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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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Cartoon illustrating different domains of Cx43 and Cx40.8 used to generate protein chimeras.(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.
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pone-0031364-g004: Cartoon illustrating different domains of Cx43 and Cx40.8 used to generate protein chimeras.(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.

Mentions: Since Cx40.8 is found in intracellular vesicles in HeLa cells but at the plasma membrane when co-transfected with Cx43 [17], we reasoned that Cx40.8 may contain an intrinsic signal regulating its subcellular localization. To test this hypothesis, multiple chimeric forms of Cx43 and Cx40.8 were generated to identify the domain responsible for the intracellular localization of Cx40.8. These chimeras were generated as EGFP fusion proteins in order to directly visualize subcellular localization. Cx43 and Cx40.8 are about 80% identical at the amino acid levels, where the sequences of the carboxy-termini are the least conserved [17]. Therefore, we subdivided the carboxy termini of Cx43 and Cx40.8 into three similarly sized regions, BCD for Cx43 and bcd for the analogous regions in Cx40.8 (Figure 4).


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)

Cartoon illustrating different domains of Cx43 and Cx40.8 used to generate protein chimeras.(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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275562&req=5

pone-0031364-g004: Cartoon illustrating different domains of Cx43 and Cx40.8 used to generate protein chimeras.(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.
Mentions: Since Cx40.8 is found in intracellular vesicles in HeLa cells but at the plasma membrane when co-transfected with Cx43 [17], we reasoned that Cx40.8 may contain an intrinsic signal regulating its subcellular localization. To test this hypothesis, multiple chimeric forms of Cx43 and Cx40.8 were generated to identify the domain responsible for the intracellular localization of Cx40.8. These chimeras were generated as EGFP fusion proteins in order to directly visualize subcellular localization. Cx43 and Cx40.8 are about 80% identical at the amino acid levels, where the sequences of the carboxy-termini are the least conserved [17]. Therefore, we subdivided the carboxy termini of Cx43 and Cx40.8 into three similarly sized regions, BCD for Cx43 and bcd for the analogous regions in Cx40.8 (Figure 4).

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