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First published online 14 August 2007
doi: 10.1242/jcs.010322

Journal of Cell Science 120, 3064-3074 (2007)
Published by The Company of Biologists 2007
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Ninein is released from the centrosome and moves bi-directionally along microtubules

David K. Moss1,*, Gemma Bellett1, Jane M. Carter1, Mirjana Liovic2,3, Jennifer Keynton1, Alan R. Prescott4, E. Birgitte Lane2 and Mette M. Mogensen1,{ddagger}

1 School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK
2 Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK
3 Medical Centre for Molecular Biology, Faculty of Medicine University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia
4 Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK


Figure 1
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  		Fig. 1. Ninein speckle relocation to apical non-centrosomal sites during inner pillar cell development. (A) Schematic diagram of microtubule (red lines) and ninein (green) organisation in a typical inner pillar (IP) cell at postnatal day 5 (P5) showing microtubule minus-ends anchored at apical sites (arrowhead). (B) IP cell at P1 showing ninein mainly confined to the centrosome (arrow) at an early stage in the assembly of the apico-basal array. (C) Two IP cells at P3 with ninein at the centrosome and as distinct speckles in the cytoplasm of the apical half. Note that some of the ninein speckles are aligned in a peripheral apical ring (arrow). (D) IP cell at P5 double-labelled for dynein (red) and ninein (green), with ninein now evident at the apical peripheral ring (arrow) as well as at the centrosome. (E) Ninein peripheral ring in an IP at P6. (F) Apical region of an IP cell at P5 with microtubules (blue) forming an apico-basal array and {gamma}-tubulin (red) concentrated at the centrosome and not evident at the apical sites (arrow). Bars, 10 µm (A-C), 5 µm (D), 2.5 µm (E,F).

 

Figure 2
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  		Fig. 2. Cytoplasmic ninein speckles are prominent in cultured epithelial cells. (A) PtK2 cell showing ninein at the centrosome (arrow) and throughout the cytoplasm as numerous speckles. (B) Merged image, with ninein (red) and microtubules (green). (C,D) Control (C) and siRNA-ninein-treated ARPE-19 cells labelled for ninein showing lack of cytoplasmic speckles and centrosomal staining in depleted cells (D) (images acquired using the same microscope exposure settings). (E) UE1 cell showing numerous ninein speckles (arrowhead; see also inset), which appear to be associated with microtubules. A few ninein speckles appear not to be associated with microtubules (arrow; see also inset). (F-H) Ninein speckle at the end of a free microtubule in a PtK2 (F) and a FHL124 (G) cell and along microtubules in an ARPE-19 cell (H). (I,J) Immuno-gold localisation of ninein cytoplasmic speckles in PtK2 cells. Gold particles (highlighted in yellow) are evident close to the wall of microtubules either singly or in groups of 3-4. Bars, 5 µm (A,B), 10 µm (C,D), 2 µm (E), 50 nm (I,J).

 

Figure 3
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  		Fig. 3. Nocodazole induces centralisation of ninein speckles and reveals colocalisation with keratin filaments. (A) Nocodazole-treated PtK2 cell showing centralisation of ninein speckles (cell periphery indicated by dots). (B) PtK2 cell treated with nocodazole and 0.5% Triton X100 in PHEMO for 30 seconds prior to fixation and labelling for tubulin (red) and ninein (green) showing centralisation of the speckles and a few speckles colocalising with nocodazole-resistant microtubules (arrow, see inset). (C) PtK2 cell showing keratin filaments (K18, red) collapsed around the nucleus following nocodazole treatment. Note that the ninein (green) speckles are retained within the keratin network. (D,E) Nocodazole removal and 15 minutes regrowth results in microtubules (E) extending to the cell periphery and ninein speckles regaining their full distribution (D) (compare with Fig. 2A,B). (F) Double labelling for keratin 18 (red) and ninein (green) in an untreated cell indicates ninein speckles associated with keratin filaments. Box shows region enlarged in inset. (G) Immuno-gold labelling for ninein showing gold particles (highlighted in yellow and arrows) associated with 15 nm diameter filaments (arrowhead indicates a microtubule). (H) Keratin filaments (K18, red) from a cell treated with nocodazole and latrunculin B showing ninein (green) speckle association. Bars, 5 µm (A,B,C), 5 µm (D,E), 5 µm (F), 250 nm (G).

 

Figure 4
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  		Fig. 4. GFP-ninein dynamics showing speckle release and movements away from the centrosome. (A-C) Numerous GFP-ninein speckles in the immediate environs of the centrosome in a PtK2 cell (A) were highly motile (see supplementary material Movie 1), and some had a string-like appearance [(B); arrows], which was also observed for endogenous ninein in an immuno-labelled non-transfected cell [(C); arrows]. (D) Image sequence (inverted) indicating initial release of string-like GFP-ninein followed by release of a speckle (arrows) from the centrosome in an ARPE-19 cell (see supplementary material Movie 4). (E,F) Image frames (inverted) taken from a time-lapse recording of a MDCKII cell showing a GFP-ninein speckle being released from the centrosome (E) and a speckle moving away from the centrosome (F), with arrows indicating the progress of the speckles at different time points (see supplementary material Movies 2 and 3). Time is shown in seconds (s). Bars, 2 µm.

