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CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment

LOGARINHO, Elsa, et al.

Abstract

Loss of spindle-pole integrity during mitosis leads to multipolarity independent of centrosome amplification. Multipolar-spindle conformation favours incorrect kinetochore-microtubule attachments, compromising faithful chromosome segregation and daughter-cell viability. Spindle-pole organization influences and is influenced by kinetochore activity, but the molecular nature behind this critical force balance is unknown. CLASPs are microtubule-, kinetochore- and centrosome-associated proteins whose functional perturbation leads to three main spindle abnormalities: monopolarity, short spindles and multipolarity. The first two reflect a role at the kinetochore-microtubule interface through interaction with specific kinetochore partners, but how CLASPs prevent spindle multipolarity remains unclear. Here we found that human CLASPs ensure spindle-pole integrity after bipolarization in response to CENP-E- and Kid-mediated forces from misaligned chromosomes. This function is independent of end-on kinetochore-microtubule attachments and involves the recruitment of ninein to residual pericentriolar satellites. Distinctively, [...]

Reference

LOGARINHO, Elsa, et al. CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment. Nature Cell Biology, 2012, vol. 14, no. 3, p. 295-303

DOI : 10.1038/ncb2423 PMID : 22307330

Available at: http://archive-ouverte.unige.ch/unige:28850

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CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment Elsa Logarinho1, Stefano Maffini1,4, Marin Barisic1, Andrea Marques1,4, Alberto Toso2,4, Patrick Meraldi2 and Helder Maiato1,3,5

Loss of spindle-pole integrity during mitosis leads to mechanism is related to multipolar-spindle formation independent multipolarity independent of centrosome amplification1–4. of centrosome amplification, for example due to premature centriole Multipolar-spindle conformation favours incorrect disengagement or loss of spindle-pole integrity1–4,15. To investigate the kinetochore–microtubule attachments, compromising faithful mechanism by which CLASPs (cytoplasmic linker-associated proteins) chromosome segregation and daughter-cell viability5,6. prevent spindle multipolarity in human cells, we used HeLa cells stably Spindle-pole organization influences and is influenced by expressing the centriole marker centrin–GFP and immunodetection kinetochore activity7,8, but the molecular nature behind this of γ-tubulin to determine the number of centrioles in each individual critical force balance is unknown. CLASPs are microtubule-, pole on CLASP1/2 depletion by RNAi. As positive controls, we used kinetochore- and centrosome-associated proteins whose cells treated with either 2 µM cytochalasin D, an inhibitor of actin functional perturbation leads to three main spindle polymerization and cytokinesis; astrin (also known as SPAG5, sperm- abnormalities: monopolarity, short spindles and associated antigen 5) short interfering RNA (siRNA), which leads to multipolarity9–13. The first two reflect a role at the premature centriole disengagement15; or TOGp (also known as CKAP5, kinetochore–microtubule interface through interaction with cytoskeleton-associated protein 5) siRNA, which perturbs spindle-pole specific kinetochore partners10,11,14, but how CLASPs prevent integrity2,3 (Fig. 1a–c and Supplementary Fig. S1a–d). All of these spindle multipolarity remains unclear. Here we found that treatments caused a significant increase in the percentage of mitotic cells human CLASPs ensure spindle-pole integrity after with multipolar spindles (Fig. 1a). Simultaneous depletion of CLASP1 bipolarization in response to CENP-E- and Kid-mediated forces and CLASP2 (Supplementary Fig. S1b) resulted in 17.3 ± 4.1% of from misaligned chromosomes. This function is independent of mitotic cells with multipolar spindles, whereas individual depletion of end-on kinetochore–microtubule attachments and involves the CLASP1 or CLASP2 caused, respectively, 6.3±0.9% and 5.1±0.2% of recruitment of ninein to residual pericentriolar satellites. multipolar mitosis (Fig. 1a). These frequencies are significantly higher Distinctively, multipolarity arising through this mechanism than in control cells (∼1%; Fig. 1a), indicating that the two human often persists through anaphase. We propose that CLASPs and CLASPs cooperate to prevent spindle multipolarity. Interestingly, ninein confer spindle-pole resistance to traction forces exerted 29.9 ± 9.5% of poles in CLASP1/2-depleted multipolar cells were during chromosome congression, thereby preventing acentriolar and 20.7 ± 8.5% had a single centriole, a phenotypic irreversible spindle multipolarity and aneuploidy. distribution that was distinct from that of cytochalasin D treatment or astrin RNAi, but resembled that of TOGp RNAi (Fig. 1b,c). However, Multipolar spindles are a hallmark of tumour cells and may arise CLASP1/2 depletion did not affect TOGp localization at spindle poles owing to supernumerary centrosomes resulting from centrosome (Supplementary Fig. S2), indicating that the observed multipolarity is overduplication or cytokinesis failure. However, multipolar mitosis independent of TOGp and is primarily caused by loss of spindle-pole is usually incompatible with cell viability and normally assumes a integrity and, to a lesser extent, due to centriole disengagement. transient nature due to the coalescence of extra centrosomes into We have previously identified astrin as part of a CLASP1 complex two functional spindle poles5,6. An alternative but less understood involved in the regulation of kinetochore–microtubule dynamics11,14.

1Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal. 2Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland. 3Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, 4200-319 Porto, Portugal. 4Present addresses: Max Planck Institute of Molecular Physiology, 44202 Dortmund, Germany (S.M.); Cell Biology, Faculty of Science, Utrecht University, 3584-CH Utrecht, The Netherlands (A.M.); Institute of Oncology Research, CH-6500 Bellinzona, Switzerland (A.T.). 5Correspondence should be addressed to H.M. (e-mail: [email protected])

Received 30 August 2011; accepted 15 December 2011; published online 5 February 2012; DOI: 10.1038/ncb2423

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a b 50 100 01 234 45 40 centrioles 80 35 n 30 60 25 20 40 15 10 20 5 Percentage of multipolar cells 0 0 Cytoch D Astrin TOGp CLASP1/2 d Percentage of poles with RNAi2 RNAi RNAi RNAi RNAi Luc RNAi Untreate TOGp Astrin RNAi CLASP1CLASP2 CytochalasinCLASP1/ D

c α-tubulin γ-tubulin Centrin–GFP DAPI α-tubulin γ-tubulin Centrin–GFP

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Figure 1 CLASP1/2 depletion causes the formation of multipolar spindles. siRNAs against astrin, TOGp and CLASPs (mean ± s.d.; n = 30 cells). HeLa cells stably expressing the centriole marker centrin–GFP were (c) Examples of cells with multipolar spindles most typically found for either treated with 2 µM cytochalasin D or transfected with the indicated each treatment. Insets are magniﬁcations of the centrin–GFP signal at the speciﬁc siRNA, and then stained for α-tubulin, γ-tubulin and DNA. indicated poles. Note that the possibility of centrosome overduplication was (a) Percentage of mitotic cells with multipolar spindles (mean ± s.d.; discarded because the frequency of CLASP1/2-depleted mononucleated n = 1,000 cells). Luciferase (Luc) RNAi was used as a negative control. cells entering mitosis with more than two centrosomes was less than (b) Percentage of poles with n centrioles found in cells with multipolar 1% (our unpublished observations). Quantitative data are means of three spindles generated by treatment with cytochalasin D (Cytoch D) or independent experiments. Scale bars, 5 µm.

To determine whether the eventual centriole disengagement observed siRNAs. We found that ∼70% of CLASP1/2-depleted cells showed in CLASP1/2-depleted multipolar spindles arises from premature normal sister-chromatid cohesion, corresponding to 5- and 15-fold the activation of separase as seen on astrin depletion15, we quantified respective percentages in astrin- or Sgo1-depleted cells (Supplementary loss of sister-chromatid cohesion in chromosome spreads from cells Fig. S3a,b). Moreover, separase depletion, which rescues spindle transfected with control, SGOL1 (which encodes a centromeric protein bipolarity on astrin RNAi (ref. 15), had no effect in the observed involved in sister-chromatid cohesion, Sgo1)16, astrin or CLASP1/2 multipolarity after CLASP1/2 depletion (Supplementary Fig. S3c).

