Connexin-specific distribution within gap junctions revealed in living cells.
To study the organization of gap junctions in living cells, the connexin isotypes alpha(1)(Cx43), beta(1)(Cx32) and beta(2)(Cx26) were tagged with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. The cellular fate of the tagged connexins was followed by high-resolution fluorescence deconvolution microscopy and time-lapse imaging. Comprehensive analyses demonstrated that the tagged channels were functional as monitored by dye transfer, even under conditions where the channels were assembled solely from tagged connexins. High-resolution images revealed a detailed structural organization, and volume reconstructions provided a three-dimensional view of entire gap junction plaques. Specifically, deconvolved dual-color images of gap junction plaques assembled from CFP- and YFP-tagged connexins revealed that different connexin isotypes gathered within the same plaques. Connexins either codistributed homogeneously throughout the plaque, or each connexin isotype segregated into well-separated domains. The studies demonstrate that the mode of channel distribution strictly depends on the connexin isotypes. Based on previous studies on the synthesis and assembly of connexins I suggest that channel distribution is regulated by intrinsic connexin isotype specific signals.
Falk
M M
MM
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. mfalk@scripps.edu
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D000072259:Connexin 26; D018031:Connexin 43 / Q000032:analysis; D017630:Connexins / Q000032:analysis* / Q000235:genetics; D006224:Cricetinae; D017629:Gap Junctions / Q000502:physiology / Q000648:ultrastructure*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013997:Time Factors; D014162:Transfection; D000097002:Gap Junction beta-1 Protein
D000072259:Connexin 26; D018031:Connexin 43; D017630:Connexins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097002:Gap Junction beta-1 Protein; C000607042:GJB2 protein, human