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First published online 16 March 2004
doi: 10.1242/jcs.01033

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Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells

Elsa Logarinho^1,2,*, Hassan Bousbaa^1,2,*, José Miguel Dias¹, Carla Lopes¹, Isabel Amorim^1,3, Ana Antunes-Martins¹ and Claudio E. Sunkel^1,4,

¹ Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
² Instituto Superior de Ciências da Saúde-Norte, Grupo de Biologia Molecular e Celular, Rua Central de Gandra 1317, 4580 Gandra PRD, Portugal
³ Departamento de Botânica da Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 1119, 4150-181 Porto, Portugal
⁴ Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo do Prof. Abel Salazar 2, 4099-003 Porto, Portugal

View larger version (73K): [in a new window]   Fig. 2. Immunolocalization of Mad2, Bub1, Bub3 and BubR1 during progression through mitosis in Drosophila S2 cells. Cells were stained for DNA (blue) and antibodies against Mad2, Bub1, Bub3 or BubR1 (red). -tubulin staining (green) was used to identify cells in prophase. Note that only Bub3 and Bub1 show kinetochore staining in prophase. All spindle checkpoint proteins show strong accumulation at kinetochores in prometaphase, weak or no staining in metaphase and are absent in anaphase. Staining of the spindle for Mad2 and Bub1 is observed during metaphase, and weak staining of centrosomes (arrowheads) is observed for all proteins at anaphase. Scale bar: 5 µm.

View larger version (45K): [in a new window]   Fig. 3. Tension across bi-oriented kinetochore pairs of S2 metaphase cells is reduced after taxol treatment. (A) Cells in prophase or metaphase were stained for CID (green), -tubulin (red) and DNA (blue). The distance between sister kinetochores in taxol-treated metaphase cells (right panel) is shorter than in control metaphase cells (middle panel) and closer to the distance in prophase cells (left panel). Boxes of identical size are used to compare interkinetochore distances between control and taxol treated cells. Tubulin staining shows that kinetochores still retain their microtubule fibers after taxol treatment. (B) Histogram showing the variation in distances between sister kinetochores in prophase, metaphase and taxol-treated metaphase cells. Interkinetochore distances in prophase cells (range: 0.48-0.92 mm). After sister kinetochores become bioriented poleward forces stretch centromeric heterochromatin and generate tension across kinetochore pairs (range: 0.88-1.59 mm). Taxol relieves tension by interfering with microtubule dynamics causing the distance between sister kinetochores to decrease (range: 0.59-1.00 mm). (C) S2 cells showing CID (red), 3F3/2 (green) and DNA (blue). After taxol treatment, all kinetochores in metaphase cells exhibit strong 3F3/2 labelling because of lack of tension (right panel). In a control metaphase cell, 3F3/2 labelling at the kinetochores is hardly detectable because of tension-sensitive dephosphorylation (left panel). Scale bars: 5 µm.

View larger version (43K): [in a new window]   Fig. 4. Effect of reduced tension on the kinetochore localization of Mad2, Bub1 and BubR1. (A) Cells were incubated with 10 nM taxol for 10 minutes and then processed for immunofluorescence with antibodies against Mad2, Bub1 or BubR1 (red) and a-tubulin (green); DNA was stained with DAPI (blue). Mad2 and Bub1 are hardly detectable at kinetochores of aligned chromosomes, whereas BubR1 consistently localizes at all kinetochores of the metaphase plate. Mono-oriented chromosomes (arrows) exhibit BubR1 staining at both kinetochores while Mad2 and Bub1 labelling is only observed at unattached kinetochores. Scale bar: 5 µm. (B) Quantification of Mad2, Bub1 and BubR1 immunoreactivity patterns at sister kinetochores of mono-oriented chromosomes: labelling at both kinetochores (+/+), labelling only at unattached kinetochores (+/–) and no labelling at either kinetochore (-/-). Note that, whereas BubR1 labelling at mono-oriented chromosomes is mainly symmetric, Mad2 and Bub1 show staining only at unattached kinetochores.

