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. Author manuscript; available in PMC: 2019 May 16.
Published in final edited form as: Neuron. 2018 May 3;98(4):767–782.e8. doi: 10.1016/j.neuron.2018.04.011

Figure 7. Fast activation of EphB2 induces filopodial retraction.

Figure 7

(A) Design of photoactivatable EphB2. Xfp: fluorescent protein (mCherry or iRFP670). (B) Western blots of HEK293T cells transfected with EphB2-CRY2 variants and treated with activated ephrin-B2 (30 mins) or blue light (440nm, 1 min) (CRY2-cherry: fEphB2-CRY2-mCherry; Oligo-cherry: fEphB2-CRY2-Oligo-mCherry; CRY2hm-cherry: fEphB2-CRY2hm-mCherry; CRY2-iRFP670: fEphB2-CRY2-iRFP670) (C) Western blots of HEK293T cells transfected with wild-type (WT) or kinase-dead (KD) versions of fEphB2-CRY2hm-iRFP670 (fEphB2-CRY2) and treated with ephrin-B2 or blue light. (D) HEK293T cells transfected with WT or KD versions of fEphB2-CRY2 and imaged every 10 seconds. Photostimulation was conducted by a single scan with the 470 nm laser. Arrows indicate filopodia retracting after photostimulation. (E) Quantification of fEphB2-CRY2 transfected filopodia remaining after photostimulation (n = 12, * p = 0.013, ** p = 0.002, *** p ≤ 0.001, paired t-test). (F) Quantification of fEphB2-KD-CRY2 transfected filopodia remaining after photostimulation (n = 13, p > 0.05, paired t-test). (G) Neuron transfected with WT or KD versions of fEphB2-CRY2 and imaged every 10 seconds. Arrows as in D. Dashed lines show the border of photostimulated region. (H) Quantification of filopodia retraction after photostimulation (PHS) (p = 0.025, ANOVA; WT-PHS+: n = 21, vs. WT-PHS-: n = 11, p = 0.019; WT-PHS+: n = 21, vs. KD-PHS: n = 14, p = 0.036, post hoc: Fisher’s LSD test). (I) Quantification of the distance filopodia moved after photostimulation (WT-PHS+: n = 50 vs. WT-PHS-: n = 25, p = 0.025, t-test). Scale bars = 2 μm. Error bars indicate SEM in Figures E, F, H, I. See also Figure S7 and Movies S12–S13.