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The Cx43-like connexin protein Cx40.8 is differentially localized during fin ontogeny and fin regeneration.

Gerhart SV, Eble DM, Burger RM, Oline SN, Vacaru A, Sadler KC, Jefferis R, Iovine MK - PLoS ONE (2012)

Bottom Line: Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration.During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration.Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, United States of America.

ABSTRACT
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.

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Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels.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) Homo sapiens (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) Danio rerio (Dr) Cx43-mApple + Dr Cx40.8-EGFP show uniform yellow distribution.
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pone-0031364-g007: Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels.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) Homo sapiens (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) Danio rerio (Dr) Cx43-mApple + Dr Cx40.8-EGFP show uniform yellow distribution.

Mentions: We previously found that Cx40.8 is capable of co-localizing to gap junction plaques in HeLa cells when co-transfected with Cx43 [17], suggesting that Cx43 and Cx40.8 could associate in common gap junction channels. Increasing the primary magnification during fluorescence microscopy can be used to further investigate this hypothesis [22]. For example, co-transfection of HeLa cells with human Cx26-EGFP and human Cx43-mApple demonstrates that these connexins occupy a common gap junction plaque (Figure 7C,D and [22]). However, discrete domains of green and red are observed, revealing that Cx26-EGFP and Cx43-mApple do not hetero-oligomerize in common connexons, nor do they establish homomeric heterotypic gap junction channels (i.e. homomeric heterotypic channels would be comprised of one Cx26-EGFP connexon and one Cx43-mApple connexon). In contrast, co-transfection of human Cx43-EGFP and human Cx43-mApple results in plaques that are uniformly yellow, suggesting that the two connexins are located in common gap junction channels (Figure 7A,B). Similarly, when zebrafish Cx43-mApple was co-expressed with zebrafish Cx40.8-EGFP, the resulting gap junction plaques were yellow (Figure 7E,F). This finding suggests that Cx43 and Cx40.8 co-assemble into common gap junction channels when co-expressed in HeLa cells (see Figure S1 for single channel images).


The Cx43-like connexin protein Cx40.8 is differentially localized during fin ontogeny and fin regeneration.

Gerhart SV, Eble DM, Burger RM, Oline SN, Vacaru A, Sadler KC, Jefferis R, Iovine MK - PLoS ONE (2012)

Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels.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) Homo sapiens (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) Danio rerio (Dr) Cx43-mApple + Dr Cx40.8-EGFP show uniform yellow distribution.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275562&req=5

pone-0031364-g007: Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels.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) Homo sapiens (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) Danio rerio (Dr) Cx43-mApple + Dr Cx40.8-EGFP show uniform yellow distribution.
Mentions: We previously found that Cx40.8 is capable of co-localizing to gap junction plaques in HeLa cells when co-transfected with Cx43 [17], suggesting that Cx43 and Cx40.8 could associate in common gap junction channels. Increasing the primary magnification during fluorescence microscopy can be used to further investigate this hypothesis [22]. For example, co-transfection of HeLa cells with human Cx26-EGFP and human Cx43-mApple demonstrates that these connexins occupy a common gap junction plaque (Figure 7C,D and [22]). However, discrete domains of green and red are observed, revealing that Cx26-EGFP and Cx43-mApple do not hetero-oligomerize in common connexons, nor do they establish homomeric heterotypic gap junction channels (i.e. homomeric heterotypic channels would be comprised of one Cx26-EGFP connexon and one Cx43-mApple connexon). In contrast, co-transfection of human Cx43-EGFP and human Cx43-mApple results in plaques that are uniformly yellow, suggesting that the two connexins are located in common gap junction channels (Figure 7A,B). Similarly, when zebrafish Cx43-mApple was co-expressed with zebrafish Cx40.8-EGFP, the resulting gap junction plaques were yellow (Figure 7E,F). This finding suggests that Cx43 and Cx40.8 co-assemble into common gap junction channels when co-expressed in HeLa cells (see Figure S1 for single channel images).

Bottom Line: Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration.During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration.Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus