Comparative analysis and application of fluorescent protein-tagged connexins.
In order to examine connexin transport, assembly, and turnover in living cells, we tagged green fluorescent protein or its color variants to several members of the connexin family of proteins. When green fluorescent protein was tagged to the carboxyl terminal end of connexin43 (Cx43-GFP), the resulting fusion protein was transported and assembled into functional gap junctions. However, when GFP was tagged to the amino terminal end of Cx43 (GFP-Cx43), this chimera was biosynthesized, transported to the plasma membrane, but failed to form gap junction channels that could transfer Lucifer yellow. Single cells that expressed Cx43-GFP were capable of transporting this fusion protein to the cell surface in the absence of cell-cell contact. Imaging of Cx43-yellow (Y)FP (Cx43-YFP) was quite efficient; however, the low quantum yield Cx43-BFP and the requirement for ultraviolet excitation made this chimera less suitable for time-lapse imaging. Cx43-cyan C(FP) (Cx43-CFP) was more suitable for imaging than Cx43-blue (B)FP and could be effectively separated from Cx43-YFP. The versatility of tagging GFP to the carboxyl terminal end of other members of the connexin family was established when Cx32-GFP and Cx26-YFP were found to assemble into gap junctions capable of transferring Lucifer yellow. Finally, we are examining the effectiveness of using a new red fluorescent protein (DsRed) fused to connexins in combination with Cx-GFP to simultaneously examine the kinetics, transport and turnover of two connexins. Together, our studies suggest that tagging fluorescent proteins to the carboxyl terminal end of connexins is an effective and valuable approach for studying the life cycle and dynamics of connexins in living cells.
Laird
D W
DW
Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1 Canada. dwlaird@julian.uwo.ca
Jordan
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K
Thomas
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T
Qin
H
H
Fistouris
P
P
Shao
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Q
D000818:Animals; D002460:Cell Line; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000295:instrumentation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D017630:Connexins; C000607042:GJB2 protein, human; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000072259:Connexin 26