Correcting Improper Chromosome–Spindle Attachments During Cell Division

print ncb1102 13/2/04 3:07 PM Page 232

LETTERS

Correcting improper chromosome–spindle attachments during cell division

Michael A. Lampson1,Kishore Renduchitala1,Alexey Khodjakov2 and Tarun M. Kapoor1,3

For accurate segregation of chromosomes during cell division, is reversible, which would allow activation of the kinase in living cells microtubule fibres must attach sister kinetochores to opposite by removal of the inhibitor. poles of the mitotic spindle (bi-orientation). Aurora kinases are To assay for Aurora kinase activity in mitotic cells, we used a known linked to oncogenesis1 and have been implicated in the substrate, histone H3 (ref. 13), whose phosphorylation during mitosis regulation of chromosome–microtubule attachments2. has been previously used to demonstrate inhibition of Aurora kinase Although loss of Aurora kinase activity causes an accumulation activity3,12.In cells treated with either of two Aurora kinase inhibitors, of mal-orientated chromosomes in dividing cells3,4, it is not chromosomes failed to align at the metaphase plate (Fig. 1c, e; com- known how the active kinase corrects improper chromosome pare with control cells, Fig.1a, b), consistent with Aurora kinase inhibi- attachments. The use of reversible small-molecule inhibitors tion by other methods14,15.As expected, histone H3 phosphorylation allows activation of protein function in living vertebrate cells was reduced by over 70% relative to control cells (Fig. 1d, f, k). with temporal control. Here we show that by removal of small- Phosphorylation was fully restored 30 min after removal of hes- molecule inhibitors, controlled activation of Aurora kinase peradin (Fig. 1h, k). Recovery from AKI-1 treatment required ~60 min during mitosis can correct chromosome attachment errors by in this assay (Fig. 1j–k). These data demonstrate that both inhibitors selective disassembly of kinetochore–microtubule fibres, are reversible in living cells at the level of kinase activity. rather than by alternative mechanisms involving initial release To examine the effects of Aurora kinase activation, we developed a of microtubules from either kinetochores or spindle poles5–7. method to accumulate chromosomes with improper attachments to Observation of chromosomes and microtubule dynamics with the spindle. In the presence of the small molecule monastrol, cells arrest real-time high-resolution microscopy showed that mal- in mitosis with monopolar spindles in which kinetochore–microtubule orientated, but not bi-orientated, chromosomes move to the fibres (K-fibres) extend in a radial array from the un-separated spindle spindle pole as both kinetochore–microtubule fibres shorten, poles and chromosomes are positioned at the microtubule plus-ends, followed by alignment at the metaphase plate. Our results away from the pole16.Many of these chromosomes are mal-orientated, provide direct evidence for a mechanism required for the as K-fibres attach both sister kinetochores to the same pole (syntelic maintenance of genome integrity during cell division. mal-orientation)17.After removal of monastrol, spindles become bipolar and chromosomes align (Fig. 2a)18.Cells were incubated with Detection of errors in chromosome attachment to the mitotic spindle monastrol to accumulate monopolar mitoses and then released into has been studied extensively. Mechanisms have been proposed that either of the two Aurora kinase inhibitors. Examination of fixed cells respond to tension at kinetochores or to accumulation of microtubule showed that after spindle bipolarization in the presence of the kinase attachments at kinetochores (reviewed in ref. 8). However, it is not inhibitor, many chromosomes remain mal-orientated with K-fibre known how improper chromosome attachments are corrected. pairs attached to the same spindle pole (syntelic; Fig. 2b, arrows; also Chromosome mal-orientations have been observed after inhibition of see Supplementary Information, Fig. S2). As the K-fibres extend away Aurora kinase activity3,4,but it has not been possible to activate the from the spindle body, both chromosomes and K-fibres could be kinase and observe the correction process directly. Two unrelated clearly distinguished. This method, which was confirmed for both small-molecule inhibitors of Aurora kinase activity have been used in inhibitors (data not shown), allows us to examine mal-orientated chro- recent studies: hesperadin3,9, and one referred to here as AKI-1 mosomes after Aurora kinase activation. (Aurora kinase inhibitor-1)10,11 from a chemical structural class previ- Aurora kinase inhibition could accumulate mal-orientated chromo- ously described12 (see Supplementary Information, Fig. S1). We used somes during mitosis by either increasing the formation, or decreasing both inhibitors in our experiments, as they target Aurora kinases but the correction, of improper chromosome attachments to the spindle. are unlikely to have overlapping off-target activities. We first tested It has not been possible to distinguish between these possibilities by whether inhibition of Aurora kinase activity by these small molecules analysis of fixed cells, which shows only the steady-state situation3.

1Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, NY 10021, USA. 2Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA. 3Correspndence should be addressed to T.M.K. (e-mail: [email protected])

Published online: 8 February 2004; DOI: 10.1038/ncb1102

232 NATURE CELL BIOLOGY VOLUME 6 | NUMBER 3 | MARCH 2004

LETTERS

ac e gi k Hesperadin AKI-1 1.5

1.0

bd f hj 0.5

0.0

Phospho-H3 (mean, s.e.m.) Inhibitor Inhibitor removed

Figure 1 Inhibition of Aurora kinase is reversible. (a–f) Cells were treated (1 h) immunofluorescence microscopy. In the top panels, tubulin is shown in green with no Aurora kinase inhibitor (a, b), 100 nM hesperadin (c, d) or 20 µM AKI- and DNA is shown in blue. Bottom panels show phospho-H3 staining. Scale 1 (e, f) before processing for immunofluorescence microscopy. (g–j) In parallel bar represents 5 µm. (k) Quantification of phospho-H3 immunofluorescence, experiments, inhibitors were removed after a 1-h treatment. After an additional normalized to controls (no inhibitor), for conditions c–j. The proteasome 30 min (hesperadin; g, h) or 60 min (AKI-1; i, j), cells were processed for inhibitor MG132 was included in all experiments to prevent mitotic exit.

Both chromosomes and individual K-fibres were examined over time ends. K-fibre pairs were observed attached to sister chromatids until in living cells expressing α-tubulin–green fluorescent protein (GFP) their length reduced by 72% (s.d. = 5%, n = 6). The proximity of using near-simultaneous, three-dimensional GFP fluorescence and chromosomes to the spindle poles limited our ability to resolve indi- differential interference contrast (DIC) microscopy. In monopolar vidual K-fibres within 1.5 µm of a pole. The average overall velocity spindles, K-fibres formed the expected radial array around the pole, during poleward movement was 1.2 µm min−1 (s.d. = 0.4 µm min−1, with chromosomes at their plus-ends (Fig. 2c, f). An Aurora kinase n = 7), consistent with movement of chromosomes attached to inhibitor was added after release of the arrest, and spindle poles sepa- K-fibre plus ends during prometaphase and metaphase20,rather than rated rapidly, as expected18 (Fig. 2d). Some chromosomes maintained the more rapid movements (~18 µm min−1) along the lateral surface their positions away from the spindle body during bipolarization of a microtubule19,20.Chromosomes that were already aligned at the (Fig. 2g, h), with both K-fibres clearly visible and attached to the same metaphase plate did not seem to be affected by Aurora kinase activa- spindle pole throughout the observation time (1 h; Fig. 2d, e; also see tion, indicating that K-fibre disassembly was selective for mal-orien- Supplementary Information, Movie 1), demonstrating that without tated chromosomes. Aurora kinase activity these mal-orientations are stable and fail to cor- K-fibres can in principle disassemble from either end, and Aurora rect.These persistent mal-orientations provide a starting point from kinase family members have been shown to localize to both kineto- which to examine their correction. chores (Aurora B) and centrosomes (Aurora A; reviewed in ref. 1), After spindle bipolarization in the presence of an Aurora kinase where K-fibre plus and minus ends are attached. It is not known inhibitor to accumulate chromosome mal-orientations, the inhibitor whether Aurora A or B is specifically inhibited by the small molecules was removed to activate the kinase and cells were imaged by 3D GFP at concentrations used in our experiments3,12.Although we cannot fluorescence and DIC microscopy (Fig. 3a). Immediately after kinase exclude a role for Aurora A kinase activity in error correction, activation, multiple mal-orientated chromosomes were apparent expected functions of Aurora A in centrosome separation21–23 were (Fig. 3b, f). A surprising observation was that the correction process not inhibited in our experiments. Furthermore, the phenotypes we began with rapid movement of mal-orientated chromosomes to the observed in the presence of Aurora kinase inhibitors are consistent pole (Fig. 3b–r). Chromosomes began poleward movement an average with loss of Aurora B function at kinetochores, as shown previously of 27 min (s.d = 7 min, n = 10) after removal of the inhibitor, consis- by RNA interference (RNAi) and other methods3,12,14,15,24.In addi- tent with the timing of kinase activation as determined by recovery of tion to chromosome misalignment and checkpoint inactivation, we histone H3 phosphorylation (Fig. 1k). From the pole, chromosomes found that BubR1, dynactin and MCAK (mitotic centromere-associ- moved only in the direction of the opposite pole, resulting in align- ated kinesin) fail to localize to kinetochores in the presence of the ment at the metaphase plate (Fig. 3n–r). As an example, one chromo- inhibitors (data not shown). Our observation of selective disassembly some (Fig. 3, arrowheads) began poleward movement 28 min after of K-fibres attached to mal-orientated, but not bi-orientated, chro- removal of the inhibitor, reached the pole by 30 min and aligned by 36 mosomes may be explained by a mechanism that responds to forces in min (Fig. 3t). This behaviour was observed for seven syntelic mal-ori- opposite directions at kinetochores (tension)25.Aurora B localizes to entated chromosomes in the two cells shown (Fig. 3; and see kinetochores and may function as the tension sensor, as has been sug- Supplementary Information, Movies 2, 3), and in additional experi- gested for the yeast Aurora homolog, Ipl1 (ref. 26). One possibility is ments (n = 19 cells) using either inhibitor (data not shown). that Aurora kinase is activated specifically at kinetochores of mal-ori- This poleward movement is reminiscent of events that have been entated chromosomes. Alternatively, Aurora activation may occur at described during early stages of mitosis, when chromosomes also make all kinetochores, but tension would prevent K-fibre disassembly at bi- direct movements to the spindle pole as a precursor to bi-orientation orientated chromosomes because forces act in opposite directions. and metaphase alignment19.For comparison with this process, we This possibility could be tested by estimating forces at kinetochores, examined the details of poleward chromosome movement after activa- as has been done by measuring distance between kinetochores for bi- tion of Aurora kinase. Our analysis revealed that both K-fibres short- orientated chromosomes27.At syntelic mal-orientated chromosomes, ened as a syntelic chromosome moved to the pole (Fig. 3u–w, arrow), however, we found that inter-kinetochore distance varies widely (ref. indicating that kinetochores remained attached to the microtubule plus 17; M.A.L. and T.M.K., unpublished observations), more likely

LETTERS

a b 1

c de

−5:00 36:50 58:09

fgh

Figure 2 Inhibition of Aurora kinase activity stabilizes chromosome mal- were imaged live by near-simultaneous 3D confocal fluorescence microscopy orientations. (a, b) Spindles bipolarize as cells recover from monastrol and DIC, respectively. Selected images from timelapse recordings are treatment. After removal of monastrol, cells were incubated with (b) or shown. Arrows and arrowheads indicate chromosome mal-orientations (b, without (a) 20 µM AKI-1 for 1 h and then processed for d–h). MG132 was included in all incubations (a–h). Time is shown in immunofluorescence microscopy. Optical sections for boxed regions (1 and minutes and seconds. Scale bars represent 5 µm. Stable chromosome mal- 2) highlight mal-orientations. (c–h) After removal of monastrol and addition orientations were also observed before anaphase onset in the absence of of 100 nM hesperadin (t = 0), GFP–tubulin (c–e) and chromosomes (f–h) MG132 (see Supplementary Information, Fig. S3).