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a d NEB Anaphase onset 1,000 1 1 1 1 1 1 1 1 2 2 2 2 100 Control 2 2 2 0:00 0:16 2 0:36 0:42

b Time (min) NEB 1 1 Pole fragmentation1 1 10 1 2 2 1 3 2 2 RNAi 1 3 3 3 1 1 2 2 2 2

CLASP1/2 RNAi 2 Control 0:00 0:17 1:06 3:06 CLASP1/2RNAi BPCLASP1/ Control + c RNAi MPMps1-IN-1 CLASP1/2RNAi MP NEB 1 1 Pole fragmentation1 1 + Mps1-IN-1 CLASP1/2 1 2 2 2 2 NEB–ANA NEB–MP RNAi 1 3 1 3 2 3 e 3 50 1 2 2 2

CLASP1/2 40 0:00 0:16 0:46 1:06 30 f RNAi Mock CLASP1/2 Nuf2 CLASP1/2 + Nuf2 20

Percentage of multipolar cells 0 -tubulin 2 α Nuf2 Mock Nuf2 CLASP1/2 CLASP1/+ Centrin–GFP + Hec1 RNAi DAPI

Figure 2 CLASPs ensure spindle-pole integrity independently of forces (n = 12 cells) with Mps1 inhibitor (Mps1-IN-1). Time from NEB to relying on end-on kinetochore–microtubule attachments. (a) HeLa cells multipolarity (NEB–MP) in CLASP1/2-RNAi cells was also measured. The stably co-expressing EGFP–centrin-2 and α-tubulin–mRFP were either mean is shown as horizontal bars and variability within the population mock transfected (control) or RNAi transfected (CLASP1/2 RNAi) for 72 h. is provided in the main text as s.d. (e) Percentages of mitotic cells with (b,c) Following CLASP1/2 depletion, spindles shorten and multipolarity multipolar spindles in the indicated RNAi depletions (mean±s.d.; three arises as a consequence of pole fragmentation without (b) or with centriole independent experiments). (f) Centrin–GFP HeLa cells were transfected disengagement (c). For each cell, magniﬁcations of the centrioles in the with control, CLASP1/2, NUF2 and CLASP 1/2 + NUF 2 siRNAs, and indicated poles are shown after contrast inversion of the EGFP–centrin-2 immunostained for α-tubulin and Hec1. Hec1 detection was used to signal (insets). Arrowheads indicate newly formed poles. Time, h:min. quantify NUF2 depletion because the stability of Hec1, a subunit of (d) Quantitative analysis of the time between NEB and anaphase onset the Ndc80 complex, depends on NUF2. Absence of end-on attachments (NEB–ANA) in control (n = 19 cells) and CLASP1/2-RNAi cells (BP, induced by NUF2 RNAi exacerbated spindle multipolarity on CLASP1/2 bipolar; MP, multipolar; n = 64 cells), untreated (n = 9 cells) or treated RNAi. Scale bars, 5 µm.

Finally, the recruitment of astrin to spindle poles was unaffected in (NEB to multipolarity: 80.9 ± 55.7 min; Fig. 2b–d). A significant CLASP1/2-depleted cells (Supplementary Fig. S2). These data indicate fraction of CLASP1/2-depleted multipolar cells (8/34) ended up that loss of spindle pole integrity and centriole disengagement observed satisfying the mitotic checkpoint and underwent a multipolar anaphase in CLASP1/2-depleted cells is independent of astrin and premature during our recordings (NEB to anaphase: 333.1±113.8 min; Fig. 2d; separase activation. Supplementary Movie S2). Inspection of spindle poles in CLASP1/2- To directly correlate centriole and mitotic spindle behaviour in depleted cells as they become multipolar revealed that in ∼70% CLASP1/2-depleted cells, we carried out live imaging of HeLa cells of the cases the newly formed poles were acentriolar and derived stably co-expressing EGFP–centrin-2 and α-tubulin–mRFP. Control from fragmentation of pre-existing poles (Fig. 2b and Supplementary cells assembled a bipolar spindle and progressed from nuclear envelope Movie S2). In the other 30%, there was centriole disengagement breakdown (NEB) into anaphase in 33.1 ± 11.3 min (Fig. 2a,d and leading to the formation of two mono-centriolar poles (Fig. 2c and Supplementary Movie S1). CLASP1/2-depleted cells established short Supplementary Movie S3). Importantly, all multipolar spindles in bipolar spindles, with 50% entering anaphase in 96.7 ± 75.8 min CLASP1/2-depleted cells arose after the establishment of a bipolar and the other 50% becoming multipolar after a variable delay spindle and their maintenance required pushing forces mediated by

LETTERS the kinesin-5 Eg5 (Supplementary Fig. S4 and Movies S4 and S5). The To directly investigate how a delay in mitotic-checkpoint satisfaction observation that 50% of CLASP1/2-depleted cells with short spindles leads to loss of spindle-pole integrity on CLASP1/2 depletion, ultimately become multipolar indicates that the relatively moderate we switched to HeLa cells stably co-expressing H2B-histone–GFP levels of multipolarity in fixed cells is an underestimation of the real and α-tubulin–mRFP to simultaneously monitor chromosome penetrance of this phenotype. and spindle behaviour. Whereas control cells rapidly completed It was recently reported that extensive metaphase delay causes chromosome congression and entered anaphase shortly thereafter ‘cohesion fatigue’, leading to precocious sister-chromatid separation, (Fig. 3a), CLASP1/2-depleted cells showed few chromosomes that were followed by centriole disengagement and spindle multipolarity in unable to congress and/or stabilize their position at the metaphase plate a separase-independent manner17,18. To rule out the possibility and were significantly delayed in mitosis (Fig. 3b and Supplementary that multipolarity in CLASP1/2-depleted cells was mainly due to Movie S6). Inspection of recorded cells assisted by four-dimensional a metaphase delay, we treated control cells with the proteasome reconstructions revealed that in 8/10 cells, loss of spindle-pole integrity inhibitor MG132 for 3 h, which blocks anaphase onset without on CLASP1/2 depletion was preceded by the presence of misaligned preventing mitotic-checkpoint satisfaction. No increase in the chromosomes along the vector of spindle-pole fragmentation (Fig. 3c normal number of cells with multipolar spindles was observed and Supplementary Movie S7). Immunofluorescence microscopy (our unpublished observations). Although we cannot exclude that analysis further revealed that misaligned chromosomes on CLASP1/2 cumulative events due to CLASP1/2 depletion result in some cohesion depletion showed at least one MAD2-positive kinetochore (Fig. 3d). fatigue (see Supplementary Fig. S3a,b), the initial spindle-pole Chromosome congression in human cells relies on the combined fragmentation events in CLASP1/2 RNAi occur at least 1.5 h before action of chromokinesins, such as Kid (also known as kinesin family the first manifestations of centriole disengagement and consequent member 22, KIF22; a kinesin-10), which generate polar ejection multipolarity normally associated with cohesion fatigue17,18. Although forces that push chromosome arms away from spindle poles20,22,23, not sufficient, a delay in mitotic-checkpoint satisfaction might be and the plus-end-directed motor CENP-E (centromere-associated necessary to cause loss of spindle-pole integrity in CLASP1/2-depleted protein E; a kinesin-7) at unattached kinetochores, which slides cells. To investigate this, we filmed CLASP1/2-depleted cells in the misaligned chromosomes along pre-existing spindle microtubules24,25. presence of an Mps1 inhibitor necessary for mitotic-checkpoint Both activities eventually contribute to microtubule capture from activity19. Under these conditions, all control and CLASP1/2-RNAi the opposing pole and consequently to chromosome bi-orientation. cells entered anaphase with bipolar spindles after 11.7 ± 2.4 min CENP-E exists in a complex with CLASPs during mitosis and is and 14.6 ± 4.7 min, respectively (Fig. 2d and Supplementary Fig. required for their proper recruitment to kinetochores11. However, S5a), indicating that spindle multipolarity observed on CLASP1/2 CENP-E is still normally targeted to kinetochores in the absence of depletion is associated with conditions that prevent mitotic-checkpoint CLASPs (ref. 11). To investigate whether loss of spindle-pole integrity satisfaction (presumably due to unattached kinetochores). on CLASP1/2 depletion was due to CENP-E- and/or Kid-mediated We next investigated whether loss of spindle-pole integrity on forces at kinetochores/chromosome arms, we co-depleted CLASPs CLASP1/2 depletion was related to a role in kinetochore–microtubule with CENP-E or Kid by RNAi. We observed that virtually all dynamics11. First, we perturbed end-on kinetochore–microtubule CLASP1/2- and CENP-E-co-depleted cells showed bipolar spindles, attachments by co-depleting CLASPs with NUF2 (ref. 8). Surprisingly, representing a 90% reduction in spindle multipolarity (Fig. 4a,b ∼20% of NUF2-depleted cells assembled multipolar spindles (Fig. 2e,f and Supplementary Fig. S1f). CLASP1/2 and Kid co-depletion also and Supplementary Fig. S1e), which was probably due to cohesion significantly reverted spindle multipolarity, albeit only in ∼50% fatigue17,18, because extensive sister-chromatid separation was observed of the cases (Fig. 4b). These results indicate that, in coordination in some, but not all, chromosomes (Supplementary Fig. S3a,b) with Kid-mediated forces on chromosome arms, CENP-E-mediated and the finding that even laterally attached kinetochores can forces at kinetochores are responsible for the observed spindle be significantly stretched20. Importantly, co-depletion of CLASPs multipolarity on CLASP1/2 depletion. This is in line with recent and NUF2 synergistically exacerbated the spindle-multipolarity data showing that Kid favours lateral kinetochore–microtubule phenotype (Fig. 2e,f and Supplementary Fig. S1e), indicating that attachments before chromosome bi-orientation20. To ascertain the the role of CLASPs in the maintenance of spindle-pole integrity is nature of the specific interplay between CLASP1/2 and CENP-E in independent of end-on kinetochore–microtubule attachments11,14 and spindle architecture, we co-depleted CENP-E with astrin or TOGp. that the multipolarity observed on CLASP1/2 and NUF2 individual Surprising, CENP-E depletion significantly rescued multipolarity depletions arises through independent mechanisms. Finally, as of astrin-depleted, but not TOGp-depleted, cells (Fig. 4c). Thus, CLASP1/2 depletion increases kinetochore-microtubule stability11, we CLASPs are not the only proteins required for spindle-pole resistance investigated whether this could explain the observed multipolarity. To to CENP-E-mediated forces. However, spindle multipolarity was do so, we hyperstabilized kinetochore microtubules with the aurora aggravated on CLASP1/2 co-depletion with astrin or TOGp, indicating B inhibitor ZM447439 (ref. 21), which in control cells recovering that these proteins affect spindle-pole integrity through cumulative from Eg5 inhibition always led to permanent bipolar spindles (8/8), mechanisms (Fig. 4c). but did not prevent multipolarization of CLASP1/2-depleted bipolar To distinguish between the role of CENP-E-mediated forces spindles (7/8; Supplementary Fig. S5b). Thus, spindle multipolarity on congressing chromosomes and other possible motor-dependent associated with CLASP1/2 depletion is not related to their impact activities of CENP-E (for example, promotion of microtubule plus-end on kinetochore–microtubule dynamics and is independent of end-on elongation associated with spindle microtubule flux26) as the cause of kinetochore–microtubule attachments. spindle multipolarity in CLASP1/2-depleted cells, we used a reversible

