Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions.
Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gap junctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.
Thomas
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Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada.
Jordan
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Laird
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DW
D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000072259:Connexin 26; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D017629:Gap Junctions / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D018031:Connexin 43; D017630:Connexins; C000607044:Gjb2 protein, rat; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D000072259:Connexin 26; D049452:Green Fluorescent Proteins; D003571:Cytochalasin B; D015739:Nocodazole