reflecting the unusual chromosome geometry rather than tension Chromosomes spend variable amounts of time at the pole; when they across kinetochores. A further molecular understanding of the correc- move, however, movement is invariably directed towards the opposite tion mechanism will require determination of which of the many pole of the bipolar spindle. This observation suggests that the K-fibre Aurora substrates (for example, ref. 28) have roles in regulating K- disassembly mechanism is active, keeping the chromosome at the pole fibre dynamics. until a microtubule attachment is formed to the opposite pole. After Experiments in model systems have suggested that syntelic chromo- this attachment, the K-fibre disassembly mechanism would be turned some mal-orientations (Fig. 4a) are corrected by K-fibre release from off, allowing movement to the metaphase plate (Fig. 4e). It has previ- either the kinetochore or the pole (Fig. 4b, c), followed by reattach- ously been shown that even a single microtubule attachment from the ment until the correct orientation is stabilized4,7.We found that syn- opposite pole is sufficient for movement29.We expect that such an telic mal-orientations can be corrected by selective K-fibre attachment will be difficult to detect near the pole, where microtubule disassembly, coupled with movement to the pole (Fig. 4d). density is high.

234 NATURE CELL BIOLOGY VOLUME 6 | NUMBER 3 | MARCH 2004

LETTERS

a 2 h 1 h Monsastrol Live imaging Aurora kinase inhibitor MG132 (proteasome inhibitor)

b c d e

5:37 13:34 23:49 27:00 fghi

jkl m

31:13 34:48 42:15 43:16 n o p q

r stChromosome 1 Chromosome 2 (arrow) (arrowhead) 20 Near pole Far pole

10 Metaphase Metaphase alignment alignment

44:55 0 Distance to pole (µm) 20 25 30 35 40 45 20 25 30 35 40 Time (min) Time (min)

u vwx y

13:12 15:12 19:2624:56 26:04

Figure 3 Correction of chromosome mal-orientations after activation of K-fibres (arrows and arrowheads) are tracked until metaphase alignment. (s, Aurora kinase. (a) A schematic representation of the experiment. Spindles t) Chromosome position relative to spindle poles (marked by circles in DIC became polar in the presence of hesperadin (100–200 nM). Hesperadin was images). (u–y) K-fibre disassembly after Aurora kinase activation in another then removed (t = 0), and GFP–tubulin (b–e, j–m and u–y) and cell (arrow shows a K-fibre pair; chromosome not shown). Time is shown in chromosomes (f–i, n–r) were imaged live by 3D confocal fluorescence minutes and seconds. Scale bars represent 5 µm. Timelapse recordings for microscopy and DIC, respectively. (b–r) Two chromosomes and associated these two cells are provided in Supplementary Information.

LETTERS

K-fibre pair

Chromosome Spindle pole

Aurora kinase activation

b c d e

~ ~ ~

K-fibre release K-fibre release Selective K-fibre from kinetochore from pole disassembly

Figure 4 Mechanisms to correct syntelic chromosome mal-orientations spindle pole (c). (d) Mal-orientated chromosomes move to the pole as K during cell division. (a) Syntelic mal-orientation arises during mitosis if -fibres selectively disassemble. K-fibres initially remain attached to both K-fibres attach sister kinetochores to the same spindle pole. (b, c) Initial kinetochores and the spindle pole. We propose that capture by K-fibre release hypothesis: Aurora kinase activity may correct mal- microtubule(s) from the opposite pole results in alignment at the orientation by K-fibre release from either the kinetochore (b) or the metaphase plate (e).

Attachment of chromosomes to the mitotic spindle is a stochastic chloride and 0.2% Triton X-100. Images were acquired on a Carl Zeiss Axiovert and dynamic process, during which errors arise (recently reviewed in 200M microscope, with either a 63× 1.4 NA objective (for presentation) or a ref. 30). These errors are corrected by a complex regulatory network 40× 0.75 NA objective (for quantification). Phospho-H3 immunofluorescence that ensures accurate chromosome segregation during cell division. intensity was quantified in mitotic cells using Metamorph software (Universal Based on direct observations of the dynamic responses of spindle Imaging, Downington, PA) and normalized to the control (no inhibitor; n ≥ 20 cells for each data point). For each spindle, Hoechst fluorescence was used to microtubules and chromosomes in living cells to reversible perturba- define the chromosome region, and phospho-H3 immunofluorescence was tions of Aurora kinases, we propose a model for the molecular basis of measured in that region. Background fluorescence, estimated from regions with correcting errors in chromosome–spindle attachments. Activation of no DNA, was subtracted. Aurora kinases selectively eliminates improper chromosome-spindle For spindle bipolarization (Fig. 2a, b), cells were first arrested in mitosis for attachments by disassembling the microtubule fibres (Fig. 4d). Such 4h by monastrol treatment. Monastrol was then removed, and MG132 was spatially specific control over cytoskeletal polymerization is likely to be added together with hesperadin or AKI-1. 1 h after removal of monastrol, cells a general mechanism for regulating intracellular transport and cell were fixed with calcium by permeabilization for 90 s at 37 °C in 100 mM PIPES migration. at pH 6.8, 1 mM magnesium chloride, 0.1 mM calcium chloride and 0.1% Triton X-100 to depolymerize non-kinetochore microtubules, followed by fixa- METHODS tion in the same buffer supplemented with 3.7% formaldehyde. Images were acquired as z-stacks on a DeltaVision Image Restoration Microscope (Applied Inhibitors. Inhibitors were synthesized according to the procedures described Precision Instruments, Issaquah, WA and Olympus, Melville, NY) using a 60 for hesperadin9 and AKI-1 (ref. 10). Chemical structures are shown in × objective and processed by iterative constrained deconvolution. Maximal inten- Supplementary Information, Fig. S1. Hesperadin was used at 100–200 nM, sity projections are shown, and optical sections (Fig. 2b, boxed regions) show AKI-1 at 20 µM. Unless otherwise stated, the proteasome inhibitor MG132 individual K-fibres more clearly. (Sigma, St Louis, MO) was included in all incubations (20 µM) with Aurora kinase inhibitors to prevent mitotic exit. The Eg5 inhibitor monastrol was used 2 at 100 µM. Live imaging. Cells were grown on 24 mm coverslips mounted in a Rose chamber for imaging using L-15 medium without phenol red. Washes were per-

Cell culture and fixed timepoint experiments. PtK2 cells expressing α-tubu- formed by flowing media through the chamber. Cells were incubated in lin–GFP18 were used for all experiments. Cells were cultured in Ham’s F-12 monastrol for 2 h or less (to prevent cells from rounding up). Monastrol was medium (Invitrogen, Carlsbad, CA), supplemented with 10% foetal bovine removed and an Aurora kinase inhibitor was added with MG132. As indicated, serum (Sigma) at 37 °C in a humidified atmosphere with 5% CO2.Antibodies the Aurora kinase inhibitor was removed after 1 h and cells were kept in used for immunofluorescence microscopy were against phospho-histone H3 MG132. Cells were imaged immediately after removal of monastrol (Fig. 2) or (Upstate Biotechnology, Lake Placid, NY) or α-tubulin (DM1α;Sigma). DNA after removal of the Aurora kinase inhibitor (Fig. 3). was labelled with Hoechst 33342 (Sigma). Confocal GFP fluorescence time-lapse sequences were acquired on a Carl For phospho-H3 immunofluorescence microscopy (Fig. 1), cells were incu- Zeiss Axiovert 200M microscope equipped with a z-motor and a 100× 1.4 NA bated with MG132 together with either hesperadin or AKI-1. Cells were fixed objective. Experiments were performed in a temperature-controlled chamber after 1 h, or the inhibitor was removed and cells were fixed after an additional (Solent Scientific, Portsmouth, UK) maintained at 37 °C. z-stacks (six sections 30–60 min in the presence of MG132. Cells were fixed at 37 °C with 3.7% separated by 1.0 µm) of GFP fluorescence were taken every 30 s with a formaldehyde in 100 mM PIPES at pH 6.8, 10 mM EGTA, 1 mM magnesium PerkinElmer Wallac (Boston, MA) UltraView confocal head with a 488-nm