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* * CLASP1/2 0:00 0:10 4:16 4:28 4:48 c

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y y y y x y x x x x z 1:17:30z 1:20:00 z z 1:22:30z 1:25:00 d ∗ CLASP1/2 RNAi ∗ ∗ ∗ ∗ ∗ ∗

Four-dimensional reconstruction ∗ ∗ x x x y y y DAPI y x α-tub z z 1:27:30 z 1:27:30 1:30:00 z 1:32:30 MAD2

Figure 3 Loss of spindle-pole integrity in the absence of CLASPs is preceded pole fragmentation occurred (asterisks). (NEB-to-anaphase in CLASP1/2 by the presence of misaligned chromosomes with unattached kinetochores. RNAi: 310.4±214.4 min; n = 13 cells.) Values indicate mean±s.d.. Three- Live imaging of control and CLASP1/2-RNAi-depleted HeLa cells stably dimensional-reconstruction image panels of the critical time frames where co-expressing H2B-histone–GFP/ α-tubulin–mRFP. (a) Control cells rapidly the presence of misaligned chromosomes preceded the pole fragmentation aligned all chromosomes at the metaphase plate (17.7 ± 2.5 min) and event are outlined in red. Time, h:min and h:min:s. (d) CLASP1/2-RNAi cells progressed normally into anaphase (NEB-to-anaphase: 33.7 ± 1.8 min; were immunostained for α-tubulin (α-tub), mitotic-checkpoint protein MAD2 n = 3 cells). (b,c) CLASP1/2-depleted cells typically exhibited a few and DNA (DAPI). Misaligned chromosomes in CLASP1/2-RNAi cells have at misaligned chromosomes (arrowheads) for a variable amount of time until least one MAD2-positive kinetochore. Scale bars, 5 µm. allosteric inhibitor of CENP-E, GSK-923295, which allows temporal mEos–α-tubulin to measure spindle microtubule flux in cells treated control of CENP-E motor activity27. First, we used GSK-923295 with GSK-923295, and found no difference relative to control cells in a series of inhibition–washout experiments in living cells stably (Supplementary Fig. S5c,d). This directly demonstrates that CENP-E co-expressing H2B-histone–GFP and α-tubulin–mRFP. All control ATPase/motor activity is not required for flux. Together, these data and more than 80% of CLASP1/2-depleted cells treated with 20 nM indicate that CENP-E-mediated traction forces during chromosome GSK-923295 remained bipolar with few misaligned chromosomes for congression are responsible for the irreversible loss of spindle-pole more than 2 h from NEB (Fig. 4d). However, whereas all control cells integrity and multipolarity in CLASP1/2-depleted cells. resumed congression and entered anaphase with bipolar spindles soon Spindle-pole integrity is ensured by motor proteins of the after drug washout, 50% of CLASP1/2-depleted cells became multipolar kinesin-14 and dynein families28 that work in concert with within 1 h (Fig. 4d and Supplementary Movies S8 and S9). Interestingly, stabilizing proteins, such as TOGp and Tpx2 (refs 2,3,29), to focus addition–washout of GSK-923295 to CLASP1/2-depleted cells that microtubule minus ends at spindle poles. However, the spindle- had already lost spindle-pole integrity did not revert multipolarity pole localization of all these proteins was unaffected in CLASP1/2- (5/5), highlighting the irreversible nature of the process (Fig. 4b,d depleted cells (Supplementary Fig. S2), indicative of an alternative and Supplementary Movie S10). Next, we used photo-conversion of mechanism. We have previously identified the core centrosomal

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a Centrin–GFP Centrin–GFP + α-tub DAPI + α-tubulin CENP-E CLASP1 CENP-E CLASP1

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RNAi Astrin + CENP- CLASPCLASP1/2 + + CE IN c 60 50 CENPE 40 30 20 CENPE Percentage of

multipolar cells 10 + 0 2 p RNAi: G Mock Astrin TOGp + astrin + TO CLASP1/ P1/2

CLASP1/2 S LA CLASP1/2 C d + CENP-E inhibitor Washout

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∗ ∗ Mock RNAi 0:00 1:00 2:08 2:45 3:10 NEB ∗ ∗ ∗ RNAi

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CLASP1/2 0:02 1:00 2:10 2:28 3:00 Multipolar ∗ ∗ ∗ ∗ ∗ RNAi ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 0:00 1:00 1:50 2:15 2:40 CLASP1/2

