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Titolo:
High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells
Autore:
Falk, MM; Lauf, U;
Indirizzi:
Scripps Clin & Res Inst, Dept Cell Biol, La Jolla, CA 92037 USA Scripps Clin & Res Inst La Jolla CA USA 92037 iol, La Jolla, CA 92037 USA
Titolo Testata:
MICROSCOPY RESEARCH AND TECHNIQUE
fascicolo: 3, volume: 52, anno: 2001,
pagine: 251 - 262
SICI:
1059-910X(20010201)52:3<251:HRFDMA>2.0.ZU;2-B
Fonte:
ISI
Lingua:
ENG
Soggetto:
PROTEIN; CHANNELS; EXPRESSION; CONNEXINS; PHOSPHORYLATION; COMMUNICATION; TRAFFICKING; MEMBRANES; VARIANTS; GENE;
Keywords:
connexins; fluorescent proteins; membrane proteins; oligomers; reporter technology;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
62
Recensione:
Indirizzi per estratti:
Indirizzo: Falk, MM Scripps Clin & Res Inst, Dept Cell Biol, MB21,10550 N Torrey Pines Rd, La Jolla, CA 92037 USA Scripps Clin & Res Inst MB21,10550 N Torrey Pines Rd La Jolla CA USA 92037
Citazione:
M.M. Falk e U. Lauf, "High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells", MICROSC RES, 52(3), 2001, pp. 251-262

Abstract

High-resolution, fluorescence deconvolution (DV) microscopy was implemented to obtain a detailed view of the organization and structural composition of gap junctions assembled from one or two different connexin isotypes in live and fixed cells. To visualize gap junctions, the structural protein components of gap junction channels, the connexin polypeptides alpha (1)(Cx43), beta (1)(Cx32), and beta (2)(Cx26), were tagged on their C-termini with the autofluorescent tracers green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants. Tagged connexins were expressed in transiently transfected HeLa cells. Comprehensive analysis including dye-transfer analysis demonstrated that the tagged connexins trafficked, assembled,and packed normally into functional gap junction channel plaques. Such gapjunction plaques were examined by single, dual, and triple-color DV microscopy. High-resolution images and three-dimensional volume reconstructions of gap junction plaques were obtained by this technique, which revealed several new aspects of gap junction structure. Specifically, the studies demonstrated that the mode of channel distribution strictly depends on the connexin isotypes. Here we present such images, and volume reconstructions in context with images obtained by other light, and electron microscopic techniques, such as laser scanning confocal, conventional wide-field fluorescence, thin section, and freeze-fracture electron microscopy. In addition, we givea simple description of the principal mechanisms of DV microscopy, name advantages and disadvantages, and discuss issues such as dual-color imaging using CFP and YFP, spatial resolution, colocalization, and avoiding imaging artifacts. Microsc. Res. Tech. 52:251-262, 2001. (C) 2001 Wiley-Liss. Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/12/20 alle ore 14:13:13