 

Figure 5
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  		Fig. 5. GFP-ninein speckle movements towards the centrosome. (A) Image sequence (inverted) taken from the same time-lapse recording as Fig. 4E,F showing a MDCKII cell expressing GFP-ninein, with a speckle (arrow) moving towards the centrosome (see supplementary material Movie 2). (B) A speckle (arrow) moving towards the centrosome in a GFP-ninein-expressing ARPE-19 cell. Time is shown in seconds (s). Bars, 2 µm.

 

Figure 6
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  		Fig. 6. GFP-ninein speckles travel along microtubules. (A,A') Tracking of GFP-ninein speckles moving away from the centrosome over successive frames reveals several speckles following the same path. The speckles are indicated by a coloured arrow in (A), and their paths are shown by the corresponding coloured lines in (A'). (B) Immuno-gold labelling for ninein in the centrosomal region showing gold particles (highlighted in yellow) along the wall of a microtubule (arrowhead) anchored to one of the subdistal appendages of the mother centriole. Also note gold particles associated with the subdistal appendices (arrows). (C) Time-lapse frames of the centrosomal region of a PtK2 cell expressing GFP-ninein (red) and YFP–{alpha}-tubulin (green) (see supplementary material Movie 5). GFP-ninein speckles appear associated with the radiating microtubules (arrows). The frames show distinct changes in ninein speckle distribution over time. Time is shown in seconds (s). Bars, 1 µm (A,A'), 100 nm (B), 2 µm (C).

 

Figure 7
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  		Fig. 7. Microtubules are required for GFP-ninein speckle dynamics. (A) Two image frames (inverted) of the centrosome region taken from a time-lapse series of a GFP-ninein-expressing PtK2 cell treated with nocodazole (see supplementary material Movie 6) showing no positional changes in the speckles. The arrow highlights one of the speckles. Time is shown in seconds (s). Scale bar: 2 µm. (B) Quantitative analyses of GFP-ninein speckle velocities for movements away from the centrosome in control (C) PtK2 cells (0.53±0.06 µm/second, n=27) and in PtK2 cells pre-treated with nocodazole (NOC) (0.09±0.05 µm/second, n=22) or latrunculin B (LB) (0.58±0.06 µm/second, n=36) and towards the centrosome in control (C) (0.37±0.05 µm/second, n=23), nocodazole (NOC) (0.04±0.02 µm/second, n=21) and latrunculin B (LB) treated (0.43±0.04 µm/second, n=35) cells. The results show that only nocodazole is able to significantly (P<0.001) inhibit ninein speckle movements both to and from the centrosome, indicating that microtubules and not actin filaments are required for speckle dynamics.

 

Figure 8
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  		Fig. 8. FRAP analyses of GFP-ninein at the centrosome in PtK2 cells. (A) Images from a time-lapse recording showing recovery of GFP-ninein at the centrosome following photobleaching (arrows). (B) Images from a time-lapse recording of a cell pre-treated with nocodazole showing no significant recovery at the centrosome (arrows) following photobleaching. (C) Graphs depicting the fluorescence intensity during recovery following photobleaching of the control cell in A (red line) and the nocodazole-treated cell in B (blue line). The fluorescence intensity at the centrosome following photobleaching was normalised to that prior to photobleaching. (D) Histogram comparing percentage centrosomal GFP-ninein fluorescence recovery 4 minutes after photobleaching in control (red; 98.48±1.52%, n=5) and nocodazole (blue; 23.83±3.68%, n=6) treated cells.

 

Figure 9
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  		Fig. 9. Ninein localisation in polarised MDCKII cells. (A) Single confocal optical section through the apical region of a polarised MDCKII cell, with ninein at the centrosome, as speckles in the cytoplasm, and at the cell periphery where it forms a distinct punctate pattern (arrow). (B) Apical view of a 3D reconstruction (based on confocal optical sections) of three polarised cells showing ninein at the centrosome and accumulated within a peripheral ring at the location of the anchoring sites. (C) Merged image, with ninein (green) and beta-catenin (red), showing regions of substantial colocalisation (yellow) at the cell periphery. (D-F) Regions of colocalisation analysed using Volocity (Improvision) software, with (F) indicating regions with strong colocalisation (yellow). (G) Lateral view of a 3D reconstruction from a confocal Z stack of a polarised cell showing the distinct apical localisation of ninein (green). The nucleus is labelled with DAPI (blue). (H) Rotated image showing an oblique view of a 3D reconstruction of a group of four cells, with ninein (green) at the centrosomes and concentrated within apical peripheral rings. The nucleus is labelled with DAPI (blue). Bars, 5 µm (A,D,E,F), 5 µm (B,C), 2 µm (G,H).

 




© The Company of Biologists Ltd 2007