Figure 4 CENP-E-mediated traction forces on misaligned chromosomes of CLASPs with astrin or TOGp. Quantitative data in b and c are are responsible for the irreversible loss of spindle-pole integrity mean ± s.d. of three independent experiments. (d) Live imaging of in CLASP1/2-depleted cells. (a) HeLa cells stably expressing control (n = 6) and CLASP1/2-depleted HeLa cells (n = 32) stably centrin–GFP were transfected with the indicated siRNA and stained co-expressing H2B-histone–GFP/α-tubulin–mRFP following addition of for α-tubulin (α-tub), CENP-E and CLASP1 72 h later. Most CENP-E inhibitor. Two hours after NEB, the drug was washed out and CLASP1/2 + CENP-E-depleted cells were bipolar. Note that CENP-E cells were imaged for another 1 h. Most CLASP1/2-depleted cells became localizes normally at kinetochores of CLASP1/2-depleted cells. multipolar only after CENP-E inhibitor washout. Addition of the drug did (b) Quantiﬁcation of spindle multipolarity and the effect of CENP-E not rescue bipolarity in cells that were already multipolar at the time of depletion/inhibition in different experimental conditions. CE IN, CENP-E drug addition. The asterisks indicate the position and number of spindle inhibitor. (c) Quantiﬁcation of spindle multipolarity on co-depletion poles. Scale bars, 5 µm. Time, h:min. proteins CPAP and ninein as CLASP1-interacting proteins during lowest accumulation of ninein on CLASP1/2 RNAi (Fig. 5a,b and mitosis11. CPAP and ninein have well-established roles in centriole Supplementary Fig. S6a,b). In agreement, even a partial depletion duplication and microtubule anchorage/nucleation at the centrosome of ninein by RNAi (Supplementary Fig. S1g) caused an increase during interphase, respectively30,31, but their functional relationship in the percentage of cells with multipolar spindles, which showed with CLASPs at mitotic centrosomes was unclear. We found a high number of poles with zero or one centriole (17.4 ± 6.7% CPAP centrosomal localization unaffected in CLASP1/2-depleted and 29.8 ± 12.2%, respectively; Fig. 5c,d). Moreover, we found cells (Supplementary Fig. S2), indicating that CLASPs may work that CLASP1/2 localization at centrosomes is unaffected by ninein downstream of CPAP to ensure spindle-pole integrity. On the other depletion, indicating that ninein may act downstream of CLASPs hand, we observed a strong correlation between CLASP1/2 and ninein (Supplementary Fig. S6c). The observation that CLASP1/2 and ninein levels at mitotic centrosomes, with multipolar spindles showing the co-depletion did not exacerbate spindle multipolarity relative to

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a Control bipolar 3 CLASP1/2 RNAi DAPI α-tubulin Centrin–GFP 5 Ninein

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30 Percentage of poles 0 CLASP1/2 30 100 7 RNAi: Ninein CLASP1/2 signal intensity DAPI 8 Centrin–GFP α 7 8 e -tubulin 25 f DAPINinein PCM-1 Ninein + PCM-1 20 15 10 5 Percentage of multipolar cells 0 2 RNAi: ock inein P-E M N SP1/ LA C

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Ninein n + 1 CENP-E Kid Eg5 CLASPs Ninein

Figure 5 CLASPs ensure spindle-pole integrity through a functional exacerbated following double depletion of CLASPs and ninein, and relationship with ninein. (a) Centrin–GFP HeLa cells were mock or multipolarity is reduced to control levels in ninein + CENPE RNAi. CLASP1/2-RNAi transfected and immunostained for α-tubulin, ninein Quantitative data in d and e are means ± s.d. of three independent and DNA. Insets are magnifications of the ninein signal at the indicated experiments. (f) Ninein co-localizes with the pericentriolar satellite poles. In control cells, ninein localizes at mitotic centrosomes (inset 1). marker PCM-1 at mitotic spindle poles. Cells were double stained with In CLASP1/2-RNAi cells with multipolar spindles, ninein is extensively mouse anti-ninein and rabbit anti-PCM-1 antibodies. Insets show a delocalized from the centrosomes (insets 4 and 5). Note that ninein higher magnification of the spindle-pole region, and those in the last localizes normally in the few control cells that sporadically form panel at the right highlight co-localization (white mask). Scale bars, multipolar spindles (inset 2), as well as in cells inefficiently depleted by 5 µm. (g) Proposed model for the role of CLASPs at spindle poles CLASP1/2 RNAi (inset 6). CLASP1/2 RNAi does not interfere with ninein in response to traction forces during chromosome alignment to the localization in interphase centrosomes (inset 3). (b) Graph representing metaphase plate. CLASPs provide a scaffolding platform at spindle poles the correlation between CLASP1/2 signal intensity at the poles, ninein by recruiting ninein to residual pericentriolar satellites. Perturbation of signal intensity and number of poles per cell. Multipolar spindles are CLASP1/2 function causes bipolar spindles to lose spindle-pole integrity mostly associated with low levels of both proteins. (c) Ninein RNAi in response to CENP-E- and Kid-mediated kinetochore traction forces increases the frequency of multipolar spindles. Insets are magnifications during congression of mono-oriented chromosomes, leading to multipolar of the centrioles in the indicated poles. (d) Similarly to CLASP1/2 RNAi, spindle formation independent of centrosome amplification. This spindle ninein RNAi results in multipolar spindles with a high percentage of conformation is not transient and cells normally enter anaphase, thus poles with 0 and 1 centrioles. (e) The multipolarity phenotype is not leading to aneuploidy.

