His6-tagged BiFC probe proteins were expressed in Sf9 cells by using the Bac-to-Bac baculovirus expression system (Invitrogen). Proteins were purified by a nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Valencia, CA, USA) column and eluted with 0.2 M imidazole buffer, as described previously (22). The buffer was changed to binding buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM MgCl2, 1 mM dithiothreitol, 5% glycerol) with a PD-10 column (GE Healthcare, Little Chalfont, UK). VN210-cofilin(WT or S3E; 3 µM) and 1.5 µM actin-VC210, or 1.8 µM VN210-Raf1-RBD and1.8 µM Ras (V12 or N17)-VC210, were incubated at 30°C. The fluorescence intensity was measured at 545 nm with excitation at 505 nm by a fluorescence spectrophotometer (F-2500; Hitachi High Technologies, Tokyo, Japan). To detect the interaction between CaM and M13, cell lysates of E. coli separately expressing VN210-M13 and CaM-VC210 were incubated at 30°C in the presence of 1 mM EDTA or 1 mM CaCl2.Results and discussion
To detect protein-protein interactions by the BiFC method, spontaneous self-assembly between the N- and C-terminal fragments of the fluorescent protein is undesirable. Prior to the development of the BiFC assay system for visualizing the cofilin-actin interaction, we constructed a series of expression plasmids coding for 13 N-terminal (VN) and 13 C-terminal (VC) fragments of Venus (Figure 1A) and analyzed the self-assembly ability between these fragments (Figure 1B). Venus fragments were designated VNx and VCy, which correspond to the amino acid sequences of 1-x and y-238, respectively. Venus was used because of its strong fluorescence intensity and its fast and efficient maturation properties (17,18). The sites of splitting were selected withinthe surface loops that connect 11 β-sheets and a fluorophore-containing α-helix region of the predicted β-barrel structure of GFP-related proteins (Figure 1A and Supplementary Figure S1) (23).
To analyze whether the pair of VN and VC fragments could self-reassemble in cells by complementation, each pair of Venus fragments was coexpressed in HeLa cells, and the recovery of Venus fluorescence in cells was analyzed by fluorescence microscopy. Of the 91 pairs of fragments examined, 44 pairs of the VN and VC fragments exhibited weak to strong fluorescence (Figure 1B). None of the VN and VC fragments exhibited detectable fluorescence when expressed alone (data not shown). This finding indicates that these 44 pairs of Venus fragments generate fluorescence by the self-assembly of the N- and C-terminal fragments without fusion of the interacting proteins. In contrast, no fluorescence signal was observed for other combinations of VN and VC fragments of Venus (Figure 1B).
Previous studies have shown that the cofilin-actin interaction is inhibited by cofilin phosphorylation at Ser-3 (24,25). Moreover, a phosphorylation-mimic cofilin mutant, cofilin(S3E), in which Ser-3 is replaced by glutamic acid, fails to bind to G- or F-actin (24,25). To visualize the specific interaction between cofilin and actin in cells, we constructed expression plasmids coding for the VN and VC fragments of Venus fused to the N or C terminus of actin and cofilin(WT or S3E). After 1456 pairs of VN- or VC-fused actin and cofilin(WT or S3E) constructs were coexpressed in HeLa cells, the fluorescence intensities in cells expressing cofilin(WT) were compared with those in cells expressing cofilin(S3E) in the corresponding pair by visual observations by eye (Figure 2A and Supplementary Table S1). Only three pairs reproducibly exhibited a distinct difference in fluorescence intensity between cells expressing cofilin(WT) and cofilin(S3E): actin-VC210 (VC210 fused to the C terminus of actin) plus VN210-cofilin (VN210 fused to the N terminus of cofilin), actin-VC24 plus VN38-cofilin, and VC101-actin plus VN128-cofilin (Figure 2A and Supplementary Table S1). The first pair, actin-VC210 and VN210-cofilin, showed the most distinct difference in fluorescence intensity between cells expressing cofilin(WT) and cofilin(S3E). A strong signal was observed in cells coexpressing actin-VC210 and VN210-cofilin(WT), but only a weak signal in cells coexpressing actin-VC210 and VN210-cofilin(S3E) (Figure 2B). Measurements of fluorescence intensity of cell lysates by a fluorometer further showed the marked difference in fluorescence intensity between cells expressing VN210-cofilin(WT) and VN210-cofilin(S3E) (Supplementary Figure S2B). Immunoblot analysis revealed the comparable level of expression of probe proteins in both cells (Supplementary Figure S2A). No fluorescence signal was observed by coexpression of nonfused VN210 and VC210 fragments (Figure 1B). The emission spectrum of the complex of actin-VC210 and VN210-cofilin(WT) was essentially identical to that of intact Venus (data not shown). These results indicate that the actin-VC210 and VN210-cofilin pair is a useful BiFC probe for visualizing the specific interaction between actin and cofilin in living cells.