Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling

John E. Rash, Naomi Kamasawa, Kimberly G V Davidson, Thomas Yasumura, Alberto E. Pereda, James I. Nagy

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of themembrane ''sidedness'' and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

Original languageEnglish (US)
Pages (from-to)333-344
Number of pages12
JournalJournal of Membrane Biology
Volume245
Issue number5-6
DOIs
StatePublished - Jun 2012

Fingerprint

Connexins
Gap Junctions
Proteins
Membranes
Freeze Fracturing
Light
Transmission Electron Microscopy
Neuroglia
Vertebrates
Immunohistochemistry
Neurons
Messenger RNA
Mutation

Keywords

  • Astrocyte
  • Ependymocyte
  • Glia
  • Neuron
  • Oligodendrocyte

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling. / Rash, John E.; Kamasawa, Naomi; Davidson, Kimberly G V; Yasumura, Thomas; Pereda, Alberto E.; Nagy, James I.

In: Journal of Membrane Biology, Vol. 245, No. 5-6, 06.2012, p. 333-344.

Research output: Contribution to journalArticle

Rash, John E. ; Kamasawa, Naomi ; Davidson, Kimberly G V ; Yasumura, Thomas ; Pereda, Alberto E. ; Nagy, James I. / Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling. In: Journal of Membrane Biology. 2012 ; Vol. 245, No. 5-6. pp. 333-344.
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