Figure 4: Vesicular recycling suppresses autonomous activation of EphA2. | Nature Communications

Figure 4: Vesicular recycling suppresses autonomous activation of EphA2.

From: Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode

Figure 4

(a) Ectopic expression of Rab11a or PTP1B suppresses autonomous activation of LIFEA2 in Cos-7 cells. For each panel: LIFEA2 fluorescence (first column), bfp–Rab11a or bfp–PTP1B fluorescence (second), overlay of LIFEA2 (green) and bfp–Rab11a or bfp–PTP1B (magenta) (third) and LIFEA2 τ-maps (fourth), color-coding, right. Right graphs: τ-histograms from n=3 experiments without (n=7 cells) or with bfp–Rab11a (upper, n=8 cells) or bfp–PTP1B (lower, n=7 cells) expression. (b) Expression level dependence of LIFEA2 fluorescence lifetime (τ) on ectopic expression of bfp–Rab11a. 2D histogram of τ versus integrated fluorescence intensity (F) for individual cells (n=54 cells) pre- (dark grey circles) and 20 min post-stimulation (light grey circles). (c) EphA2 phosphorylation is increased by Rab11a knockdown Left: western blot of Cos-7 lysates 72 h post-transfection with non-targeting siRNA (Ctrl, first column) or Rab11a siRNA (siRab11a, second column). Blots were probed for phospho-tyrosines (PY72, first row), EphA2 (second row), Rab11a (third row) and GAPDH (fourth row). Graph: ratio of Rab11a to GAPDH (n=5), and PY72 to total EphA2 (n=4) [mean±s.e.m., **P<0.01; ****P<0.0001; unpaired t-test]. (d) Expression level dependence of LIFEA2 fluorescence lifetime (τ) on Rab11a knockdown in Cos-7 cells. 2D histogram of LIFEA2 τ versus its integrated fluorescence intensity (F) for individual cells (n=49 cells) with non-targeting siRNA (Ctrl) or Rab11a siRNA (siRab11a). The two distributions fitted with an exponential function (black and blue lines) showed a statistical difference with P value=0.034 [*P<0.05; Kolmogorov–Smirnov test]. (e) Interaction of EphA2-mCitrine with substrate-trapping mutant PTP1B D/A-mCherry detected by FLIM–FRET in the absence (upper panel) or presence (lower panel) of bfp–Rab11a. First column: fluorescence images of EphA2-mCitrine, second: fluorescence images of mCherry–PTP1B D/A, third: τ (ns; color-coding right) of EphA2-mCitrine, fourth: fluorescence image of bfp–Rab11a. Graph: average fluorescence lifetime of EphA2-mCitrine (τ)±s.e.m. versus its fluorescence intensity (FL) in absence (black, n=55 cells) or presence (red, n=50 cells) of bfp–Rab11a expression. The percentage of EphA2-mCitrine/PTP1B-D/A-mCherry interactions in the vicinity of Rab11a-positive endosomes (iii) was retrieved from the overlap between non-cell contact areas with low τ values (i, green) and high intensity areas in bfp–Rab11a images (ii, blue). Scale bars, 10 μm.

Back to article page