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

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 alpha1(Cx43), beta1(Cx32), and beta2(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 gap junction 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 give a 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.

Evidence that Bone Marrow Cells Do Not Contribute to the Alveolar Epithelium

“…Moreover, serial sections have been demonstrated to be inadequate for excluding overlapping leukocytes (19). Deconvolution microscopy offers superior resolution to conventional wide-field fluorescent microscopy, and when combined with three-dimensional reconstruction, has been shown to resolve overlapping structures located 100 nm apart (20). Another effective microscopy tool would be confocal microscopy, used by Spyridonidis and colleagues to demonstrate both true colonic epithelial chimerism and false-positives in sex-mismatched transplant patients (19).…”

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

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Non-invasive molecular imaging and reporter genes

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