Comparative analysis and application of fluorescent protein-tagged connexins

In order to examine connexin transport, assembly, and turnover in living ce lls, we tagged green fluorescent protein or its color variants to several m embers of the connexin family of proteins. When green fluorescent protein w as tagged to the carboxyl terminal end of connexin43 (Cx43-GFP), the result ing fusion protein was transported and assembled into functional gap juncti ons. However, when GFP was tagged to the amino terminal end of Cx43 (GFP-Cx 43), this chimera was biosynthesized, transported to the plasma membrane, b ut failed to form gap junction channels that could transfer Lucifer yellow. Single cells that expressed Cx43-GFP were capable of transporting this fus ion protein to the cell surface in the absence of cell-cell contact. Imagin g of Cx43-yellow (Y)FP (Cx43-YFP) was quite efficient; however, the low qua ntum yield Cx43-BFP and the requirement for ultraviolet excitation made thi s 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 te rminal end of other members of the connexin family was established when Cx3 2-GFP and Cx26-YFP were found to assemble into gap junctions capable of tra nsferring Lucifer yellow. Finally, we are examining the effectiveness of us ing 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 p roteins to the carboxyl terminal end of connexins is an effective and valua ble approach for studying the life cycle and dynamics of connexins in livin g cells. Microsc. Res. Tech. 52:263-272, 2001. (C) 2001 Wiley-Liss, Inc.