Extracellular-loop peptide antibodies reveal a predominant hemichannel organization of connexins in polarized intestinal cells.
Shigella, the causative agent of bacillary dysentery, invades colonic epithelial cells to elicit an intense inflammatory reaction leading to destruction of the mucosa. ATP-dependent paracrine signalling induced by connexin (Cx) hemichannel opening was previously shown to favor Shigella flexneri invasion and dissemination in transfectants of HeLa cells [G. Tran Van Nhieu, C. Clair, R. Bruzzone, M. Mesnil, P. Sansonetti and L. Combettes. (2003). Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat. Cell Biol. 5, 720-726.]. However, although Cxs have been described in polarized epithelial cells, little is known about their structural organization and the role of hemichannels during S. flexneri invasion. We show here that polarized Caco-2/TC7 cells express significant amounts of Cx26, Cx32 and Cx43, but that unexpectedly, cell-cell coupling assessed by dye-transfer experiments is inefficient. Consistent with a predominant Cx organization in hemichannels, dye loading induced by low calcium was readily observed, with preferential loading at the basolateral side. Antibodies (Abs) against connexin extracellular loop peptides (CELAbs) demonstrated the importance of hemichannel signalling since they inhibited dye uptake at low calcium and at physiological calcium concentrations during S. flexneri invasion. Importantly, CELAbs allowed the visualization of hemichannels at the surface of epithelial cells, as structures distinct from gap intercellular junctions.
Clair
Caroline
C
Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris Cedex 15, France.
Combettes
Laurent
L
Pierre
Fabienne
F
Sansonetti
Philippe
P
Tran Van Nhieu
Guy
G
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D018938:Caco-2 Cells; D002118:Calcium / Q000494:pharmacology; D016764:Cell Polarity / Q000187:drug effects*; D000072259:Connexin 26; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology* / Q000187:drug effects; D007546:Isoquinolines / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012763:Shigella flexneri / Q000166:cytology / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D000097002:Gap Junction beta-1 Protein
D000906:Antibodies; D002118:Calcium; D000072259:Connexin 26; D018031:Connexin 43; D017630:Connexins; D007546:Isoquinolines; D010455:Peptides; D012333:RNA, Messenger; D000097002:Gap Junction beta-1 Protein; C000607042:GJB2 protein, human; C000607044:Gjb2 protein, rat; C017475:lucifer yellow