View larger version (42K): [in a new window]   Fig. 5. Phosphorylation of 3F3/2 epitopes in S2 lysed cells. In all images DNA is shown in blue and 3F3/2 antibody staining in red. (A) S2 cell lysed in the absence of the phosphatase inhibitor microcystin (T-Mc). 3F3/2 epitopes are only detectable at the spindle poles (arrowhead). (B,C) S2 cells lysed in the presence of microcystin (T+Mc). Prometaphase cells (B) exhibit strong 3F3/2 staining at the kinetochores and spindle poles (arrowhead). At metaphase (C), 3F3/2 kinetochore labelling decreases significantly and is often undetectable. (D,E,F) S2 cells lysed in the absence of microcystin and then incubated with ATP plus microcystin (ATP+Mc). Kinetochores are strongly labelled with 3F3/2 antibody even at metaphase (E), but not at anaphase (F). (G) Dephosphorylated S2 cell (lysed with T-Mc) and incubated with ATP alone (ATP-Mc). 3F3/2 epitopes are not phosphorylated in the absence of microcystin. Scale bar: 5 µm.

View larger version (35K): [in a new window]   Fig. 6. Localization of Mad2 and Bub1 checkpoint proteins is not affected by kinetochore dephosphorylation. S2 cells were lysed with detergent either in the presence (T+Mc) or in the absence (T-Mc) of microcystin, fixed and immunostained. (A) Immunostaining with the anti-Mad2 (green) and 3F3/2 (red) antibodies shows that Mad2 labelling remains unchanged after dephosphorylation. (B) Immunostaining with the anti-Bub1 (green) and 3F3/2 (red) antibodies shows that Bub1 still localizes normally after dephosphorylation. Scale bar: 5 µm.

View larger version (37K): [in a new window]   Fig. 7. Dephosphorylation of kinetochore phosphoepitopes releases BubR1 and Bub3. S2 cells were lysed with detergent either in the presence (T+Mc) or in the absence (T-Mc) of microcystin, fixed and immunostained. (A) Immunostaining with anti-BubR1 (green), 3F3/2 (red) and anti-CID (pink) antibodies shows that BubR1 staining is no longer detectable after dephosphorylation. CID localization is shown as a control and is not affected by dephosphorylation. (B) Immunostaining with anti-Bub3, 3F3/2 and anti-CID antibodies shows that Bub3 staining is lost after dephosphorylation, while CID labelling remains. Scale bar: 5 µm.

View larger version (43K): [in a new window]   Fig. 8. BubR1 and Bub3 loss from kinetochores is specifically due to 3F3/2 epitope dephosphorylation. Condensed chromosomes were purified from S2 colchicine-arrested cells in the presence of microcystin to maintain kinetochore phosphoepitopes. In all preparations DNA was stained with DAPI (blue). (A,E) Isolated chromosomes were fixed and immunostained with 3F3/2, anti-BubR1 and anti-Bub3 antibodies. Note the strong kinetochore labelling for all antibodies. (B,F) Isolated chromosomes were incubated with lambda phosphatase (PPase) leading to complete dephosphorylation of the 3F3/2 epitopes and to loss of BubR1 and Bub3 from kinetochores. (C,G) Chromosomes incubated with lambda phosphatase buffer alone ( buffer) retain 3F3/2 phosphoepitopes, as well as BubR1 and Bub3 proteins. (D,H) Isolated chromosomes were first incubated with the 3F3/2 antibody and then treated with lambda phosphatase before immunostaining for BubR1 or Bub3. Preincubation with 3F3/2 antibodies blocks the accessibility of the phosphatase to the 3F3/2 phosphoepitopes causing BubR1 and Bub3 to remain at kinetochores. Scale bar: 5 µm.

