Figure 6. Endocytosis of guidance sensors upon growth cone desensitization.
(A) SNAP–ephrin-A5 surface signal is strongly reduced on GCs growing on either homogeneous EphA3-Fc (A') or ephrin-A5-Fc (A'') compared to controls (on Fc), indicating clearance of sensors from the surface upon co-adaptation. Outlines of GCs drawn in the GFP (transfection marker) channel are indicated as white, dotted lines. Scale: 10 µm. (A''') displays a cartoon of the experiment in (A–A'') with axonal SNAP surface labeled ephrin-A5 in magenta and with the corresponding guidance cues (Fc, gray; EA3, blue and eA5, red). (B) Quantification of SNAP–ephrin-A5 surface signal intensities. Relative signal intensity within the GC outline normalized to the control situation is given as fold change (on Fc: 1; on EA3: 0.29; on eA5: 0.32). (C) Effects of Pitstop2 on adaptation towards soluble ephrin-A5. Pitstop2 (P; carrier DMSO) prevents temporal GCs from desensitizing to 0.25 µg/ml ephrin-A5-Fc (eA5+DMSO 120 min: 15.8% collapsed; eA5+P 120 min: 74.3% collapsed). The initial response of GCs towards eA5 is unaltered in the presence of P (eA5+DMSO 20 min: 85.4% collapsed; eA5+P: 83.1% collapsed). Neither P itself, nor its carrier DMSO induces a collapse (Fc+DMSO 20 min: 13.6% collapsed; Fc+P 20 min: 7.5% collapsed). (D) Effects of Pitstop2 on adaptation towards soluble EphA3. Soluble EphA3-Fc (EA3; 15 µg/ml) triggers a collapse on nasal or temporal GCs (EA3 20 min: 64.4% collapsed; Fc [4.2 µg/ml] 20 min: 24.3% collapsed). GCs desensitize towards EA3 within 120 min (EA3 120 min: 22.6% collapsed), but not in the presence of 30 μM Pitstop2 (EA3+P 120 min: 49.8% collapsed). P does not impede GCs from recovering their morphology in general (EA3 20 min, then Fc+P 120 min: 19.3% collapsed). Combined data from nasal and temporal GCs. (B, C, D) N: number of independent experiments, number of analyzed GCs in brackets; error bars represent standard deviations. T-test with n.s.: α ≥0.05, *: α <0.05, ***: α <0.001.
DOI: http://dx.doi.org/10.7554/eLife.25533.016
Figure 6—figure supplement 1. SNAP–ephrin-A5 expression constructs pSNAP–ephrin-A5–IRES-FP contains the coding sequence of a fusion protein consisting of the signal sequence of chick ephrin-A5 (60 bp), the SNAP-tag and full-length chick ephrin-A5 downstream of the CAG enhancer/promoter and upstream of an IRES-FP sequence.
