FIGURE 5:

FLIM-FRET reveals that MCAK activity is spatially controlled in migrating cells. (A) Schematic illustration of the MCAK-FRET biosensor in which mEmerald is fused to the N-terminus and mCherry is fused to the C-terminus of MCAK. MCAK in solution is in a closed conformation, which correlates with active MCAK and a short lifetime for the FLIM-FRET sensor. Phosphorylation of MCAK by Aurora A/B changes its conformation from closed to open, which correlates with the inactive MCAK and a longer lifetime for the FLIM-FRET sensor. (B) Representative mEmerald fluorescence intensity images (left) and mEmerald average amplitude weighted fluorescence lifetime measurements (right) of RPE-1 cells expressing the “donor-only” control, em-MCAK (top), or the FLIM-FRET sensor, em-MCAK-mCh (bottom). The color-coded mEmerald fluorescence lifetime scale is shown at the right. Scale bar, 10 µm. (C) Three ROIs were drawn at the leading edge and at the rear of the cell body to calculate the average lifetime in each region. (D) Dot plots showing the average mEmerald fluorescence lifetimes (ns) found at the leading edge (left) and the trailing edge (right) of cells as calculated in B and C. Lines represent mean ± SEM. n = 69 cells for em-MCAK and 72 cells for em-MCAK-mCh from five independent experiments. (E) Dot plots showing the average mEmerald fluorescence lifetimes (ns) for the em-MCAK-mCh sensor found at the leading edge (left) and the trailing edge (right) of cells with the indicated treatment. Lines represent mean ± SEM. n = 97 cells for control, 44 cells for 40 nM Aurora A inhibitor, and 36 cells for 80 nM Aurora A inhibitor from at least three independent experiments. p values were determined by the Welch’s t test and the two-tailed Student’s t test. ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.