Meyer, Rita A. and Laird, Dale W. and Revel, Jean-Paul and Johnson, Ross G. (1992) Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies. Journal of Cell Biology, 119 (1). pp. 179-189. ISSN 0021-9525 http://resolver.caltech.edu/CaltechAUTHORS:20120329-083618973
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We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.
|Additional Information:||© 1992 The Rockefeller University Press. Received for publication 4 September 1991 and in revised form 15 June 1992. Thanks are due to S. B. Yancey and D. A. Goodenough for providing anti-bodies and S. G. Remington for critical review of the manuscript. This work was supported by National Institutes of Health grants CA-28548, GM46277, and HL37109. D. W. Laird was supported in part by an American Heart Association Fellowship.|
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|Deposited By:||Tony Diaz|
|Deposited On:||17 Apr 2012 21:16|
|Last Modified:||26 Dec 2012 15:00|
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