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Files in this Data Supplement:
Fig. S1. Latrunculin B does not affect GFP-ninein speckle distribution or dynamics. GFP-ninein expressing PtK2 cells treated with 2 μM latrunculin B. (A) Two image frames taken from a movie recorded at 2 second intervals showing a ninein speckle (arrow) progressing towards the centrosome. (B) Cell showing actin depolymerisation and formation of aggregates (red) and preservation of the radial microtubule (green) organisation. The inset shows GFP-ninein at the centrosome and speckles in the cytoplasm. Bars: A=2 μm; B=10 μm, inset=2 μm.
Fig. S2. GSK3β inhibition does not affect GFP-ninein speckle release or dynamics. Image sequences taken from a time-lapse of a GFP-ninein expressing MDCKII cell treated with the GSK3β inhibitor SB415286 at 10 μM for 24 hours prior to imaging showing speckles being released from the centrosome (0s-2s, arrows) and a speckle moving towards the centrosome (3s-6s, arrows). The velocities of moving speckles were estimated to be 0.83 μm/s (±0.15 μm/s, n=9) towards the centrosome and 0.81 μm/s (±0.11 μm/s, n=11) away from the centrosome in control MDCKII cells and 0.76 μm/s (±0.16 μm/s, n=5) towards the centrosome and 0.64 μm/s (±0.11 μm/s, n=9) away from the centrosome in GSK3β inhibited MDCKII cells. This was not significant. Bar: 2 μm.
Fig. S3. Ninein and E-cadherin localisation in polarised MDCKII cells. Apical view of a 3D reconstruction from a confocal Z stack of four polarised cells labelled for ninein (green) and E-cadherin (red) showing ninein at the centrosome, as speckles in the cytoplasm and within a ring at the cell periphery in close proximity to E-cadherin containing junctions (see also inset). However, ninein localisation appears distinct from that of E-cadherin suggesting no or minimal co-localisation (yellow). Bar: 5 μm.
Movie 1. Movie showing GFP-ninein dynamics in centrosome region − short range movements. Movie of the centrosomal region of a PtK2 cell expressing GFP-ninein showing numerous GFP-ninein speckles moving to and from the centrosome. Note that some of the GFP-ninein has a string-like appearance (see also Fig. 4A for details). The images were recorded at 2 second intervals for a total of 2 minutes and the movie shows10 frames/second.
Movie 2. Movie showing release of GFP-ninein speckles and bi-directional speckle movements within the cytoplasm − long range movements. Movie of a MDCKII cell expressing GFP-ninein showing speckle release from the centrosome (see also Movie 3 for close up of speckle release) and bi-directional movements of speckles throughout the cytoplasm (see also Figs 4EF,5A). Images were taken at 1 second intervals for a total of 77 seconds and the movie shows 5 frames/second.
Movie 3. Movie showing release of GFP-ninein speckles from the centrosome. Movie of the centrosomal region generated from Movie 2 showing GFP-ninein speckles being released from the centrosome (see also Fig. 4E). Images were taken at 1 second intervals for a total of 30 seconds and the movie shows 5 frames/second.
Movie 4. Movie showing release of string-like GFP-ninein from the centrosome. Movie of the centrosomal region of an ARPE-19 cell expressing GFP-ninein showing string-like ninein and speckles being released from the centrosome (see also Fig. 4D). Also note speckles moving towards the centrosome. Images were taken at 1.4 second intervals for a total of 102 seconds and the movie shows 5 frames/second.
Movie 5. Movie showing GFP-ninein speckles travelling along microtubules. PtK2 cell expressing GFP-ninein (red) and YFP-α-tubulin (blue) showing GFP-ninein speckles being released from the centrosome and travelling along centrosome anchored microtubules (see also Fig. 6C). Images were taken at 10 second intervals for a total of 2 minutes and the movie shows 5 frames/second.
Movie 6. GFP-ninein speckle dynamics is inhibited by nocodazole. PtK2 cell expressing GFP-ninein treated with nocodazole showing no significant GFP-ninein speckle movements (see also Fig. 7). Images were taken at 2 second intervals for a total of 30 seconds and the movie shows 10 frames/second.
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