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First published online August 16, 2005
doi: 10.1242/10.1242/jcs.02504

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Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends

Nikola S. Dzhindzhev¹, Stephen L. Rogers^2,*, Ronald D. Vale² and Hiroyuki Ohkura^1,

¹ The Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, EH9 3JR, UK
² The Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA

View larger version (52K): [in a new window]   Fig. 1. Localisation of CLIP-190 in the cell cycle. S2 cells were immunostained with CLIP-190 and -tubulin antibodies, DNA was counterstained with DAPI. (A) In interphase cells, CLIP-190 is mainly localised to microtubule plus-ends. (B) Magnified view of the region marked by the rectangle in A. Three arrowheads indicate the variation in CLIP-190 localisation at microtubule plus-ends, which may reflect differences in microtubule dynamics. (C) In prometaphase, CLIP-190 is localised to kinetochores. (D) In metaphase, CLIP-190 disappears from kinetochores although it still associates weakly with spindle microtubules. Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 2. Localisation of CLIP-190 to unattached kinetochores. S2 cells were immunostained without (A,C) or after (B) incubation with colchicine. The right panels show magnified images of the regions marked with squares in the left panels. (A) Kinetochore localisation of CLIP-190. Staining of CLIP-190 overlaps with the one of BubR1, a known kinetochore protein. (B) Microtubule-independent localisation of CLIP-190 to kinetochores. CLIP-190 and BubR1 both localise to primary constrictions of all chromosomes in the absence of microtubules. Note that CLIP-190 localises to the outer regions of kinetochores relative to BubR1. (C) Localisation of CLIP-190 to unattached kinetochores. Chromosomes were visualised with an antibody against phosphorylated histone H3. Only one of the sister kinetochores, the one facing away from the pole, has CLIP-190. Bars, 10 µm.

View larger version (79K): [in a new window]   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 (p150Glued), 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.

View larger version (72K): [in a new window]   Fig. 4. The plus-end localisation of CLIP-190 depends on EB1. S2 cells were immunostained with antibodies against EB1, CLIP-190 and -tubulin. (A) Colocalisation of CLIP-190 and EB1 to the microtubule plus-ends during interphase. Both proteins accumulate near microtubule plus-ends and weakly localise along the microtubules. The region marked with a rectangular box is magnified below each panel. (B) Distinct localisation of CLIP-190 and EB1 in mitosis. CLIP-190 localises to the kinetochores at prometaphase, whereas EB1 localises to spindle and aster microtubules without accumulation at kinetochores. (C) Conserved domain structure of CLIP-190. CLIP-190 has two CAP-Gly domains in its N-terminus, followed by a coiled-coil region and two zinc finger motifs in its C-terminus. Subregions of CLIP-190 shown here were expressed in bacteria as GST-fusion proteins. (D) Physical interaction between CLIP-190 and EB1. GST-tagged subdomains of CLIP-190 and GST alone were expressed in bacteria and bound to glutathione beads. After washing, the beads were incubated in bacterial lysates containing MBP-EB1 or MBP alone (to control for the MBP-part of the fusion protein). The glutathione beads were washed again and analysed by immunoblotting using an MBP antibody. The N-terminal domain (CAP-Gly domain) of CLIP-190 directly interacts with EB1. (E) Delocalisation of CLIP-190 in EB1-depleted cells. S2 cells were immunostained for CLIP-190, -tubulin and EB1 after EB1 depletion. The region marked with a box is magnified below each panel. Localisation of CLIP-190 to microtubule plus-ends was abolished (compare with A). (F) The proportion of cells with CLIP-190 at microtubule plus-ends after control and EB1 RNAi (n=100 cells scored each). Grey boxes indicate robust localisation of CLIP-190 to microtubule plus-ends, whereas open boxes indicate a greatly reduced signal. (G) The localization of CLIP-190 to unattached kinetochores is unaffected in EB1 RNAi. S2 cells were immunostained for DNA, CLIP-190 and EB1 after EB1 depletion. CLIP-190 was still be seen on unattached kinetochores in EB1 RNAi (arrowheads; compare with B). Bars, 10 µm.

View larger version (49K): [in a new window]   Fig. 5. The cell-cycle regulation of microtubule plus-end localisation. (A,B) S2 cells were stained simultaneously for EB1 and CLIP-190. (A) The CLIP-190 signal at microtubule plus-ends (either interphase or astral microtubules) is strong in interphase but very weak in mitosis. By contrast, the EB1 signal is constant in both mitosis and interphase. Bar, 10 µm. (B) Higher magnification images of interphase (upper panels) and mitotic cells (lower) marked by the squares in A. (C) Cell-cycle change of CLIP-190 and EB1 localisation to microtubule plus-ends. The plus-end signal-intensity relative to background was measured for each cell-cycle stage (a total of ten microtubules from three different cells were scored for each mitotic stage) and is shown as a circle with the standard deviation represented by vertical bars. CLIP-190 dissociates from microtubule plus-ends during mitosis. (D) Levels of EB1 and CLIP-190 at microtubule plus-ends in mitotic cells after control (n=30 microtubules from three different cells) and Dhc (n=18 microtubules from three different cells) RNAi. In Dhc-depleted cells, CLIP-190 does not localise to kinetochores, but still dissociates from microtubule plus-ends during mitosis. (E) Cell-cycle regulation of CLIP-190 localisation. Molecular requirements for CLIP-190 localisation during the cell cycle are illustrated. During interphase, CLIP-190 localises to microtubule plus-ends in an EB1-dependent manner. The association of CLIP-190, but not EB1, to microtubule ends is greatly reduced during mitosis. Instead, CLIP-190 is localised to unattached kinetochores in mitosis. This localisation depends on dynein-dynactin and Lis1. Dynein-dynactin and Lis1 localisation are mutually dependent on each other, and both depend on Rod. Upon attachment to microtubules, CLIP-190 is removed from kinetochores by the motor-activity of dynein.