Figure 2.
Figure 2. Ectopic axonal localization of Golgi outposts and hTfR-positive vesicles results from Khc E177 charge-reversal mutations. (A) Schematic of a class IV sensory neuron. In the VNC, multiple axons form a ladder-like pattern. (B) Golgi outposts labeled by ManII::GFP normally localize to dendrites. CD4::Tomato illuminates neuron morphology. Orange bracket indicates axon, and blue arrows indicate Golgi outposts. Bars: (main images) 50 µm; (inset) 20 µm. (C) Colocalization of Golgi outposts and Klc in dendrites. Individual video frames show a ManII- and Klc-positive punctum moving retrograde. Arrowheads mark start point (blue) and moving outpost (purple). Bars: (left) 10 µm; (right) 2 µm. (D) Quantification of RFP::Klc-positive Golgi outposts. 32% of moving ManII::GFP-positive outposts (27/83) colocalize with RFP::Klc. Of these RFP::Klc-positive outposts (n = 27), 81% move retrograde at speeds consistent with kinesin-mediated transport (n = 83 outposts and 75 RFP::Klc puncta in 24 neurons from 14 animals). RFP::Klc-only puncta may represent vesicles, which move faster than some organelles. (E) Golgi outposts mislocalize to axons in KhcE177K/− and Khc-RNAi neurons (unbranched axons were selected for clarity). Bar, 10 µm. Arrows indicate Golgi outposts. (F) Quantification of Golgi outposts (mean ± SD) in the proximal 75 µm of axons in 11 (KhcE177A/−), 12 (KhcE177K/−), 14 (KhcE177R/−), and 15 (control, Khc-RNAi, + Khc = KhcE177K/− ppk-Gal4 UAS-Khc::BFP) neurons from at least five larvae. (G) Unlike control neurons, ManII::GFP accumulates in the axon terminals of KhcE177K/− and Khc-RNAi neurons. Bushy CD4::Tomato signal flanking axon terminals results from nonspecific transgene expression. Bar, 25 µm. Dashed orange lines demarcate axon terminal borders. (H) Quantification of ManII::GFP signal (mean ± SD) for 25 VNC segments from five larvae of each genotype. (I) hTfR::GFP normally localizes predominantly to dendrites. Bars: (main image) 50 µm; (inset), 20 µm. (J) Representative images of hTfR::GFP in control and mutant axons. Bar, 10 µm. (K) hTfR::GFP accumulates at the axon terminals of KhcE177K/− and Khc-RNAi neurons. Bar, 25 µm. Arrows indicate hTfR::GFP puncta (I and J), and dashed lines indicate axon terminal borders (K). (L) Quantification of hTfR::GFP signal (mean ± SD) in 25 VNC segments from five larvae of each genotype. *, P = 0.05–0.01; **, P = 0.01–0.001; ****, P < 0.0001 in comparison with control and evaluated by one-way ANOVA and Tukey post hoc test (F, H, and L).

Ectopic axonal localization of Golgi outposts and hTfR-positive vesicles results from Khc E177 charge-reversal mutations. (A) Schematic of a class IV sensory neuron. In the VNC, multiple axons form a ladder-like pattern. (B) Golgi outposts labeled by ManII::GFP normally localize to dendrites. CD4::Tomato illuminates neuron morphology. Orange bracket indicates axon, and blue arrows indicate Golgi outposts. Bars: (main images) 50 µm; (inset) 20 µm. (C) Colocalization of Golgi outposts and Klc in dendrites. Individual video frames show a ManII- and Klc-positive punctum moving retrograde. Arrowheads mark start point (blue) and moving outpost (purple). Bars: (left) 10 µm; (right) 2 µm. (D) Quantification of RFP::Klc-positive Golgi outposts. 32% of moving ManII::GFP-positive outposts (27/83) colocalize with RFP::Klc. Of these RFP::Klc-positive outposts (n = 27), 81% move retrograde at speeds consistent with kinesin-mediated transport (n = 83 outposts and 75 RFP::Klc puncta in 24 neurons from 14 animals). RFP::Klc-only puncta may represent vesicles, which move faster than some organelles. (E) Golgi outposts mislocalize to axons in KhcE177K/− and Khc-RNAi neurons (unbranched axons were selected for clarity). Bar, 10 µm. Arrows indicate Golgi outposts. (F) Quantification of Golgi outposts (mean ± SD) in the proximal 75 µm of axons in 11 (KhcE177A/−), 12 (KhcE177K/−), 14 (KhcE177R/−), and 15 (control, Khc-RNAi, + Khc = KhcE177K/− ppk-Gal4 UAS-Khc::BFP) neurons from at least five larvae. (G) Unlike control neurons, ManII::GFP accumulates in the axon terminals of KhcE177K/− and Khc-RNAi neurons. Bushy CD4::Tomato signal flanking axon terminals results from nonspecific transgene expression. Bar, 25 µm. Dashed orange lines demarcate axon terminal borders. (H) Quantification of ManII::GFP signal (mean ± SD) for 25 VNC segments from five larvae of each genotype. (I) hTfR::GFP normally localizes predominantly to dendrites. Bars: (main image) 50 µm; (inset), 20 µm. (J) Representative images of hTfR::GFP in control and mutant axons. Bar, 10 µm. (K) hTfR::GFP accumulates at the axon terminals of KhcE177K/− and Khc-RNAi neurons. Bar, 25 µm. Arrows indicate hTfR::GFP puncta (I and J), and dashed lines indicate axon terminal borders (K). (L) Quantification of hTfR::GFP signal (mean ± SD) in 25 VNC segments from five larvae of each genotype. *, P = 0.05–0.01; **, P = 0.01–0.001; ****, P < 0.0001 in comparison with control and evaluated by one-way ANOVA and Tukey post hoc test (F, H, and L).

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