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CLASP1/2 depletion and that multipolarity in ninein RNAi was provided invaluable reagents. E.L. is supported by Programa Ciência funded extensively reverted by co-depletion with CENP-E (Fig. 5e) further by Programa Operacional Potencial Humano (POPH)/QREN, as well as grant PTDC/SAU-OBD/100261/2008 from Fundação para a Ciência e a Tecnologia of supports this conclusion. Finally, we mapped ninein localization on Portugal (COMPETE-FEDER). S.M. held a fellowship from the Fundação para a mitotic centrosomes and found that it extensively co-localizes (Pearson Ciência e a Tecnologia (FCT) of Portugal (SFRH/BPD/26780/2006). P.M. holds correlation = 0.89) with the pericentriolar satellite marker PCM-1 an SNF-Professorship and a EURYI award. Work in the laboratory of H.M. is financially supported by grants PTDC/SAU-GMG/099704/2008 and PTDC/SAU- (Fig. 5f; ref. 32). Overall, these data support that CLASPs ensure ONC/112917/2009 from Fundação para a Ciência e a Tecnologia of Portugal spindle-pole integrity by recruiting ninein to residual pericentriolar (COMPETE-FEDER), the Human Frontier Research Program and the seventh satellites during mitosis. framework programme grant PRECISE from the European Research Council. Molecular perturbations that lead to loss of spindle-pole integrity are AUTHOR CONTRIBUTIONS normally associated with misaligned chromosomes and a significant E.L. designed, carried out and analysed most experiments. S.M. and A.M. carried mitotic delay2,3,7,15,29,33–36. The findings reported in this work assign out initial phenotypic characterization of CLASP1/2 RNAi. M.B. carried out flux measurements. A.T. and P.M. provided the EGFP–centrin-2/α-tubulin–mRFP to multiple forces required for normal chromosome alignment an stable HeLa cell line. H.M. designed experiments, analysed the data, coordinated active role as the cause for the loss of spindle-pole integrity on certain the work and wrote the manuscript with contributions from E.L. and S.M. pole perturbations (Fig. 5g). Interestingly, a similar force balance COMPETING FINANCIAL INTERESTS between CENP-E and spindle poles has recently been reported on The authors declare no competing financial interests. disruption of the Ndc80 complex37, but it remained unclear how this Published online at http://www.nature.com/naturecellbiology perturbation sensitizes spindle poles. Our data indicate that, contrary Reprints and permissions information is available online at http://www.nature.com/ to loss of CLASP1/2 function at spindle poles, this is probably due to a reprints prolonged mitotic delay associated with cohesion fatigue. Furthermore, 1. Brenner, S., Branch, A., Meredith, S. & Berns, M. W. The absence of centrioles from our work implicates CLASPs in the recruitment of ninein to residual spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division pericentriolar satellites during mitosis. Pericentriolar satellites are in vitro. J. Cell Biol. 72, 368–379 (1977). electron-dense granules at the centrosome periphery involved in 2. Gergely, F., Draviam, V. M. & Raff, J. W. The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells. Genes Dev. 17, the recruitment of centrosomal proteins, including ninein, during 336–341 (2003). interphase32,38, but their roles in mitosis remained mysterious. Recently, 3. Cassimeris, L. & Morabito, J. TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle other pericentriolar satellite components were shown to be required assembly. Mol. Biol. Cell 15, 1580–1590 (2004). for spindle-pole integrity34,35. CEP90 prevents the fragmentation of 4. Keryer, G., Ris, H. & Borisy, G. G. Centriole distribution during tripolar mitosis in 34 Chinese hamster ovary cells. J. Cell Biol. 98, 2222–2229 (1984). pericentriolar material in response to ‘forces during prometaphase’ 5. Ganem, N. J., Godinho, S. A. & Pellman, D. A mechanism linking extra centrosomes and CEP72 interacts with the Plk1 target Kizuna, which ensures to chromosomal instability. Nature 460, 278–282 (2009). spindle-pole integrity in part due to Kid-mediated forces36. Thus, a role 6. Silkworth, W. T., Nardi, I. K., Scholl, L. M. & Cimini, D. Multipolar spindle pole coalescence is a major source of kinetochore mis-attachment and chromosome for pericentriolar satellites during mitosis is emerging as a structural mis-segregation in cancer cells. PLoS ONE 4, e6564 (2009). mechanism of spindle-pole resistance to CENP-E- and Kid-mediated 7. Gordon, M. B., Howard, L. & Compton, D. A. Chromosome movement in mitosis requires microtubule anchorage at spindle poles. J. Cell Biol. 152, forces exerted during chromosome alignment. 425–434 (2001). Finally, we demonstrate that mammalian CLASPs contribute to 8. Manning, A. L. & Compton, D. A. Mechanisms of spindle-pole organization are influenced by kinetochore activity in mammalian cells. Curr. Biol. 17, mitotic fidelity not only by regulating kinetochore–microtubule 260–265 (2007). attachments9,12,14, but also by preventing irreversible spindle multi- 9. Maiato, H. et al. Human CLASP1 is an outer kinetochore component that regulates polarity through distinct molecular partnerships at kinetochores and spindle microtubule dynamics. Cell 113, 891–904 (2003). 10. Maiato, H., Khodjakov, A. & Rieder, C. L. Drosophila CLASP is required for the centrosomes. For instance, incomplete CLASP1/2 depletion by RNAi, incorporation of microtubule subunits into fluxing kinetochore fibres. Nat. Cell Biol. which does not remove a stable centrosome-associated CLASP1/2 7, 42–47 (2005). 11. Maffini, S. et al. Motor-independent targeting of CLASPs to kinetochores by 11,12 9 pool , or injection of anti-CLASP1 antibodies , leads mostly to CENP-E promotes microtubule turnover and poleward flux. Curr. Biol. 19, short bipolar or monopolar spindles, which might be explained by 1566–1572 (2009). 12. Pereira, A. L. et al. Mammalian CLASP1 and CLASP2 cooperate to ensure preferential loss of CLASP1/2 function at kinetochores. Importantly, mitotic fidelity by regulating spindle and kinetochore function. Mol. Biol. Cell 17, the irreversible nature of spindle multipolarity arising through the 4526–4542 (2006). mechanism reported here is in marked contrast with the transient 13. Mimori-Kiyosue, Y. et al. Mammalian CLASPs are required for mitotic spindle organization and kinetochore alignment. Genes Cells 11, 845–857 (2006). 5,6 multipolar configuration due to supernumerary centrosomes , un- 14. Manning, A. L. et al. CLASP1, astrin and Kif2b form a molecular switch that covering an alternative route to aneuploidy. On the other hand, regulates kinetochore–microtubule dynamics to promote mitotic progression and fidelity. EMBO J. 29, 3531–3543 (2010). as multipolar anaphase often results in daughter-cell lethality in 15. Thein, K. H., Kleylein-Sohn, J., Nigg, E. A. & Gruneberg, U. Astrin is required for several tumour-derived cell lines5, CLASPs may represent potential the maintenance of sister chromatid cohesion and centrosome integrity. J. Cell Biol. 178, 345–354 (2007). chemotherapeutic targets in human cancers. 16. McGuinness, B. E., Hirota, T., Kudo, N. R., Peters, J. M. & Nasmyth, K. Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells. METHODS PLoS Biol. 3, e86 (2005). Methods and any associated references are available in the online 17. Daum, J. R. et al. Cohesion fatigue induces chromatid separation in cells delayed at metaphase. Curr. Biol. 21, 1018–1024 (2011). version of the paper at http://www.nature.com/naturecellbiology 18. Stevens, D., Gassmann, R., Oegema, K. & Desai, A. Uncoordinated loss of chromatid cohesion is a common outcome of extended metaphase arrest. PLoS ONE 6, Note: Supplementary Information is available on the Nature Cell Biology website e22969 (2011). 19. Kwiatkowski, N. et al. Small-molecule kinase inhibitors provide insight into Mps1 ACKNOWLEDGEMENTS cell cycle function. Nat. Chem. Biol. 6, 359–368 (2010). The authors would like to thank J. Macedo and P. Sampaio for technical 20. Magidson, V. et al. The spatial arrangement of chromosomes during prometaphase help, A. Pereira for expertise in statistical analysis and all colleagues that facilitates spindle assembly. Cell 146, 555–567 (2011).

LETTERS

21. Cimini, D., Wan, X., Hirel, C. B. & Salmon, E. D. Aurora kinase promotes turnover of 30. Bornens, M. Centrosome composition and microtubule anchoring mechanisms. Curr. kinetochore microtubules to reduce chromosome segregation errors. Curr. Biol. 16, Opin. Cell Biol. 14, 25–34 (2002). 1711–1718 (2006). 31. Bettencourt-Dias, M. & Glover, D. M. Centrosome biogenesis and function: 22. Levesque, A. A. & Compton, D. A. The chromokinesin Kid is necessary for Centrosomics brings new understanding. Nat. Rev. 8, 451–463 (2007). chromosome arm orientation and oscillation, but not congression, on mitotic 32. Dammermann, A. & Merdes, A. Assembly of centrosomal proteins and microtubule spindles. J. Cell Biol. 154, 1135–1146 (2001). organization depends on PCM-1. J. Cell Biol. 159, 255–266 (2002). 23. Levesque, A. A., Howard, L., Gordon, M. B. & Compton, D. A. A functional 33. Gruber, J., Harborth, J., Schnabel, J., Weber, K. & Hatzfeld, M. The mitotic-spindle- relationship between NuMA and kid is involved in both spindle organization and associated protein astrin is essential for progression through mitosis. J. Cell Sci. 115, chromosome alignment in vertebrate cells. Mol. Biol. Cell 14, 3541–3552 (2003). 4053–4059 (2002). 24. Kapoor, T. M. et al. Chromosomes can congress to the metaphase plate before 34. Kim, K. & Rhee, K. The pericentriolar satellite protein CEP90 is crucial for integrity biorientation. Science 311, 388–391 (2006). of the mitotic spindle pole. J. Cell Sci. 124, 338–347 (2011). 25. Cai, S., O’Connell, C. B., Khodjakov, A. & Walczak, C. E. Chromosome congression 35. Oshimori, N., Li, X., Ohsugi, M. & Yamamoto, T. Cep72 regulates the localization in the absence of kinetochore ﬁbres. Nat. Cell Biol. 11, 832–838 (2009). of key centrosomal proteins and proper bipolar spindle formation. EMBO J. 28, 26. Sardar, H. S., Luczak, V. G., Lopez, M. M., Lister, B. C. & Gilbert, S. P. Mitotic 2066–2076 (2009). kinesin CENP-E promotes microtubule plus-end elongation. Curr. Biol. 20, 36. Oshimori, N., Ohsugi, M. & Yamamoto, T. The Plk1 target Kizuna stabilizes 1648–1653 (2010). mitotic centrosomes to ensure spindle bipolarity. Nat. Cell Biol. 8, 27. Wood, K. W. et al. Antitumor activity of an allosteric inhibitor of centromere- 1095–1101 (2006). associated protein-E. Proc. Natl Acad. Sci. USA 107, 5839–5844 (2010). 37. Mattiuzzo, M. et al. Abnormal kinetochore-generated pulling forces from expressing 28. Compton, D. A. Focusing on spindle poles. J. Cell Sci. 111, 1477–1481 (1998). a N-terminally modiﬁed Hec1. PLoS ONE 6, e16307 (2011). 29. Garrett, S., Auer, K., Compton, D. A. & Kapoor, T. M. hTPX2 is required for normal 38. Kubo, A., Sasaki, H., Yuba-Kubo, A., Tsukita, S. & Shiina, N. Centriolar spindle morphology and centrosome integrity during vertebrate cell division. Curr. satellites: Molecular characterization, ATP-dependent movement toward centrioles Biol. 12, 2055–2059 (2002). and possible involvement in ciliogenesis. J. Cell Biol. 147, 969–980 (1999).

