Fig. 3. Wdr8 is required to recruit katanin to the cortical nucleation sites.

a, c Time-lapse confocal microscopy images of katanin recruitment to the branching nucleation sites at the release of daughter microtubules in wild-type (a) and wdr8 (c) cells. Katanin (magenta circles in the diagrams) and microtubules (green lines) are labeled by GFP-KTN1 and mCherry-TUB6, respectively. Open and closed white arrowheads respectively indicate the absence and the presence of KTN1 particles, whereas yellow and white arrows show the plus and minus ends of daughter microtubules. Closed blue arrowheads point to KTN1 particles at the microtubule crossover sites. Kymographs track the dotted blue lines. Microtubule nucleation events set the zero time points. Rectangle regions boxed by red lines on the left kymographs are enlarged on the right where dotted white lines mark the minus ends of released daughter microtubules. Bars, 2 µm. The images in a are representative of 90 similar events, whereas those in c represent 29 similar events. b Time-lapse images of katanin (magenta) and microtubules (green) at 2 s intervals in wild-type cells. Microtubule nucleation occurs at 0 s, whereas detachment of the daughter microtubule minus end is recognizable at 172 s. White arrowheads indicate the katanin particles dislodged from the branch base. A yellow square at −2 s shows ROI (5 pixel × 5 pixel). Bar, 1 μm. The images are representative of 30 similar events. d Distribution of GFP-KTN1 signal intensities at the branching nucleation sites (384 ROIs from 6 wild-type cells and 464 ROIs from 4 wdr8 cells) and the non-nucleating regions of the mother microtubule lattice (272 ROIs from 6 wild-type cells and 320 ROIs from 4 wdr8 cells). MT, microtubule. e Katanin accumulates at the branching nucleation sites prior to severing in wild-type cells (green line; 24 events from 6 cells) but not in wdr8 cells (magenta line; 29 events from 4 cells). Detachment of the minus ends of daughter microtubules from the mother microtubules were visually detected from the confocal images and was set to the time zero. The error bars indicate SD. f, g Time-lapse confocal microscopy images of katanin recruitment to the bundle-forming nucleation sites at the release of daughter microtubules in wild-type (f) and wdr8 (g) cells. Similar events were observed in nine wild-type cells (f) and three wdr8 cells (g). Kymographs on the right track the dotted blue lines. Symbols are the same as in a and c. Bars, 2 µm.