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. 2020 Jul 13;9:e58943. doi: 10.7554/eLife.58943

Figure 7. Microtubules within the soma grow preferentially towards the axon and are excluded from entering dendrites in a Kinesin-2-dependent manner.

(A,B) Widefield fluorescent images from movies showing the somas of class I da neurons expressing UAS-EB1-GFP and either γ-tubulin-37C RNAi (control) (A) or UAS-Kap3 RNAi (B). Manually assigned multi-colour tracks of EB1-GFP comets are drawn over each image (filled circle = last location of comet). Time in min:s from the start of the movie is shown. (C) Graph shows the % of comets that approach either the axon (green) or the dendrites (magenta) in either control (666 comets analysed across 13 movies) or Kinesin-2 RNAi (1058 comets analysed across nine movies) class I da neuron somas, as indicated. (D) Graph shows the % of comets that enter the axon (green) or the dendrites (magenta) as a proportion of those that had approached the axon in either control or Kinesin-2 RNAi class I da neuron somas, as indicated. (E) Graph shows the % of applicable comets that display turning events within either control (n = 257 comets across 13 movies) or Kinesin-2 RNAi (n = 386 comets across nine movies) class I da neuron somas, as indicated. (F) Graph shows the % of comets that are anterograde in the proximal primary dendrite (before any branches) in either control (n = 252 comets across 13 movies) or Kinesin-2 RNAi (n = 338 comets across nine movies) class I da neurons, as indicated. Error bars in (C–F) show the 95% confidence intervals.

Figure 7—source data 1. Calculating percentages of comets aproaching and entering axons and dendrites, turning events, and proximal dendrite polarity in control neurons.
Figure 7—source data 2. Calculating percentages of comets aproaching and entering axons and dendrites, turning events, and proximal dendrite polarity in Kap3 RNAi neurons.
elife-58943-fig7-data2.xlsx (103.1KB, xlsx)
Figure 7—source data 3. Calculating percentages of comets entering dendrites in Klp64D RNAi neurons.

Figure 7.

Figure 7—video 1. Microtubules turn towards the axon and are excluded from entering dendrites.
Download video file (3.5MB, mp4)
The Movie was taken using single-Z time-lapse epifluorescence microscopy and shows a control class I da neuron expressing 221-Gal4 > UAS-EB1-GFP and 221-Gal4 > UAS-γ-tubulin-37c-RNAi. The left panel shows the GFP channel, while the right panel shows the GFP channel with the ImageJ-generated EB1-GFP comet tracks drawn onto the images. The axon (A) and dendrites (D) are labelled in the right panel. The tracks are multi-coloured, as is default for ImageJ, and the colours have no reference to comet type. Comets display turning events, indicating that the growing plus ends of microtubules can be guided along pre-existing microtubules. Comets that approach the axon frequently enter and grow down the axon; In contrast, comets that approach a dendrite entry site do not enter (quantified in Figure 7). Images were collected every 5 s and the Movie plays at 10 frames/second.
Figure 7—video 2. Kinesin-2 is required for growing microtubules to turn within the soma and to be excluded from entering dendrites.
Download video file (6.6MB, mp4)
The Movie was taken using single-Z time-lapse epifluorescence microscopy and shows a Kinesin-2 RNAi class I da neuron expressing 221-Gal4 > UAS-EB1-GFP and 221-Gal4 > UAS-Kap3-RNAi. The left panel shows the GFP channel, while the right panel shows the GFP channel with the ImageJ-generated EB1-GFP comet tracks drawn onto the images. The axon (A) and dendrites (D) are labelled in the right panel. The tracks are multi-coloured, as is default for ImageJ, and the colours have no reference to comet type. Comets can be seen emerging from multiple locations within the soma but unlike in control neurons they do not turn unless they encounter the nuclear envelope or cell cortex, suggesting that Kinesin-2 is required to guide growing microtubules towards the axon. Comets that approach the axon still frequently enter and grow down the axon, and, in contrast to control neurons, comets that approach dendrites also readily enter (quantified in Figure 7). This shows that Kinesin-2 is also required for excluding growing microtubules from entering dendrites. Images were collected every 5 s and the Movie plays at 10 frames/second.
Figure 7—video 3. Knockdown of Klp64D supports the conclusion that Kinesin2 is required for growing microtubules to be excluded from entering dendrites.
Download video file (4.5MB, mp4)
The Movie was taken using single-Z time-lapse epifluorescence microscopy and shows a Kinesin-2 RNAi class I da neuron expressing 221-Gal4 > UAS-EB1-GFP and 221-Gal4 > UAS-Klp64D RNAi. Images were collected every 5 s and the Movie plays at 10 frames/second.