Fig. 3.

Spindly is required for sorting of microtubules in axons. (A) Representative still images of EB1-mCherry–expressing primary neurons cultured for 48 h. Magenta and green arrows indicate the direction of EB1 comets in the axons (plus-end-in and plus-end-out, respectively) (Movie S3). Kymographs of EB1 comets are shown below corresponding images. Dashed yellow lines define the area of the axon used for plotting EB1-GFP kymographs. (Horizontal scale bars, 10 µm in the main panels and 5 µm in the kymographs.) Vertical bars in the kymographs represent time (60 s). (B) Representative Z-stack images of class IV sensory neurons of control (ppk-Gal4; Left) or ppk > DHC RNAi (Middle) or ppk > Spindly RNAi (Right). Membrane and microtubule plus-ends were labeled with mCD8-GFP and KHC(1-576)-RFP, respectively. (Scale bar, 20 µm.) (C) Graph depicting the fraction of axonal EB1-GFP comets directed toward the tip of the process (plus-end-out) or the cell body (plus-end-in). Data obtained from three individual experiments (control, n = 33 axons; DHC RNAi, n = 26 axons; Spindly RNAi, n = 31 axons). Error bars represent SD. *P = 0.0134; ****P < 0.0001. (D) Classification of sensory neurons based on the KHC(1-576)-RFP signal intensity in the somas (control, n = 29 sensory neurons; DHC RNAi, n = 16 sensory neurons; Spindly RNAi, n = 20 sensory neurons). (E) Schematic cartoon showing the full-length Drosophila Spindly protein and variants used for Spindly RNAi rescue assays in flies. Note that all these Spindly proteins are resistant to the shRNA sequence used to knock down Spindly. Adapted from ref. 16, which is licensed under CC BY 4.0. (F) Fraction of microtubules with plus-end-in found in Spindly RNAi axons from neurons expressing full-length Spindly and variants (S234A, ΔSB, and ΔC). Data obtained from at least three independent experiments (number of quantified axons for each genotype ranged between 17 and 54). n.s., not significant; *P = 0.011; ****P < 0.0001. (G) Confocal image of a fixed third-instar eye disk expressing elav > GFP-SpindlyFL (Left) and immunostained with Fas-II (Right). Spindly is enriched in both somas and axons of the photoreceptor neurons. (Scale bar, 10 µm.) (H) Sensory neurons expressing elav > GFP-SpindlyFL. Note that Spindly localizes primarily in the somas and axons. A small fraction of Spindly can be found in dendrites. (Scale bar, 20 µm.)