Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed‐tagged proteins¹

Abstract

A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12^16 h post transfection, much earlier than previously reported, and could readily be differentiated from co-expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein. ß

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion

“…In the fusion construct, both partners must retain their functional integrity under the desired experimental conditions. Especially, the tendency of anthozoan FPs to form tetramers and aggregate can be detrimental for many applications (27,28). Functional expression of EosFP and its dimeric variants works very well in mammalian cells at 37°C without any signs of aggregation (see Fig.…”

Specific amino-acid residues in the N-terminus and TM3 implicated in channel function and oligomerization compatibility of connexin43

“…Thus, we co-expressed transport-deficient DsRed-tagged Cx43 or DsRed-tagged Cx32, respectively (Lauf et al, 2001), with the GFP-tagged substitution variants and assessed the interaction and assembly of the tagged proteins by the appearance of DsRed-tagged connexins within connexin clusters at cell-cell appositions (see Discussion). As expected, all Cx43 variants oligomerized with wt Cx43, as suggested by the presence of DsRed-tagged Cx43 in connexin clusters that were visible in the GFP, and the DsRed channels (Fig.…”

“…However, co-expression of wt or GFP-tagged connexinpolypeptides together with transportdeficient DsRed-tagged connexins will recover trafficking of DsRed-tagged connexin polypeptides that then localize within connexin clusters at cell-cell appositions. Successful trafficking of DsRed-tagged connexins appears to result from the co-oligomerization of untagged or GFP-tagged connexin subunits with DsRed-tagged connexins into mixed connexons (Lauf et al, 2001). Comparable approaches that either deployed transport-deficient connexins tagged with β-galactosidase, or connexins tagged with endoplasmic reticulum retention signals have also been developed recently and have been used to investigate intracellular interaction and co-assembly of coexpressed connexin isoforms (Das Sarma et al, 2001;Das Sarma et al, 2002).…”

Cross-linking ATP synthase complexes in vivo eliminates mitochondrial cristae

“…By contrast, expression of an equivalent subunit γ-GFP fusion did not impair growth of cells, or result in mtATPase oligomerisation (Gavin et al, 2002;Prescott et al, 2003). DsRed is known to form tetramers in vitro and in living cells (Baird et al, 2000), a property that can cause oligomerisation of proteins to which it is fused (Gavin et al, 2002;Lauf et al, 2001;Mizuno et al, 2001). Thus, we concluded that strong interactions between the DsRed moieties of individual mtATPase complexes incorporating the γ-DsRed fusion led to stable associations between multiple adjacent mtATPase complexes.…”