Baetscher, Manfred and Pumplin, David W. and Bloch, Robert J. (1986) Vitronectin at sites of cell-substrate contact in cultures of rat myotubes. Journal of Cell Biology, 103 (2). pp. 369-378. ISSN 0021-9525. http://resolver.caltech.edu/CaltechAUTHORS:BAEjcb86
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Affinity-purified antibodies to the serum glycoprotein, vitronectin, were used to study sites of cell-substrate contact in cultures of rat myotubes and fibroblasts. Cells were removed from the substrate by treatment with saponin, leaving fragments of plasma membrane attached to the glass coverslip. When stained for vitronectin by indirect immunofluorescence, large areas of the substrate were brightly labeled. The focal contacts of fibroblasts and the broad adhesion plaques of myotubes appeared black, however, indicating that the antibodies had failed to react with those areas. Contact sites within the adhesion plaque remained unlabeled after saponin-treated samples were extracted with Triton X-100, or after intact cultures were sheared with a stream of fixative. These procedures expose extracellular macromolecules at the cell-substrate interface, which can then be labeled with concanavalin A. In contrast, when samples were sheared and then sonicated to remove all the cellular material from the coverslip, the entire substrate labeled extensively and almost uniformly with anti- vitronectin. Extracellular molecules associated with substrate contacts were also studied after freeze-fracture, using a technique we term "post-release fracture labeling." Platinum replicas of the external membrane were removed from the glass with hydrofluoric acid to expose the extracellular material. Anti-vitronectin, bound to the replicas and visualized by a second antibody conjugated to colloidal gold, labeled the broad areas of close myotube-substrate attachment and the nearby glass equally well. Our results are consistent with the hypothesis that vitronectin is present at all sites of cell-substrate contact, but that its antigenic sites are obscured by material deposited by both myotube and fibroblast cells.
|Additional Information:||Copyright © 1986 by The Rockefeller University Press. Received for publication 30 July 1985, and in revised form 11 March 1986. M. Baetscher has been the recipient of a postdoctoral fellowship from the Muscular Dystrophy Association of America. Our research has been supported by grants to D.W. Pumplin from the National Institutes of Health (NS 15513) and the Muscular Dystrophy Association, and to R.J. Bloch from the March of Dimes, the NIH (NS 17282), and the Muscular Dystrophy Association. R.J. Bloch was also supported by a McKnight Scholar's Award and a Research Career Development Award (NS 00679).|
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|Deposited On:||21 May 2008|
|Last Modified:||26 Dec 2012 10:02|
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