Development of Cell Surface Linkage Complexes in Cultured Fibroblasts

• Published in: The Journal of cell biology 1985-04, Vol.100 (4), p.1103-1114
• Main Authors: Chen, Wen-Tien, Hasegawa, Etsuko, Hasegawa, Takayuki, Weinstock, Catherine, Yamada, Kenneth M.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antibodies, Monoclonal; Antigens; Biological and medical sciences; Cell Adhesion; Cell coat. Cell surface; Cell Membrane - metabolism; Cell membranes; Cell structures and functions; Cells; Cells, Cultured; Chick Embryo; Cultured cells; Cytoskeleton - metabolism; Extracellular Matrix - metabolism; Fibroblasts; Fibroblasts - metabolism; Fibroblasts - ultrastructure; Fibronectins - metabolism; Focal adhesions; Fundamental and applied biological sciences. Psychology; Histocytochemistry; Immunologic Techniques; Membrane Proteins - metabolism; Molecular and cellular biology; Molecular Weight; Monoclonal antibodies; Solar fibrils; Space life sciences
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.100.4.1103

The possible role of a 140K membrane-associated protein complex (140K) in fibronectin-cytoskeleton associations has been examined. The 140K was identified by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140K showed identical patterns of binding to the cell membranes of fixed and permeabilized chicken embryonic fibroblasts; localization was diffuse, but with marked concentration in cell-to-extracellular matrix contact sites. Correlative localization with interference reflection microscopy and double-label or triple-label immunofluorescence showed that 140K co-distributed with extracellular fibronectin fibrils and intracellular α-actinin in microfilament bundles at extracellular matrix contact sites but tended not to co-localize with tropomyosin present in bundles at sites farther from adhesion sites. In addition, binding of antibodies to 140K, α-actinin, and fibronectin was excluded from vinculin-rich focal adhesion sites at the cellular periphery. A progressive development of cell surface α-actinin-140K-fibronectin associations was observed in early spreading cells. The anti-140K monoclonal antibody JG22E inhibited the attachment and spreading of both normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts to a fibronectin substratum. However, the anti-140K monoclonal antibody became a positive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our results provide the first description of a membrane-associated protein complex that co-localizes with fibronectin and microfilament bundles, and they suggest that the 140K complex may be part of a cell surface linkage between fibronectin and the cytoskeleton.