METHODS DOI: 10.1038/ncb2423

METHODS of at least 45 cm onto cleaned glass coverslips. For phenotypic analysis, 150–300 Cell culture, RNAi and western blot analysis. HeLa cell lines stably expressing cells were quantified in each of three independent experiments, unless otherwise centrin–GFP (provided by A. Khodjakov, Wadsworth Center, Albany, USA), indicated. EGFP–centrin-2/α-tubulin–mRFP and H2B-histone–GFP/α-tubulin–mRFP were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Image acquisition and time-lapse microscopy. Image acquisition for immunoflu- 10% fetal bovine serum (FBS; Invitrogen) and selective antibiotics (G418 and orescence microscopy analysis was carried out in a Zeiss AxioImager Z1 equipped ◦ puromycin) at 37 C in a humidified atmosphere with 5% CO2. For RNAi, we used with a Axiocam MR. Images were subsequently blind deconvolved with Autoquant previously validated siRNA oligonucleotides against human CLASP1 and CLASP2 X (Media Cybernetics). Adobe Photoshop CS4 (Adobe Systems) was used for (ref. 13), astrin33, TOGp (ref. 3), Sgo1 (ref. 16), NUF2 (ref. 8), ninein32, CENP-E image processing. CLASP1/2 and ninein levels at the centrosomes were measured (ref. 11), KID (ref. 39) and separase15. Cells were transfected 1 h after plating using MATLAB (The Mathworks) by quantification of the pixel grey levels of the with Lipofectamine RNAiMAX–siRNA complexes according to the manufacturer’s focused z plane within a region of interest. The background signal was measured instructions (Invitrogen). Phenotypes were analysed and quantified 36 h or 72 h later within a neighbouring region and was subtracted from the measured fluorescence depending on RNAi depletion efficiency as monitored by western blotting using the intensity inside the region of interest. Co-localization analysis of ninein and PCM-1 following antibodies: rat anti-CLASP1 1:100 and rat anti-CLASP2 1:100 (ref. 11), immunostainings (respectively mouse and rabbit antibodies) was carried out using mouse anti-astrin 1:1,000 (provided by M. S. Chang, Mackay Memorial Hospital, the co-localization plugin package in ImageJ (http://rsbweb.nih.gov/). For time- Taiwan), rabbit anti-TOGp 1:2,000 (provided by L. Cassimeris, Lehigh University, lapse microscopy, cells were plated in 35 mm glass-bottom dishes (14 mm, No 1.5 PA, USA; ref. 3), mouse anti-Hec1 1:1,000 (clone 9G3, Abcam), rabbit anti-ninein coverglass; MatTek Corporation), transfected and/or treated with chemical reagents ◦ 1:2,000 (provided by Y-R. Hong, Kaohsjung Medical University, Taiwan), sheep and imaged in a heated chamber (37 C and 5% CO2). Four-dimensional data sets anti-CENP-E 1:500 (provided by W. C. Earnshaw, Wellcome Trust Centre for were collected with a Revolution spinning-disc confocal system (Andor) equipped Cell Biology, Edinburgh, UK) and mouse anti-α-tubulin 1:3,000 (clone B 5-1-2, with an electron-multiplying CCD (charge-coupled device) iXonEM + camera and Sigma). HRP-conjugated secondary antibodies (Amersham) were visualized using a Yokogawa CSU-22 unit based on an Olympus IX81 inverted microscope. Two the ECL system (Pierce). A GS-800 calibrated densitometer (BioRad) was used for laser lines at 488 and 561 nm were used for the excitation of GFP and mRFP and quantitative analysis of protein levels on RNAi. the system was driven by IQ software (Andor). z stacks (0.8–1.0 µm) covering the entire volume of the mitotic apparatus were collected every 1.5–2.5 min according Drug treatments. HeLa centrin–GFP cell cultures were incubated for 24 h in media to the experiments with a PLANAPO 60× NA 1.40 objective. All images represent containing 2 µM cytochalasin D (Sigma-Aldrich) to induce cytokinesis failure. To maximum-intensity projections of all z planes. ImageJ was used to process all movies. inhibit the proteasome and induce a metaphase arrest, cells were treated with 5 µM Images for four-dimensional-reconstruction analysis were deconvolved using the MG132 (Calbiochem). For Eg5 inhibition, 5 µM STLC (Sigma-Aldrich) was added classical maximum-likelihood algorithm of Huygens Professional 4.0 (Scientific to the media and cells were either immediately filmed or fixed after 1 h. STLC Volume Imaging) and then analysed with the open-source software 3D Viewer of washout was carried out by rinsing cells four times with fresh medium. Cells were Fiji (www.fiji.sc). then filmed for ∼2 h (Supplementary Fig. S4 and Movies S4 and S5), or alternatively filmed for ∼4 h following the addition of medium containing 2 µM ZM447439 Photoconversion and measurement of microtubule flux. For photoconversion (AstraZeneca) +5 µM MG132 (Calbiochem; Supplementary Fig. S5b). For mitotic- assays, U2OS cells stably expressing mEos–tubulin (gift from S. Geley, Innsbruck checkpoint inhibition, cells were treated before mitotic entry with 5 µM Mps1-IN-1 Medical University, Innsbruck, Austria) were cultivated on glass-bottomed dishes (ref. 19). For CENP-E inhibition, 20 nM GSK-923295 (MedChemexpress) was (MatTek). Imaging was carried out using a PLANAPO 100× NA 1.40 DIC objective added to the media and cells were either immediately filmed or fixed after 1 h. on a Nikon TE2000U inverted microscope equipped with a Yokogawa CSU-X1 GSK-923295 washout was carried out 2 h later by rinsing cells four times with fresh spinning-disc confocal head containing two laser lines (488 nm and 561 nm) and medium. a Mosaic (Andor) photoactivation system also containing two laser lines (405 nm and 488 nm). Photoconversion was carried out in late prometaphase/metaphase Immunofluorescence microscopy. HeLa centrin–GFP cells were processed for cells, identified by green fluorescent tubulin signals, by using two line-like regions immunofluorescence microscopy as described previously9. Primary antibodies used of interest, placed perpendicular to the main spindle axis on both sides of the were: rat anti-CLASP1 1:20 (ref. 11), mouse or rat anti-α-tubulin respectively metaphase plate. Activation was carried out by one 500 ms pulse from a 405 nm 1:2,000 and 1:100 (Sigma-Aldrich), rabbit anti-γ-tubulin 1:2,000 (Sigma-Aldrich), laser. Photoconverted red signals as well as green fluorescence signals were then rabbit anti-MAD2 1:400 (Bethyl), rabbit anti-CENP-E 1:400 (Santa Cruz), mouse followed over using 561 nm and 488 nm lasers and an iXonEM +electron-multiplying anti-astrin 1:500, rabbit anti-ninein 1:1,000 (provided by Y-R. Hong, Kaohsjung CCD camera every 3 s for 5 min. Microtubule flux (µm min−1) was quantified by Medical University, Taiwan), rabbit and mouse anti-ninein respectively 1:1,000 and tracking photoconverted mEos–tubulin over time using a Matlab algorithm for 1:1 (provided by E. Nigg, Biozentrum, University of Basel, Switzerland), rabbit kymograph generation and analysis40. Generated kymographs were collapsed and anti-PCM-1 1:400 (provided by A. Merdes, CNRS, Toulouse, France)32, mouse the fluorescence intensity curve was created. Fluorescence intensity peaks of each anti-Hec1 1:1,000 (9G3, Abcam), rabbit anti-TOGp 1:200 (ref. 3), rabbit anti-NuMA time frame were defined by manually tracking regions of the highest fluorescence 1:1,000 (ref. 8), rabbit anti-HSET 1:1,000 (ref. 8), rabbit anti-TPX2 1:1,000 (ref. 8), intensities followed by automatized curve fitting. Distances between measured peaks rabbit anti-Kid 1:500 (ref. 23; all provided by D. A. Compton, Dartmouth Medical were then exported into an Excel table and microtubule flux values were calculated School, Hanover, USA) and mouse anti-CPAP 1:300 (provided by T. K. Tang, both from the distances between two activated regions and from the distances Institute of Biomedical Sciences, Taipei, Taiwan)28. Secondary antibodies used between each region and the mitotic pole of its associated half-spindle. were Alexa Fluor 488, 568 and 647 diluted 1:1,500 (Invitrogen) and DNA was −1 counterstained with DAPI (1 µg ml ). For chromosome spreads, HeLa cells were 39. Tokai-Nishizumi, N., Ohsugi, M., Suzuki, E. & Yamamoto, T. The chromokinesin Kid 17 −1 treated as described previously with 300 ng ml nocodazole for 1 h, and then is required for maintenance of proper metaphase spindle size. Mol. Biol. Cell 16, swollen in a 1:1 mixture of medium and double-distilled H2O plus nocodazole for 5455–5463 (2005). 20 min at 37 ◦C. Mitotic cells were collected by shake off, suspended and washed 40. Pereira, A. J. & Maiato, H. Improved kymography tools and its applications to mitosis. by centrifugation in 3:1 methanol/acetone, and finally dropped from a height Methods 51, 214–219 (2010).

