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Correlative Confocal and Electron Microscopy of the Connexin43 Gap Junction Protein in NRK Cells: Balancing Fixation Conditions, Cell Permeabilization, Antigen-Antibody Interaction and Cell Ultrastructure.

Published online by Cambridge University Press:  02 July 2020

Gina Sosinsky ,
Maryann Martone ,
Galen Handel ,
Linda Musil  and
Mark Ellisman
Gina Sosinsky
Affiliation:
Dept. of Neurosciences, University of California ,San Diego, 9500 Gilman Drive, La Jolla, CA, 92093- 0608
Maryann Martone
Affiliation:
Dept. of Neurosciences, University of California ,San Diego, 9500 Gilman Drive, La Jolla, CA, 92093- 0608 National Center for Microscopy and Imaging Research at San Diego, University of California ,San Diego, 9500 Gilman Drive, La Jolla, CA, 92093- 0608
Galen Handel
Affiliation:
Dept. of Biology, University of California ,San Diego, 9500 Gilman Drive, La Jolla, CA, 92093- 0608
Linda Musil
Affiliation:
Vollem Inst, for Advanced Biomedical Research, L474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201.
Mark Ellisman
Affiliation:
Dept. of Neurosciences, University of California ,San Diego, 9500 Gilman Drive, La Jolla, CA, 92093- 0608 National Center for Microscopy and Imaging Research at San Diego, University of California ,San Diego, 9500 Gilman Drive, La Jolla, CA, 92093- 0608
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Extract

Synthesis and assembly of gap junction proteins into intercellular channels is an important biological problem with direct bearing on embryonic development. In particular, transgenic mice Cx43 gap junction protein knock-outs 1 and mutations in Cx43 sequence in human Viscera Hyper Taxia patients result in severely deformed hearts.

NRK cells are one of the best studied systems for gap junction synthesis, transport and assembly. Gap junctions contain numerous cell-cell channels which are composed of two paired oligomers (called a connexon or hemichannel) each contributed from the plasma membrane of apposing cells. Although biochemical studies have shown indirectly that unpaired Cx43 connexons are in the plasma membrane, direct hemichannel visualization has not been demonstrated. Musil and Goodenough have shown that oligomerization occurs in the trans-Golgi network and the connexon is transported to the plasma membrane.

Type
Unique Approaches in Imaging, Computation and Communication for Characterization of the 3D Cell & Organelles I
Information
Microscopy and Microanalysis , Volume 4 , Issue S2: Proceedings: Microscopy & Microanalysis '98, Microscopy Society of America 56th Annual Meeting, Microbeam Analysis Society 32nd Annual Meeting, Atlanta, Georgia July 12-16, 1998 , July 1998 , pp. 450 - 451
Copyright
Copyright © Microscopy Society of America

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References

References:

1.Reaume, A.G., de Souza, P.A., Kulkarni, S., Langille, G.L., Zhu, D., Davies, T.C., Junega, S.C., Kidder, G.M., and Rossant, J., (1995) Science, 267: 1831–1834.CrossRefGoogle Scholar
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3.Musil, L.M. and Goodenough, D.A., (1993) Cell, 74: 1065–1077.CrossRefGoogle Scholar
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5. The authors wish to thank Dr. Dan Goodenough for his gift of the Cx43 antisera.Google Scholar