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).