SUPPLEMENTARY INFORMATION

DOI: 10.1038/ncb2423

a Cytokinesis failure Premature centriole Loss of pole integrity and/or disengagement MT minus end attachment ex: cytochalasin D ex: Astrin RNAi ex: TOGp RNAi

centrin-GFP microtubules

majority of poles majority of poles increased number of with 2 centrioles with 1 centriole poles with 0 centrioles

RNAi b c RNAi

WB Ab: ControlCLASP1CLASP2CLASP1+2 WB Ab: Control Astrin 150 CLASP1 150 Astrin α-tubulin 50 α-tubulin 50 Depletion % 86

CLASP2 150 e RNAi

α-tubulin 50

Depletion % 85 80 92 CLASPs WB Ab: Control Nuf2 CLASPs & Nuf2 150 d CLASPs RNAi 100 Hec1 WB Ab: ControlTOGp 250 α 50 TOGp -tubulin α-tubulin 50 Depletion % 85 90 80/89

Depletion % 83

f RNAi g RNAi

Control CLASPsCENP-ECLASPs + CENP-EkDa WB Ab: ControlNinein CENP-E 250 250 Ninein CLASPs 150 α-tubulin 50 α-tubulin 50 Depletion % 70 Depletion % 89 80 80/91 Figure S1 - Logarinho et al.,

Figure S1 Use of Centrin-GFP centriole marker as readout for possible causes be induced by Astrin RNAi, increasing the number of poles with one centriole. of spindle multipolarity and quantification of RNAi efficiency. (a) Scheme Loss of spindle pole integrity can be induced by TOGp RNAi, increasing the representing how multipolar spindles may arise from different cellular events number of acentriolar poles. (b-g) Determination of RNAi depletion efficiencies that can be distinguished by the number of centrioles per pole. Cytokinesis by Western blot. Total cell extracts from HeLa Centrin-GFP cells either mock failure can be induced by cytochalasin D treatment leading to multipolar or RNAi treated were run on SDS-PAGE and immunoblotted with specific spindles with two centrioles per pole. Premature centriole disengagement can antibodies as indicated. Detection of a-tubulin was used as loading control.

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CLASPs RNAi CLASPs RNAi control short spindle multipolar spindle α-tubulin centrin-GFP centrin-GFP centrin-GFP DAPI

TOGp TOGp TOGp

Astrin Astrin Astrin

NuMA NuMA NuMA

HSET HSET HSET

TPX2 TPX2 TPX2 1 1 1 2

1 1

2 1

2 2 3 2 2 3 CPAP CPAP CPAP

Figure S2 - Logarinho et al.,

Figure S2 CLASPs depletion does not affect the polar localization stained with DAPI. For CLASPs RNAi cells, representative images of of TOGp, Astrin, NUMA, HSET, TPX2 or CPAP. HeLa Centrin-GFP short or multipolar spindles are shown. For CPAP immunostaining, cells either mock transfected or RNAi depleted for CLASPs were magnifications of the indicated poles are shown. Scale bar, immunostained for a-tubulin and the indicated protein. DNA was 5 mm.

SUPPLEMENTARY INFORMATION

a mock Sgo 1 Astrin

CLASPs Separase

CLASPs + Separase Nuf2 Ninein

b Quantification of sister-chromatid separation (SCS) in chromosome spreads of RNAi depleted cells RNAi No SCS (%) Total SCS (%) Mild SCS (%) n

Mock 89.7 8.6 1.7 58

Sgo1 4.8 92.9 2.4 42

Astrin 13.0 80.4 6.5 184

CLASPs 69.2 17.7 13.1 130

Separase 97.6 2.4 0 42

CLASPs+Separase 61.4 24.2 14.4 132

Nuf2 23.7 23.0 53.4 266

Ninein 48.5 33.7 17.8 163

c 40

10 % of multipolar cells

0 1 2 3 4 RNAi: Mock SeparaseCLASPsSeparase

+CLASPs Figure S3 - Logarinho et al.,

Figure S3 Comparative analysis of sister chromatid separation in distinct analysis depicted in (b). Scale bar, 5 mm. (b) Sister-chromatid separation RNAi conditions. (a) Chromosome spreads were prepared from mitotic HeLa (SCS) was quantified for each condition. Percentages of cells with no, total Centrin-GFP cells either mock transfected or RNAi depleted for Sgo1, Astrin, and mild SCS from a total number (n) of cells quantified are indicated. (c) CLASPs, Separase, CLASPs+Separase, Nuf2 and Ninein. Representative Separase RNAi depletion does not revert SCS nor spindle multipolarity of pictures of spreads from each sample are shown accordingly to quantitative CLASPs RNAi depletion.

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a DAPI αtubulin γtubulin MERGE

rotibihni 5gE rotibihni

tuo Control RNAi

hsaW

rotibihni 5gE rotibihni

iANR sPSALC iANR

tuo

hsaW

b 120 Monopolar 100 Bipolar Multipolar 80

0 Control CLASPs Control CLASPs Control CLASPs RNAi RNAi RNAi RNAi RNAi RNAi untreated STLC washout

rotibihni 5gE c rotibihni

iANR sPSALC iANR

0:00 0:15 0:32 0:30 0:52 1:00 1:38

tuo

hsaW

0:00 0:30 1:00 1:30 Figure S4 - Logarinho et al.,

Figure S4 Eg5/kinesin-5-mediated pushing forces are required to sustain cells became bipolar whereas CLASPs RNAi cells became both bipolar and multipolarity in CLASPs-depleted cells. (a) Centrin-GFP HeLa cells transfected multipolar. (b) Quantitative analysis of spindle conformation in control and with Control RNAi or CLASPs RNAi were incubated for 4 h with the Eg5 CLASPs RNAi cells untreated, treated with STLC and washed out from STLC. inhibitor STLC. Following drug treatment, cells were either immediately fixed Percentages correspond to mean±s.d. from three independent experiments. (c) or fixed 1 h after incubation in fresh medium without STLC (drug wash out), Stills from movies of cells co-expressing EGFP-Centrin-2 and a-tubulin-mRFP and then stained for a- and g-tubulin. Similarly to control, CLASPs RNAi that were transfected with CLASPs RNAi and live imaged following addition of cells were mostly monopolar following drug treatment. After washout, control STLC into the media or after drug wash out. Time, h:min. Scale bars, 5 mm.

