FIGURE 1.

Heterodimerization of EBc domains. A, a schematic diagram shows FRET of dimeric EBc domains that are N-terminal-tagged with fluorescence proteins. CFP absorbs light at 434 nm and emits light at 477 nm. YFP absorbs light at 514 nm and emits light at 527 nm. When brought in close proximity, for example, through the coiled-coil domains of EBc dimers, energy absorbed by CFP is transferred to YFP through nonradiative FRET and emitted by YFP. B, shown are fluorescence emission spectra obtained by mixing equimolar amounts of CFP-EB1c and YFP-EB1c (10 μm each) at 37 °C and recorded at 60-min time intervals with excitation at 434 nm. Red and green indicate spectra obtained after 0- and 600-min incubation times, respectively. Note that the fluorescence emission signal at 527 nm observed at time 0 is due to background FRET in the sample (supplemental Fig. 3). a.u., arbitrary units. C, shown is time-dependent increase of the fluorescence signal at 527 nm (excitation at 434 m) after mixing equimolar amounts (10 μm each) of CFP-EB1c and YFP-EB1c (1c), CFP-EB2c and YFP-EB2c (2c), CFP-EB3c and YFP-EB3c (3c), CFP-EB1c and YFP-EB2c (1c/2c), CFP-EB1c and YFP-EB3c (1c/3c), CFP-EB2c and YFP-EB3c (2c/3c), and CFP-EB1c(I224A) and YFP-EB1c(I224A) (1c(I224A)) at 37 °C. D, shown is titration of 10 μm CFP-EB1c with increasing amounts of YFP-EB3c. The data show the loss of intensity of the fluorescence signal at 477 nm due to the dequenching of CFP-EB1c homodimers because of EB1c·EB3c heterodimer formation. Samples were incubated at 37 °C for 16 h before each measurement.