Fig. 3. CLIP-190 localisation to kinetochores requires the dynein complex. (A) The proportion of cells having CLIP-190 on at least one kinetochore after depletion of Drosophila Mad1 (n=100 cells). Mad1 was depleted by RNAi and cells were immunostained with CLIP-190 before and after colchicine treatment. A ²-test indicated no significant difference between Mad1-depleted cells and mock-depleted cells. Mad1-mediated spindle checkpoint is not required for the localisation of CLIP-190 to unattached kinetochores. (B) Kinetochore localisation of CLIP-190 after control or BubR1 RNAi. Cells were immunostained for BubR1 and CLIP-190 after colchicine treatment. Both BubR1 and CLIP-190 localise to kinetochores in control RNAi. After BubR1 RNAi, BubR1 signals on kinetochores were undetectable, whereas CLIP-190 localises robustly to kinetochores. (C) S2 cells depleted of Dhc were immunostained with CLIP-190 after colchicine treatment. Chromosomes marked by arrowheads are magnified in the top right corners. CLIP-190 was not localised to kinetochores in Dhc depleted cells. (D) The proportion of mitotic cells that have CLIP-190 on at least one kinetochore (n=100 cells scored in each category) after depletion of the following proteins; Ctrl (control, ß-lactamase), Dhc (dynein heavy-chain), Dic (dynein intermediate-chain), Lis1, p150 (p150^Glued), Cenp-M (Cenp-meta), NudC, NudC-L (NudC-like: CG31251), and NudE (CG8104). Cells were immunostained with CLIP-190 after colchicine treatment at day 5 of RNAi. All residual CLIP-190 kinetochore signals were weak in Rod-depleted cells. (E) Localisation dependency among CLIP-190, dynein-dynactin complex and Rod. Kinetochore localisation of CLIP-190, Dic and Rod were examined after depletion of each protein by RNAi. + indicates that kinetochore localisation is retained, while – indicates kinetochore localisation is abolished or greatly reduced. Dic after p150 depletion gave reduced but consistent signals on kinetochores and is therefore marked with ±. (F) Metaphase cells without (upper panel) or with (lower panel) vanadate treatment, an inhibitor of dynein-motor-activity. S2 cells were treated with vanadate before immunostaining for Dic, CLIP-190 and DNA. Dynein and CLIP-190 is lost from kinetochores of metaphase chromosomes without vanadate treatment, whereas both proteins accumulated on the kinetochores as well as spindle microtubules and poles after vanadate treatment. Bars, 10 µm.