Cytoplasmic Relaxation of Active Eph Controls Ephrin Shedding by ADAM10 (original) (raw)
Figure 5
FLIM analysis shows EphA3 activation accompanies extension of the cytoplasmic domain.
(A) Schematic of the FRET-assay; FRET (yellow arrow) reflects the proximity between the GFP on the EphA3 C-terminus and tkRasRFP on the inner plasma membrane (Kin, kinase domain). (B) Confocal FLIM time-series of EphA3-GFP in tkRasRFP co-transfected COS7 cells at indicated times (min) after ephrin-A5 stimulation. Upper row: EphA3-GFP fluorescence intensity images. Middle row: tkRasRFP fluorescence intensity images. Lower row: fluorescence lifetime images of EphA3-GFP; the colour bar inset indicates the fluorescence lifetime range in ns. (C) Upper panel: 2D-histograms of fluorescence rate τ−1 versus tkRasRFP acceptor intensities for 0 min (red) and 20 min (blue) of the EphA3-GFP confocal FLIM series. Linear fitting of the time series 2D-histograms are indicated by solid lines. Lower panel: acceptor normalized energy transfer rate, kT/acceptor, for selected time points after EphA3 stimulation as derived from the slope of the fits to the 2D-histograms. (D) Upper panel: 2D-histograms of τ−1 tkRasRFP acceptor intensities for 0 min (red) and 20 min (blue) of a 2YE-EphA3-GFP confocal FLIM time series after ephrin-A5 stimulation (see also Figure S6F). Lower panel: acceptor normalized energy transfer rate, kT/acceptor, of the time series as in (C). (E) Upper panel: 2D-histograms of τ−1 tkRasRFP acceptor intensities for 0 min (red) and 20 min (blue) of a 3YF-EphA3-GFP confocal FLIM time series after ephrin-A5 stimulation. Lower panel: acceptor normalized energy transfer rate, kT/acceptor, of the time series as in (C). (F) Left panel: 2D-histograms of fluorescence rate, τ−1, versus acceptor intensities for EphA3 [2YE], [3YF], and [2YE-KM] obtained from at least 16 fluorescence lifetime/acceptor intensity images obtained with wide-field frequency-domain FLIM. Right panel: linear fitting of these data showed a significant difference between the slopes (kT/acceptor) for EphA3-GFP-[2YE] and EphA3-GFP-[3YF].