236 NATURE CELL BIOLOGY VOLUME 6 | NUMBER 3 | MARCH 2004

LETTERS

excitation filter and an argon ion laser (Melles Griot, Carlsbad, CA). A single BubR1, Mad2 and Cenp-E to kinetochores. J. Cell Biol. 161, 267–280 (2003). DIC image was taken immediately after each GFP stack. Images were acquired 13. Hsu, J. Y. et al. Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, with an Orca ER cooled CCD camera (Hamamatsu, Hamamatsu City, Japan) 279–291 (2000). with 2 × 2 pixel binning. Metamorph software (Universal Imaging) was used 14. Murata-Hori, M. & Wang, Y. L. The kinase activity of aurora B is required for kineto- for acquisition and analysis. Maximal intensity projections of GFP–tubulin z- chore–microtubule interactions during mitosis. Curr. Biol. 12, 894–899 (2002). stacks and single DIC images are shown for selected timepoints. A 2 × 2 low- 15. Kallio, M. J., McCleland, M. L., Stukenberg, P. T. & Gorbsky, G. J. Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and per- pass filter was applied to all images for presentation. turbs microtubule dynamics in mitosis. Curr. Biol. 12, 900–905 (2002). 16. Mayer, T. U. et al. Small molecule inhibitor of mitotic spindle bipolarity identified in Note: Supplementary Information is available on the Nature Cell Biology website. a phenotype-based screen. Science 286, 971–974 (1999). 17. Kapoor, T. M., Mayer, T. U., Coughlin, M. L. & Mitchison, T. J. Probing spindle assem- ACKNOWLEDGEMENTS bly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, We acknowledge A. North and The Rockefeller University Bioimaging Facility. We Eg5. J. Cell Biol. 150, 975–988 (2000). thank W. Brinkley for the generous gift of CREST antiserum. This work was 18. Khodjakov, A., Copenagle, L., Gordon, M. B., Compton, D. A. & Kapoor, T. M. Minus- supported by National Institutes of Health grants GM65933 (T.M.K.) and end capture of preformed kinetochore fibers contributes to spindle morphogenesis. J. GM59363 (A.K.). M.A.L. is a Goelet fellow. Cell Biol. 160, 671–683 (2003). 19. Rieder, C. L. & Alexander, S. P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. COMPETING FINANCIAL INTERESTS J. Cell Biol. 110, 81–95 (1990). The authors declare that they have no competing financial interests. 20. Skibbens, R. V., Skeen, V. P. & Salmon, E. D. Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a Received 19 December 2003; accepted 13 January 2004 push-pull mechanism. J. Cell Biol. 122, 859–875 (1993). Published online at http://www.nature.com/naturecellbiology. 21. Glover, D. M., Leibowitz, M. H., McLean, D. A. & Parry, H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81, 1. Giet, R. & Prigent, C. Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic 95–105 (1995). serine-threonine kinases. J. Cell Sci. 112, 3591–3601 (1999). 22. Hannak, E., Kirkham, M., Hyman, A. A. & Oegema, K. Aurora-A kinase is required for cen- 2. Biggins, S. et al. The conserved protein kinase Ipl1 regulates microtubule binding to trosome maturation in Caenorhabditis elegans. J. Cell Biol. 155, 1109–1116 (2001). kinetochores in budding yeast. Genes Dev 13, 532–544 (1999). 23. Roghi, C. et al. The Xenopus protein kinase pEg2 associates with the centrosome in a 3. Hauf, S. et al. The small molecule Hesperadin reveals a role for Aurora B in correcting cell cycle-dependent manner, binds to the spindle microtubules and is involved in kinetochore-microtubule attachment and in maintaining the spindle assembly check- bipolar mitotic spindle assembly. J. Cell Sci. 111, 557–572 (1998). point. J. Cell Biol. 161, 281–294 (2003). 24. Murata-Hori, M., Tatsuka, M. & Wang, Y. L. Probing the dynamics and functions of 4. Tanaka, T. U. et al. Evidence that the Ipl1–Sli15 (Aurora kinase–INCENP) complex aurora B kinase in living cells during mitosis and cytokinesis. Mol. Biol. Cell 13, promotes chromosome bi-orientation by altering kinetochore-spindle pole connec- 1099–1108 (2002). tions. Cell 108, 317–329 (2002). 25. Li, X. & Nicklas, R. B. Mitotic forces control a cell-cycle checkpoint. Nature 373, 5. Nicklas, R. B. How cells get the right chromosomes. Science 275, 632–637 (1997). 630–632 (1995). 6. Ault, J. G. & Rieder, C. L. Chromosome mal-orientation and reorientation during mito- 26. Biggins, S. & Murray, A. W. The budding yeast protein kinase Ipl1/Aurora allows the sis. Cell Motil. Cytoskel. 22, 155–159 (1992). absence of tension to activate the spindle checkpoint. Genes Dev. 15, 3118–3129 7. Nicklas, R. B. & Ward, S. C. Elements of error correction in mitosis: microtubule cap- (2001). ture, release, and tension. J. Cell Biol. 126, 1241–1253 (1994). 27. Waters, J. C., Chen, R. H., Murray, A. W. & Salmon, E. D. Localization of Mad2 to 8. Rieder, C. L. & Salmon, E. D. The vertebrate cell kinetochore and its roles during kinetochores depends on microtubule attachment, not tension. J. Cell Biol. 141, mitosis. Trends Cell Biol. 8, 310–318 (1998). 1181–1191 (1998). 9. Walter, R. et al. in PCT Int. Appl. 112 ((Boehringer Ingelheim Pharma K.-G., 28. Cheeseman, I. M. et al. Phospho-regulation of kinetochore-microtubule attachments Germany, WO, 2002). by the Aurora kinase Ipl1p. Cell 111, 163–172 (2002). 10. Mortlock, A. A. & Jung, F. H. in PCT Int. Appl. 62 ((Astrazeneca AB, Sweden; 29. McEwen, B. F., Heagle, A. B., Cassels, G. O., Buttle, K. F. & Rieder, C. L. Astrazeneca UK Limited, WO, 2001). Kinetochore fiber maturation in PtK1 cells and its implications for the mechanisms of 11. Straight, A. F. et al. Dissecting temporal and spatial control of cytokinesis with a chromosome congression and anaphase onset. J. Cell Biol. 137, 1567–1580 (1997). myosin II Inhibitor. Science 299, 1743–1747 (2003). 30. Cleveland, D. W., Mao, Y. & Sullivan, K. F. Centromeres and kinetochores: from epige- 12. Ditchfield, C. et al. Aurora B couples chromosome alignment with anaphase by targeting netics to mitotic checkpoint signaling. Cell 112, 407–421 (2003).

