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. 2020 Jul 13;9:e56547. doi: 10.7554/eLife.56547

Figure 3. γ-TuRC localizes to RAB-11.1 endosomes.

(A) Electron micrograph of the dendritic growth cone. Red arrows, MTs; white arrowhead, clear-core vesicle; scale bar, 0.2 μm. (B) Reconstruction of serial electron microscopy sections through the anterior dendrite. Yellow lines, MTs in the distal dendrite; green lines, MTs crossing the predicted MTOC region; red lines, MTs in the proximal region; white circles, clear-core vesicles; blue circles, dense-core vesicles; scale bar, 1 μm. (C) MT number distribution determined by serial electron microscopy in the anterior dendrite growth cone region in three PVD neurons during development. (D) GIP-2 and RAB-11.1 colocalization in the dendritic growth cone region. Scale bar, 5 μm. (E) Quantification of GIP-2 and RAB-11.1 fluorescence overlap in the growth cone region (n = 11 individual animals). ****p<0.0001, Brown-Forsythe and Welch ANOVA test, error bars represent SEM. (F) Normalized intensity of GIP-2 and RAB-11.1 from the dendritic tip along the dendrite shaft to the cell body at different time points. Black arrow, direction of GIP-2 and RAB-11.1 movement. Vertical dashed line, the center of GIP-2 and RAB-11 endosomes at t=0s. (G) Kymograph of GIP-2::GFP and mCherry::RAB-11.1 in the growth cone region. Horizontal scale bar, 2 μm; vertical scale bar, 10 s. (H) GIP-2 localization in worms overexpressing RAB-11.1(S25N) dominant negative mutant. (I) GIP-2 localization in rab-11PVD(-) worms (Punc-86::Cre; rab-11.1(wy1444[lox])): multiple dim GIP-2 puncta in cell body (i1 and i2), dispersed dim GIP-2 puncta along the dendrite shaft (i3). Dashed white lines: PVD outline; white arrows, GIP-2 puncta; gray arrows, unrelated signal from gut granules. (J) Quantification of GIP-2::GFP class of localization in wt worms, worms overexpressing a RAB-11, RAB-11(S25N) dominant negative mutant or rab-11PVD(-) worms (Punc-86::Cre; rab-11.1(wy1444[lox])). Scale bar, 5 μm. A, anterior; P, posterior. Images in D and H were taken in the glo-1(zu391) mutant background to reduce the gut granule signal.

Figure 3—source data 1. Quantification data for Figure 3 and Figure 3—figure supplement 1.

Figure 3.

Figure 3—figure supplement 1. Synaptic vesicles and Golgi stacks and outposts do not localize to the growth cone region.

Figure 3—figure supplement 1.

(A–C) GFP::RAB-3 (A), mCherry::RER-1 and GFP::RAB-6.2 (n = 13 animals) (B), and AMAN-2::GFP and mCherry::RAB-6.2 (n = 7 animals) (C) localization in outgrowing anterior PVD dendrite. White arrows: Golgi stack in cell body; white arrowheads: RAB-6.2 in growth cone. (D) GFP::RAB-11.1 and mCherry::RAB-6.2 localization in outgrowing dendrite (n = 20 animals). White arrowhead: GFP::RAB-11.1 and mCherry::RAB-6.2 in growth cone. (E) Kymograph of GFP::RAB-11.1 and mCherry::RAB-6.2 in growth cone region. Horizontal scale bar, 2 μm; vertical scale bar, 10 s. (F–G) Frame to frame colocalization analysis between GIP-2::GFP and mCherry::RAB-11.1 in growth cone (F) and cell body (G) (100 frames in one animal). (H) Kymograph of EBP-2::GFP in outgrowing anterior dendrite of a worm expressing RAB-11.1(S25N). h1, worm with an almost reversed MT polarity; h2, worm with a mixed MT polarity. All images: Scale bar, 5 μm (horizontal) (except E);10 s (vertical); white dashed line, outline of PVD; anterior to the left and posterior to the right. Images in D were taken in the glo-1(zu391) mutant background to reduce the gut granule signal.