The Cx43-like Connexin Protein Cx40.8 Is Differentially Localized during Fin Ontogeny and Fin Regeneration

Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration

Voltage dependent conductances are similar between Cx43 and Cx40.8-cd in transiently transfected N2a cells.

<p>(A) Transjunctional current family from a Cx43 expressing cell pair. V_j was varied ±180 mV in 30 mV steps. (B) Currents from a Cx40.8 expressing pair. (C) Normalized steady state G_j measures as a function of V_j show similar inactivation properties between the two constructs. Error bars are SEM.</p

Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels.

<p>High resolution fluorescence microscopy was used to provide evidence for co-association of Cx43-mApple and Cx40.8-EGFP in common gap junction channels. Constructs that were co-transfected in HeLa cells are indicated to the left of the panels (A, B) <i>Homo sapiens</i> (Hs) Cx43-mApple + Hs Cx43-EGFP show uniformly yellow plaques, suggesting co-asociation. (C, D) Hs Cx43-mApple + Hs Cx26-EGFP show discrete green and red domains, revealing a lack of co-association. Arrows indicate green Hs Cx26-EGFP localization and arrowheads indicate red Hs Cx43-mApple localization. (E, F) <i>Danio rerio</i> (Dr) Cx43-mApple + Dr Cx40.8-EGFP show uniform yellow distribution.</p

Cartoon illustrating different domains of Cx43 and Cx40.8 used to generate protein chimeras.

<p>(A) Full length sequences of Cx43 and Cx40.8. The amino terminus through the end of the fourth transmembrane-spanning domain was not modified. Swaps of the entire carboxy termini were generated, as well as swaps between domains B/b, C/c, and D/d. Uppercase refers to the Cx43 domains; lowercase refers to the Cx40.8 domains. (B) Amino acid sequence of Cx43-B vs. Cx40.8-b domains.</p

Cx40.8 co-localizes with the GalT-GFP transgene found in the Golgi during regeneration.

<p>(A–C) Co-localization in untreated regenerating fins. (D–F). Co-localization in regenerating fins treated with 0.1% DMSO carrier. (G–I) Fins injected with10 µg/ml BFA/0.1% BFA to disrupt the Golgi show dispersal of both Cx40.8 and GalT-GFP signals. In D-I, nuclei (blue) are stained with TO-PRO3 detected in the far red channel. Arrows point to areas of overlap in F (intact Golgi) and I (remnants of intact Golgi). Scale bars in C and F, 10 µm.</p

The Cx40.8 antibody is specific.

<p>(A) An antibody raised against a Cx40.8 carboxy-terminal peptide detects a single major band from regenerating fin lysates (arrowhead). Non-competed (B) and competed (C) anti-Cx40.8 antibody was used to detect bacterially expressed GST-Cx40.8CT. Decreasing volumes of a concentrated lysate were loaded in each lane on two identical gels. When the antibody was pre-incubated with the Cx40.8 target sequence (i.e. competed), antibody binding was reduced in all lanes compared with non-competed antibody. Arrowhead points to the full length product. Bands at molecular weights smaller than the full-length fusion protein represent degradation products.</p

The Cx40.8-b domain is responsible for the intracellular localization of Cx40.8.

<p>HeLa cells were singly transfected with EGFP fusions of each construct. (A) Cx43-BCD-EGFP, (B) Cx40.8-bcd-EGFP, (C) Cx43-bcd-EGFP, (D) Cx40.8-BCD-EGFP, (E) Cx43-bCD-EGFP, (F) Cx40.8-Bcd-EGFP, (G) Cx43-BcD-EGFP, (H) Cx40.8-bCd-EGFP, (I) Cx43-BCd-EGFP, (J) Cx40.8-bcD-EGFP, (K) Cx43-bBCD-EGFP, (L) Cx40.8-cd-EGFP. Asterisks denote constructs in which the localization depends on a component of the carboxy terminus. Arrows identify gap junction plaques.</p