Figures & data
Figure 1 Domain structure of Cx43 and ZO-1 constructs. (A) Full-length Cx43, with or without EGFP fused to its C-terminus. The PDZ2 binding region spans the last ∼ 20 residues in Cx43, which include the C-terminal DLEI consensus sequence, as well as upstream residues that mediate non-canonical PDZ2 interactions. TM, transmembrane domain. (B) DsRed was fused to the N-terminus of full-length ZO-1 and mutant ZO-1 lacking PDZ2. SH3, Src homology domain; GUK, guanylate kinase domain.
Figure 2 Full-length ZO-1 targets to edge domains of gap junctions composed of either Cx43 or Cx43-GFP. (A) Immunofluorescence of Cx43 (red) and endogenous ZO-1 (green) in HeLa Cx43 cells. HeLa Cx43 (B) and HeLa Cx43-GFP (C) cells transiently expressing DsR-ZO-1, with Cx43 (red) detected by immunofluorescence (B) or GFP fluorescence (C), and ZO-1 (green) by DsRed fluorescence (B, C). Note that both endogenous ZO-1 (A, inset) and DsR-ZO-1 (B) display stretches of continuous overlap with the edges of Cx43 plaques, whereas DsR-ZO-1 associated with Cx43-GFP GJs manifests as punctate rather than diffuse accumulations along plaque edges that exhibit minimal overlap with Cx43. All images are maximum projections of z-series acquired by laser scanning (A) or spinning disk (B, C) confocal microscopy. Scale bars, 5 μ m.
Figure 3 PDZ2-mediated interactions are not required for ZO-1 targeting to Cx43 gap junctions. HeLa Cx43 (A) and HeLa Cx43-GFP (B) cells transiently expressing mutant ZO-1 lacking PDZ2 (DsR-ZO-1Δ PDZ2), with Cx43 (red) detected by immunofluorescence (A) or GFP fluorescence (B), and mutant ZO-1 (green) by DsRed fluorescence (A, B). Despite the absence of a PDZ2 domain, DsR-ZO-1Δ PDZ2 localizes at the periphery of Cx43 plaques (A), with some areas of edge overlap similar to that seen with full-length ZO-1 (A, arrowheads). DsR-ZO-1Δ PDZ2 also targets to the edges of Cx43-GFP plaques (B); however, 3D rotations show that the mutant ZO-1 accumulates in punctae juxtaposed but not overlapping with plaque edges (B, arrowheads). All images are maximum projections of z-series acquired by spinning disk confocal microscopy. Z-series were rendered in 3D and rotated to provide en face plaque perspectives and reveal the extent of true colocalization (3D panels). Scale bars, 5 μ m.
Figure 4 ZO-2 colocalizes with ZO-1 at the periphery of Cx43 and Cx43-GFP gap junctions. (A) Immunofluorescence of Cx43 (cyan) and endogenous ZO-2 (red) in HeLa Cx43 cells, showing ZO-2 at the periphery of Cx43 plaques. (B) HeLa Cx43-GFP cells transiently expressing DsR-ZO-1, with Cx43 (cyan) detected by GFP fluorescence, ZO-1 (green) by DsRed fluorescence, and ZO-2 (red) by immunofluorescence. Note that the majority of ZO-2 at the edges of Cx43-GFP plaques is colocalized with ZO-1 (B, arrowheads), whereas some ZO-1 at edges does not overlap significantly with ZO-2 (B, arrow). (C) Z-series (same as shown in B) was rendered in 3D and rotated to show the relative distributions of DsR-ZO-1 and ZO-2 at plaque edges from an en face perspective. (Note: rotation of cytoplasmic structures into the foreground creates the false impression of ZO-1 and ZO-2 localization in the plaque interior.) All images are maximum projections of z-series acquired by laser scanning (A) or spinning disk (B, C) confocal microscopy. Scale bars, 5 μ m.