1 1

2 2

Figure S2. Kinetochore staining demonstrates syntelic chromosome mal- orientations upon inhibition of Aurora kinase. Spindles bipolarized as cells recovered from monastrol treatment. After monastrol removal, cells were incubated 1 hr with 100 nM hesperadin and processed for immunofluorescence. Optical sections for boxed regions (1 and 2) highlight two chromosomes with syntelic mal-orientations. MG132 was included with hesperadin to prevent mitotic exit. Tubulin (green), DNA (blue), CREST (red) labels kinetochores. Scale bars 3 µm. Figure S1. Chemical structures of Aurora kinase inhibitors.

WWW.NATURE.COM/NATURECELLBIOLOGY 1

abc

10:00 20:01 33:54

d e f

Figure S3. Syntelic attachments are persistent in the absence of MG132, if and tubulin dynamics. As expected, after 30 minutes cells enter anaphase Aurora kinase is inhibited. Spindles bipolarized as cells recovered from with improper chromosome attachments due to inhibition of Aurora kinase. monastrol treatment, in the presence of an Aurora kinase inhibitor. Arrows Scale bar 5 µm. show persistent syntelic attachments in live-cell recordings of chromosome

Movie 1. Movie corresponds to images shown in Fig 2c-h. Timestamp (min:s) starts ~5 min after monastrol washout. GFP-tubulin (a), DIC (b). Movie 2. Movie corresponds to images shown in Fig 3b-r. Timestamp (min:s) starts ~5 min after hesperadin washout. GFP-tubulin (a), DIC (b). Movie 3. Movie corresponds to images shown in Fig 3u-y. Timestamp (min:s) starts ~5 min after hesperadin washout. GFP-tubulin (a), DIC (b).

2 WWW.NATURE.COM/NATURECELLBIOLOGY