Assembly and function of Cx46 but not Cx43 is dependent on a C-terminal domain. (A, B) Wild-type Cx46, like Cx50, produced strong macular staining in HeLa cells and robust neurobiotin transfer. (C, D) Deletion of its ultimate C-terminal residue (Cx46Δ417) resulted in a major reduction but not complete loss of macular localization and a complementary reduction of dye transfer. (E, F) FLAG-tagged Cx46 truncated at residue 290 neither formed immunofluorescent puncta nor induced coupling in HeLa cells. (G, H) Wild-type Cx43 expression induced robust dye coupling, which was not affected by severe C-terminal truncation at residue 258.

Seven C-terminal amino acids constituting a PDZ-binding motif are sufficient to confer normal assembly and function on severely truncated Cx50. (A–D) Although FLAG-tagged Cx50 truncated at residue 290 (Cx50Δ290-440+FL) exhibited neither macular localization (A, N) nor conferred dye coupling (B, N), the addition of the ultimate seven amino acids (Cx50Δ290-440+FL+RSDDLTI) was sufficient to restore normal behavior (C, D, O). (E–M) More than seven residues did not increase the level of coupling above WT, while fewer than seven residues did not restore coupling at all. (P, Q) Fusion of RSDDTLI to Cx50 truncated at residue 245 restored WT levels of plaques and dye coupling. (R) Coexpression of Cx50Δ290-440 with a full-length, soluble C-terminal fragment did not restore function. Thus, unlike Cx43, the efficient assembly of Cx50 into gap junctions requires a PDZ-binding motif at its ultimate C-terminus. E, extracellular domain; FL, FLAG tag; IM, intracellular membranes; M, membrane domain; ND, not determined.

Cx50Δ440 homozygote lenses are small, with nuclear cataracts, and display abnormal connexin distribution. (A) A comparison of Cx50Δ440 knock-in lenses (middle, right) and littermate controls (left) at P28. Knock-in lenses were smaller and developed nuclear cataracts, similar to Cx50-null mouse lines (White et al., 1998; Rong et al., 2002). (B–J) An immunofluorescence comparison of connexin distribution in P12 knock-in and littermate control lenses. (B) Cx50Δ440 was only evident in superficial cortical fibers and accumulated in large linear or irregular patches. Intracellular signal in epithelium (arrow) is higher than in control. (C) Cx46 distribution in the knock-in was abnormal in anterior epithelium and superficial fibers, but deeper fibers displayed typical plaques. (G, F, H) Cx50 and Cx46 were evenly distributed in small puncta in all fibers of the control. (I) High levels of intracellular Cx50Δ440 were evident in bow region fibers of the knock-in. (J) Intracellular connexin is not obvious in control bow region.

Construction of the Cx50Δ440 knock-in mouse. The Cx50 coding sequence is contained in a single exon following a 5.5-kb intron. The targeting vector added a floxed PGK–neomycin resistance cassette inside the intron and a PGK–diphtheria toxin A chain cassette outside the 5′ homology and contained a three-base deletion in the coding sequence deleting the ultimate C-terminal residue. Initial screening for homologous recombinants used PCR amplification with primers outside the 5′ homology arm (1) and in the neo cassette (2). A secondary screen verified 3′ structure using primers in the neo cassette (3) and outside the 3′ homology arm (4). Because the Cx50Δ440 mutation eliminated an Nde1 site, the presence of the mutation was confirmed by restriction digestion. In addition to fully recombined clones, several were identified with partial recombination, resulting in homologous integration of the neo cassette but leaving the native coding sequence, providing an ideal negative control. Recombinant ES clones were then transfected with cre recombinase and screened for neo excision using primers P7 and P8.

The PDZ-domain scaffolding protein ZO-1 is required for normal assembly and function of Cx50. (A–C) Double-label immunofluorescence for Cx50 (red) and ZO-1 (green) in Cx50-transfected HeLa cells. Nuclear signal is a nonspecific background staining. Cx50 and ZO-1 were often closely associated. (D) Western blot for ZO-1 and actin in HeLa cells treated with different siRNAs. Three nonoverlapping anti–ZO-1 siRNAs were equally effective at reducing steady-state protein levels (lanes 2–4), whereas a scrambled control (lane 1) had little effect. Actin served as a loading control. (E–G) Double immunofluorescence labeling for Cx50 and ZO-1 in siRNA-treated cells. ZO-1 staining was greatly reduced (compare E and A), and Cx50 puncta were generally not observed, although intracellular Cx50 signal was abundant (compare F and B). The boxed area in E and F (shown at higher magnification in G) shows an area where ZO-1 expression was retained. Note that Cx50 puncta are observed only in this region. (H) Neurobiotin transfer (red) was not observed in ZO-1–depleted, Cx50-transfected cells. (I–K) Mouse ZO-1 transfection fully restored both plaque formation (J) and dye transfer (K) to siRNA-treated HeLa cells expressing Cx50. Arrow indicates the injected cell.

The ultimate C-terminal residue of Cx50 is required for efficient assembly of gap junctions and formation of active intercellular channels. (A) Transfection of wild-type Cx50 into connexin-deficient, communication-incompetent HeLa cells produced strong punctate immunofluorescence staining at cell–cell interfaces, a pattern commonly observed with other connexins and indicative of gap junction formation. Some intracellular signal, likely from Golgi and other intracellular membranes, was also evident. (B) In contrast, Cx50Δ440, which lacked only the ultimate C-terminal isoleucine, displayed no macular localization, increased intracellular staining, and occasional diffusely distributed signal at or near the plasma membrane (arrow). (C–H) Microinjection of neurobiotin was used to assess junctional coupling. A fluorescent marker (Cerulean or Venus) was incorporated into the expression vector so that injections could be targeted to clusters of transfected cells. For WT Cx50 (C–E), neurobiotin is evident in a large number of cells surrounding the injected cell (arrow), indicating robust coupling by gap junctions (E). In contrast, Cx50Δ440 (F–H) induced no detectable tracer coupling (H), indicating that functional intercellular channels did not form. More extensive truncations were similarly nonfunctional (I–Q), but a modest level of coupling was retained after nearly complete removal of the C-terminal cytoplasmic domain (R). E, extracellular domain; IM, intracellular membranes; M, membrane domain; ND, not determined; PM, plasma membrane.