Figure 7.

Strip affects Rab5 activity in axon elongation

(a–c) Representative image of Rab5² DL1 PNs (a, green) and Rab5² DL1 PNs expressing YFP fused Rab5 (b,c, green). Magenta: Bruchpilot staining. Asterisk: Cell body. Yellow and white dotted circles indicate the mushroom body (MB) and the lateral horn (LH), respectively. Scale bar: 25 μm. (d) Immunoprecipitation of S2 lysates expressing c-myc tagged Spri (Spri-myc) and YFP-tagged Rab5 (YFP-Rab5WT), YFP-tagged dominant negative form of Rab5 (YFP-Rab5DN) or YFP-tagged constitutively active form of Rab5 (YFP-Rab5CA). Spri-myc and Strip were co-immunoprecipitated when YFP-Rab5 WT/CA/DN were precipitated by the anti-GFP antibody. (e) Immunoprecipitation of S2 lysates. The anti-Strip antibody co-immunoprecipitated Strip and endogenous Rab5. (f) The expression level of Spri-myc in control and strip dsRNA-treated S2 cells. Knockdown efficiency was confirmed by anti-Strip antibody and anti-Histone H3 was used as a loading control. (g) Densitometric analysis of Spri-myc in (f). The intensity of Spri-myc was measured by using Image J software and normalized against the intensity of Histone H3 and showed in graph. (h–k) Representative image of DL1 wild-type (WT, h), strip^dogi PN (i), strip^dogi PN expressing constitutively active form of Rab5 (Rab5CA, Rab5Q88L, j), and strip^dogi PN expressing dominant-negative form of Rab5 (Rab5DN, Rab5S43N, k) labelled by mCD8RFP (green). Magenta: Bruchpilot staining. Yellow and white dotted circles indicate the mushroom body (MB) and the lateral horn (LH), respectively. Scale bar: 25 μm. (l) The axons of DL1 PNs were categorized into three classes according to the extent of elongation in a blind test. Class 1 axons reached the end of the LH, while class 3 axons terminated near the mushroom body (l, arrowheads). The percentage of PNs in each category is shown in (l).