View larger version (47K): [in a new window]   Fig. 9. BubR1 is recruited to phosphorylated but not to unphosphorylated kinetochores. All cells were initially lysed in the absence of microcystin to become dephosphorylated and, after different treatments, fixed and stained for 3F3/2, BubR1, CID and DNA (blue). (A) Cells treated with ATP in the absence of microcystin (ATP-Mc) retain normal CID staining but BubR1 and 3F3/2 labelling is abolished. (B) Cells treated with ATP plus microcystin (ATP+Mc) show CID and 3F3/2 staining but endogenous BubR1 is depleted during extraction. (C) Cells rephosphorylated with ATP but without microcystin and subsequently incubated with S2 mitotic extract (M ext). Note that 3F3/2 epitopes are not rephosphorylated and that exogenous BubR1 is not recruited to kinetochores. (D) Cells rephosphorylated in the presence of microcystin and subsequently incubated with S2 mitotic extract show rephosphorylated 3F3/2 epitopes and strong accumulation of BubR1 at kinetochores. (E) Cells rephosphorylated in the presence of microcystin and incubated with an S2 mitotic extract mostly depleted of BubR1, show strongly labelled 3F3/2 phosphoepitopes but only weak accumulation of BubR1. Scale bars: 5 µm.

View larger version (56K): [in a new window]   Fig. 1. Classification of Bub1-like proteins in Drosophila. Protein sequence analyses were done using Clustal X (http://www-igbmc.u-strasbg.fr/BioInfo/ClustalX/). Dm, Drosophila melanogaster; Hs, Homo sapiens; m, Mus musculus; X, Xenopus laevis; Sc, Saccharomyces cerevisiae; Sp, Saccharomyces pombe; An, Anopheles gambiae; Ce, Caenorhabditis elegans. (A) Sequence alignment of the KEN box motif uniquely present in the N-termini of BubR1 and Mad3 proteins. Note that DmBub1 (165 kDa) previously reported as Bub1 contains the KEN box motif similarly to other BubR1 proteins. (B) Sequence alignment of the C-terminal Ser/Thr kinase domains of Bub1 and BubR1 proteins. The 12 subdomains conserved among protein kinases (Hanks and Hunter, 1995) are underlined and the amino acids conserved within these subdomains are in red. In comparison with BubR1 kinases from other organisms, Drosophila BubR1 (DmBub1-165kDa) contains a much more conserved kinase domain. (C) Schematic representations of the Mad3, Bub1 and BubR1 proteins showing the relative positions of the KEN box motif present in Mad3 and BubR1 (crossed hatched box), the conserved Cdc20- and Bub3-binding domains present in all of them (black and grey boxes, respectively), and the conserved Ser/Thr kinase domain in Bub1 and BubR1 (vertical hatched box). Species that contain Mad3 and Bub1 or Bub1 and BubR1 are indicated on the right. (D) Phylogeny of the complete sequences of Bub1 and Mad3/BubR1 proteins.

View larger version (30K): [in a new window]   Fig. 10. Kinetochore localization of spindle checkpoint proteins is differentially regulated by spindle assembly events. (A) The conditions underlying binding to unattached kinetochores are similar for all proteins and require phosphorylated kinetochores. (B) Once chromosomes become mono-oriented, the attached kinetochore retains little or no Mad2 and Bub1, generating an asymmetrical localization pattern. BubR1 and Bub3 levels are only slightly asymmetrical with the attached kinetochore exhibiting reduced staining because of some tension generated from anti-poleward forces (arrows). (C) Under reduced tension from taxol treatment mono-oriented chromosomes show symmetrical BubR1/Bub3 staining while Mad2/Bub1 labelling remains asymmetrical. (D) When bipolar attachment is achieved, Mad2/Bub1 no longer localize to kinetochores, 3F3/2 epitopes are dephosphorylated because of tension and BubR1 and Bub3 are released. (E) Disruption of tension with taxol leads to recruitment of BubR1 and Bub3 to kinetochores because 3F3/2 epitopes become rephosphorylated. However, Mad2 and Bub1 cannot accumulate at kinetochores because of microtubule occupancy.

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