Figure 6. Somatic Golgi stacks nucleate microtubules.

(A) Widefield fluorescent images from a movie showing the soma of a class I da neuron expressing EB1-GFP (greyscale) and ManII-mCherry (magenta). Manually assigned multi-colour tracks of EB1-GFP comets were drawn over each image (filled circle = last location of comet). Arrow indicates a Golgi stack from which sequential EB1-GFP comets emerge; dotted lines show an example of an angle measurement of a comet’s initial growth relative to the axon entry point (orange circle). Time in min:s from the start of the movie is shown. (B) Graph shows a frequency distribution of the initial growth angle (before turning) relative to the axon entry site (as indicated in (A)) of EB1-GFP comets that emerged from Golgi stacks. Negative angles were made positive so as not to distinguish between comets growing to the left or right of the axon. 163 comets from 59 Golgi stacks from seven neurons were analysed. (C) Scatter graph shows the position of the normalised resultant comet vectors (see methods) for each of 44 Golgi stacks from seven neurons. Circle position is representative of the average angle from the axon and how similar the angles were (as indicated). Circle size reflects the number of comets analysed per Golgi stack (as indicated below). (D) Graph shows a frequency distribution of the resultant vector lengths for the comet data (blue) and for 3 sets of data produced using randomly generated angles (grey shades). (E) Widefield fluorescent images from a movie showing the soma of class I da neuron expressing UAS-EB1-GFP (greyscale) and UAS-ManII-mCherry (magenta). The sample was subjected to a warm-cool-warm temperature cycle to induce depolymerisation (5°C; blue outline) and then repolymerisation (20 °C; red outline) of dynamic microtubules. Tracks were manually colour coded: green = comets emerging from Golgi stacks; purple = comets emerging from elsewhere. Time in min:s from the 5°C to 20°C transition is shown.

Figure 6—video 1. EB1-comets emerge from somatic Golgi stacks and initially grow preferentially towards the axon.

Download video file ^{(5.1MB, mp4)}

The Movie was taken using single-Z time-lapse epifluorescence microscopy and shows a class I da neuron expressing 221-Gal4 > UAS-EB1-GFP and 221-Gal4 > UAS ManII-mCherry. The left panel shows an overlay of the GFP and mCherry channels, while the right panel shows the same overlay 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 Golgi stacks and this can occur repeatedly from the same stack (see bottom right stack). Images were collected every 3 s and the Movie plays at 10 frames/second.

Figure 6—video 2. The somatic Golgi stacks nucleate microtubules.

Download video file ^{(6.8MB, mp4)}

The Movie was taken using single-Z time-lapse epifluorescence microscopy and shows a class I da neuron expressing 221-Gal4 > UAS-EB1-GFP and 221-Gal4 > UAS ManII-mCherry. The sample was subjected to a warming-cooling-warming (20 °C-5°C-20°C) cycle, as indicated by the red (20 °C) and blue (5 °C) borders. The left panel shows an overlay of the GFP and mCherry channels, while the right panel shows the same overlay 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 have been manually post-coloured such that green represent comets emerging from Golgi stacks and purple represent comets emerging from elsewhere within the soma. Comets can be seen emerging from Golgi stacks before cooling, and then all comets stop during the 3 min cooling period. After warming, comets can be seen emerging from the Golgi stacks, including comets that emerge >1 min after warming. Images were collected every 3 s and the Movie plays at 10 frames/second.