TABLE 3.

FRET analysis of Rif-mDia1 interaction

N1E115 cells transfected with controls, mRFP-RifQL and EYFP-mDia1, or mRFP-RifQL and EYFP-mDia2 were fixed at 24 h post-transfection. Fluorescence intensities of selected regions of interest were monitored in both mRFP and EYFP channels for the entire duration of the experiment. mRFP was bleached using a 561-nm laser once base-line intensities of both mRFP (acceptor) and EYFP (donor) fluorescence were established. Subsequent changes in these fluorescence intensities were measured and expressed as % FE, and the correlation between the rates of change of these values is expressed as CC (see “Experimental Procedures” for details). Positive FRET was defined as % FE > 3% with CC values of −1.0 to −0.7. Data are presented as mean ± S.D. (for mRFP-EYFP, n = 14; mRFP/EYFP-mDia1, n = 7; mRFP/EYFP-mDia2, n = 6; mRFP-RifQL/EYFP-mDia1 in filopodia-like projections, n = 9; mRFP-RifQL/EYFP-mDia1 in ruffles, n = 7; mRFP-RifQL/EYFP-mDia2 in filopodia-like projections, n = 5; mRFP-RifQL/EYFP-mDia2 in cell edge, n = 5).

FRET pairs	% FE	CC
Controls
mRFP-EYFP (tandem positive control)	17.8 ± 3.6	−0.97 ± 0.04
mRFP and EYFP-mDia1 (negative control)	4.0 ± 2.3	−0.41 ± 0.42
mRFP and EYFP-mDia2 (negative control)	1.4 ± 1.7	0.05 ± 0.37
Rif and mDia1
mRFP-RifQL and EYFP-mDia1 (filopodia)	11.3 ± 5.0	−0.94 ± 0.07
mRFP-RifQL and EYFP-mDia1 (ruffles)	11.7 ± 3.4	−0.95 ± 0.04
Rif and mDia2
mRFP-RifQL and EYFP-mDia2 (filopodia)	4.8 ± 5.0	−0.18 ± 0.45
mRFP-RifQL and EYFP-mDia2 (cell edge)	2.8 ± 4.7	−0.11 ± 0.50