Epidermal Growth Factor Receptors Associated to Cytoskeletal Elements of Epidermoid Carcinoma (A431) Cells

• Published in: The Journal of cell biology 1986-07, Vol.103 (1), p.87-94
• Main Authors: Fred A. C. Wiegant, Blok, Freda J., Libert H. K. Defize, Wilbert A. M. Linnemans, Verkley, Arie J., Boonstra, Johannes
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies, Monoclonal; Biological and medical sciences; carcinoma; Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - ultrastructure; Cell growth; Cell Line; Cell membranes; Cell receptors; Cell structures and functions; Cell surface receptors; Cells; Cytoskeleton; Cytoskeleton - metabolism; epidermal growth factor; ErbB Receptors; Fixatives; Fundamental and applied biological sciences. Psychology; Hair cells; Hepatocytes; Humans; Microscopy, Electron - methods; Molecular and cellular biology; PC12 cells; Polyethylene Glycols; Receptors; Receptors, Cell Surface - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.103.1.87

The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membrane-associated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The co-localization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular-located filaments. Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X-100 insoluble cytoskeleton.