QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online December 31, 2003
doi: 10.1242/10.1242/jcs.00889

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doble, B. W. Articles by Kardami, E. Search for Related Content PubMed PubMed Citation Articles by Doble, B. W. Articles by Kardami, E. Social Bookmarking                         What's this?

Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes

Bradley W. Doble^1,3,*, Xitong Dang^2,3, Peipei Ping^4,, Robert R. Fandrich^2,3, Barbara E. Nickel³, Yan Jin¹, Peter A. Cattini¹ and Elissavet Kardami^1,2,3,

² Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg MB R3E 3J7, Canada
¹ Physiology, Faculty of Medicine, University of Manitoba, Winnipeg MB R3E 3J7, Canada
³ Institute of Cardiovascular Sciences, SBGH Research Centre 3008, 351 Tache Avenue, Winnipeg MB R2H 2A6, Canada
⁴ Department of Medicine, University of Louisville, Louisville, KY 40292, USA

View larger version (39K): [in a new window]   Fig. 1. Effect of FGF2 and PMA on Cx43 phosphorylation on S262. Western blots of total lysates from cardiomyocyte cultures (20 µg/lane) probed with antibodies recognizing (A) total, or (B) P-262-Cx43, as indicated. Cultures were stimulated with FGF2 (10 ng/ml) or PMA (100 nM) for 15-60 minutes, as indicated.

View larger version (55K): [in a new window]   Fig. 2. Detection of overexpressed wt, 262A- and 262D-Cx43. (A) Western blot of lysates from vector-infected (10 µg/lane) or Cx43 (wt and mutant) overexpressing cultures (1 µg/lane; as indicated) probed with rabbit polyclonal anti-Cx43 antibodies. (B-D) Triple-fluorescence labelling, using rabbit polyclonal anti-Cx43 antibodies (green), mouse monoclonal anti-Cx43 antibodies (red) and Hoechst 33342 (blue). Myocyte cultures were infected with (B) vector, (C) 262A-Cx43 or (D) 262D-Cx43. Scale bar: 50 µm. Arrows point to areas of cell-cell contact.

View larger version (130K): [in a new window]   Fig. 3. Effect of wt or mutant Cx43 overexpression on cardiomyocyte anti-BrdU labelling. Myocyte cultures, infected with adenoviral vectors expressing wt-, 262A- and 262D-Cx43 (as indicated) were labelled simultaneously for BrdU (nucleus) and Cx43 (cell membrane), using monoclonal antibodies specific for each antigen. One colour is sufficient to detect both antigens, since they are found in different cellular locations. Arrows and arrowheads point to BrdU-positive and negative nuclei, respectively. Scale bar: 50 µm.

View larger version (81K): [in a new window]   Fig. 4. Effect of 262A- and 262D-Cx43 overexpression on cardiomyocyte anti-Ki-67 labelling. Myocyte cultures infected with adenoviral vectors expressing (A) 262A-Cx43 or (B) 262D-Cx43 were labelled for N-cadherin (green; rabbit polyclonal antibodies to delineate cell-cell contact cardiomyocyte areas), Ki-67 (green; rabbit polyclonal to detect nuclei of proliferating cells; elicits speckled nuclear staining) and Cx43 (red; monoclonal antibodies). Arrowheads, non-proliferating myocytes; arrows, proliferating myocytes. Scale bar: 50 µm.

View larger version (17K): [in a new window]   Fig. 5. Effect of wt-, 262A- and 262D-Cx43 on myocyte DNA synthesis. (A) BrdU labelling index (LI) in semi-confluent myocyte cultures infected with adenoviral vectors expressing wt-, 262A- and 262D-Cx43, as indicated (n=4-6). (B) Ki-67 LI in sparse cultures infected with adenoviral vectors expressing wt-, 262A- and 262D-Cx43, as indicated (n=4). Bars indicate s.e.m.

View larger version (34K): [in a new window]   Fig. 6. Effect of Cx43 and S262 mutations on dye coupling. (A,B) Representative micrographs of dye migration (green), assessed by scrape-loading, in confluent cultures (seen by simultaneous phase contrast photography) expressing (A) 262A- or (B) 262D-Cx43. Arrowheads indicate length of dye spreading. In C dye spreading is plotted as a function of overexpressing wt-, 262A- (+/–PMA) and 262D-Cx43 (+/–PMA), compared to vector-infected cultures, as indicated (n=4). Bars indicate s.e.m.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter