling experiments on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the three proteins fibronectin, vinculin, and a-actinin with respect to these sites.(a) Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the two surfaces, and the presence of submembranous densities. These types were: (i) focal adhesions (FA); (ii) close contacts (CC); and (iii) extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed us to recognize and assign distinctive immunolabeling patterns for the three proteins to each type of site on the frozen sections.(b) FA sites were immunolabeled intracellularly for vinculin and a-actinin, with vinculin labeling situated closer to the membrane than a-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum, or between two cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites.(c) CC sites were labeled intracellularly for a-actinin but not vinculin and were labeled extracellularly for fibronectin.(d) ECM sites were characterized by large separations (often >100 nm) between the cell and substratum or between two cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of two kinds, one labeled intracellularly for both aactinin and vinculin, the other for a-actinin alone.(e) From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin, and a-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.The nature and molecular structure of the sites of contact of fibroblastic cells with one another and with the substrata on which they grow are subjects of much recent interest. Three major types of investigation have been pursued in this area: (a) at the biochemical level, extracellular matrix molecules such as fibronectin (for review, see reference 42) and intracellular cytoskeletal molecules such as a-actinin (50,55) (12,26) have been isolated and implicated in the formation of such contacts; (b) at the light microscopic level, interference reflection microscopy has been used to define different sites of contact of cells to substrata (20,46,47), and immunofluorescence micro...
Abstract. A major collagen-binding heat shock protein of molecular mass 47,000 D was found to bind to collagen by a pH-dependent interaction; binding was abolished at pH 6.3. Native 47-kD protein could therefore be purified from chick embryo homogenates in milligram quantities by gelatin-affinity chromatography and gentle acidic elution. Rat monoclonal and rabbit polyclonal antibodies were generated against the purified 47-kD protein. Immunofluorescence microscopy of cultured chick embryo fibroblasts with these antibodies revealed bright, granular perinuclear staining as well as a weaker reticular network structure towards the cell periphery, suggesting that this protein was located in the endoplasmic reticulum. No immunofluorescence staining was detected on the cell surface. Doublestaining experiments with these antibodies and fluorescently labeled wheat-germ agglutinin suggested that the 47-kD protein was absent from the Golgi apparatus. Localization of the 47-kD protein in the endoplasmic reticulum but not in the Golgi complex was confirmed by immunoelectron microscopy. In vivo localization studies using immunohistochemistry of cryostat sections of chick liver revealed that the 47-kD protein was present in fibrocytes, Kupffer cells, and smooth muscle cells. It was absent from hepatocytes and the epithelia of bile ducts or sinusoidal endothelium. This major transformation-and heat shockregulated glycoprotein is thus localized intracellularly, is expressed in only certain cells, and functions in a pH-regulated manner. These findings suggest that this glycoprotein is not likely to be a general cell-surface collagen receptor, but may instead play roles in intracellular protein processing or translocation.ECENT studies have characterized a major collagenbinding protein of Mr 47,000 D that is present in a wide variety of cultured cell types (12,15,16,21). This membrane-associated glycoprotein binds to gelatin (denatured collagen types I and ~ and to native collagen types I, M, and IV (12,15,16,21). It represents the major protein besides fibronectin that binds to collagen-affinity columns under physiological salt conditions, and it can be the major concanavalin A-binding protein of membrane preparations (6,12,15,16,21).Previously, we reported (16) that this 47-kD collagen/ gelatin-binding protein corresponds to a previously described membrane protein (6,19), and that its synthesis and phosphorylation are regulated in opposing directions by an oncogene product. In addition, we found that this protein is a novel heat-shock protein (15). Studies of its specificity of binding indicated that it is relatively specific for the collagens, although it can bind to fetuin (16; Nagata, K., S. Saga, and K. M. Yamada, unpublished); whether such interactions with noncollagenous proteins provide a mechanism for reguDr. Saga's present address is The Second Department of Pathology, Nagoya University School of Medicine, Nagoya 466, Japan. Dr. Nagata's present address is Chest Disease Research Institute, Kyoto University, Kyoto 606, J...
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