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a NEB Anaphase onset Control + Mps1-IN-1

0:00 0:06 0:12 0:16 0:27 + Mps1-IN-1 CLASPs RNAi

0:00 0:06 0:14 0:22 0:34

STLC washout Spindle bipolarization Misaligned chromosomes Chromosome alignment DNA decondensation b +ZM 447439 +MG132 Control

0:00 0:40 1:20 2:00 3:50 STLC washout Spindle bipolarization Misaligned chromosomes Multipolar spindle DNA decondensation +ZM 447439 +MG132 Short spindle * *

* * * * * * CLASPs RNAi 0:00 0:30 1:00 1:05 3:50 c Pre-conversion (phase) Photo-conversion Control

00:00 00:30 01:00 02:00

CENP-E inhibitor 00:00 00:30 01:00 02:00 d Poleward Microtubule Flux Rates in mEos-α-tubulin U2OS Cells Control CENP-E inhibitor Mark to pole (μm/min) 0.61 ± 0.28 (9) 0.64 ± 0.19 (12; 0.79) Mark away from mark on 0.63 ± 0.22 (9) 0.64 ± 0.19 (12; 0.89) opposing KT MTs/2 (μm/min) Values show mean ± standard deviation; n and p versus control are given in parentheses. FIgure S5 - Logarinho et al.,

Figure S5 Effects of Mps1, Aurora B and CENP-E inhibition on spindle spindles for > 3h (8/8 cells), CLASPs-depleted cells became multipolar multipolarity. (a) HeLa cells stably co-expressing EGFP-Centrin-2 and a-tubulin- following mitotic arrest in the presence of misaligned chromosomes for > mRFP were either mock transfected (Control) or RNAi transfected (CLASPs 3h (7/8 cells). Note that Eg5 inhibition is known to generate a mixture of RNAi) and treated immediately before imaging with the small molecule inhibitor monotelic and syntelic attachments. Time, h:min. (c) Representative phase Mps1-IN-1 to abrogate the mitotic checkpoint. Both control and CLASPs contrast images showing control and CENP-E inhibited (GSK-923295-treated) RNAi imaged cells entered anaphase shortly after NEB, always with bipolar U2OS cells. Images were captured before (Pre-conversion) and several times spindles. Time, h:min. (b) HeLa cells stably co-expressing H2B-Histone-GFP/a- after photoconversion of mEos-a-tubulin. Microtubule flux was measured by tubulin-mRFP were incubated with Eg5 inhibitor/STLC for 30 min and then photoconversion of two line-like regions in mEos-tubulin cells and tracking of immediately live imaged following STLC washout and replacement with fresh photoconverted mEOS-tubulin over a time interval of 2 min (arrows). Time, media containing the Aurora B inhibitor ZM 447439 and the proteasome min:sec. Scale bars, 5 mm. (d) mEos-tubulin U2OS cells in late prometaphase/ inhibitor MG132. Whereas control cells typically bipolarized with misaligned metaphase treated with GSK-923295 display no significant reduction of chromosomes within 40 min and remained arrested in mitosis with bipolar kinetochore microtubule poleward flux in comparison with controls.

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centrin-GFP + α-tubulin a DAPI centrin-GFP + α-tubulin Ninein CLASPs + Ninein + CLASPs 1

2 G2

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Ninein RNAi CLASPs RNAi 4 3 3 Figure S6 - Logarinho et al.,

Figure S6 Ninein localization during the cell cycle. (a) Centrin-GFP significantly reduced in multipolar CLASPs-depleted cells. (c) Ninein HeLa cells were stained for a-tubulin, Ninein and CLASP1. Ninein immunostaining is specifically abolished following Ninein RNAi, and CLASP1 co-localize partially at the pericentriolar regions during whereas CLASPs localization remains unchanged in Ninein-depleted both interphase and mitosis. Two different anti-Ninein antibodies multipolar cells. Insets are magnifications of the indicated poles. Scale were used which gave identical results. (b) Ninein accumulation is bars, 5 mm.

SUPPLEMENTARY INFORMATION

Supplementary movie legends

Movie S1 Mitotic progression in a control HeLa cell stably co-expressing EGFP-Centrin-2 and a-tubulin-mRFP. Images were acquired on a spinning disk confocal microscope in Z-axis sections (0.8-1.0 um steps) using 150 s intervals.

Movie S2 Loss of spindle pole integrity in a CLASPs RNAi depleted cell stably co-expressing EGFP-Centrin-2 and α-tubulin-mRFP. Images were acquired on a spinning disk confocal microscope in Z-axis sections (0.8-1.0 um steps) using 150 s intervals. Arrowheads indicate spindle pole fragmentation events leading to the formation of a tripolar spindle consisting of two pre-existing poles with two centrioles each (green asterisks) and one newly formed pole with no centrioles (white asterisks).

Movie S3 Loss of spindle pole integrity with premature centriole disengagement in a CLASPs RNAi depleted cell stably co-expressing EGFP-Centrin-2 and α-tubulin-mRFP. Images were acquired on a spinning disk confocal microscope in Z-axis sections (0.8-1.0 um steps) using 150 s intervals. Arrowheads indicate spindle pole fragmentation events. In one cell (top right) a tripolar spindle is formed (green asterisks) consisting of one pre-existing pole with two centrioles and two poles with one centriole. In another cell (center), spindle pole fragmentation leads to centriole disengagement. One of the poles with only one centriole merges with the normal pole and the other pole with only one centriole moves apart (green asterisk).

Movie S4 Maintenance of multipolar spindles in CLASPs-depleted cells requires Eg5/kinesin-5-mediated pushing forces. CLASPs-depleted multipolar spindle cells stably co-expressing EGFP-Centrin-2 and α-tubulin-mRFP were imaged immediately following addition of a Kinesin-5/Eg5 small molecule inhibitor. Images were acquired on a spinning disk confocal microscope in Z-axis sections (0.8-1.0 um steps) using 120 s intervals. Two poles are acentriolar (white asterisks) and the other two contain two centrioles each (green asterisks). Multipolar spindles resolve into monopolar spindles upon Kinesin-5/Eg5 inhibition.

Movie S5 CLASPs-depleted cells reestablish multipolar spindles following washout from a Kinesin-5/Eg5 inhibitor treatment. CLASPs-depleted cells stably co-expressing EGFP-Centrin-2 and α-tubulin-mRFP were imaged immediately following washout from a Kinesin-5/Eg5 small molecule inhibitor treatment. Images were acquired on a spinning disk confocal microscope in Z-axis sections (0.8-1.0 um steps) using 120 s intervals. Monopolar spindles resolve into multipolar spindles upon drug washout with two acentriolar poles (white asterisks) and two poles with two centrioles each (green asterisks).

Movie S6 Loss of spindle pole integrity in the absence of CLASPs is preceded by the presence of misaligned chromosomes (arrowheads). CLASPs RNAi depleted cell stably expressing H2B-Histone-GFP/α-tubulin-mRFP was imaged on a spinning disk confocal microscope. Images were acquired in Z-axis sections (0.8-1.0 um steps) using 150 s intervals. Pole fragmentation events are indicated (asterisks).

Movie S7 Loss of spindle pole integrity in the absence of CLASPs is preceded by the presence of misaligned chromosomes (arrowheads). CLASPs RNAi depleted cellstably expressing H2B-Histone-GFP/α-tubulin-mRFP was imaged on a spinning disk confocal microscope as described in Movie S6. The pole disintegration event is indicated (asterisks) leading to formation of a tetrapolar spindle that eventually resolves into a multipolar anaphase and cytokinesis. Critical time frames of this movie were processed into 4D reconstruction images (Figure 3c).

Movie S8 Reversible inhibition of CENP-E motor activity in control cells. CENP-E inhibitor GSK-923295 was added into control HeLa cells stably expressing H2B-Histone-GFP/α-tubulin-mRFP (final concentration: 20 nM) and cells entering mitosis were immediately imaged in 150 s time intervals for 2 h in the presence of the drug. Note the presence of misaligned chromosomes that fail to congress into the metaphase plate. GSK-923295 was then washed out and fresh medium added. Cells aligned all chromosomes and exited mitosis within 1 h.

Movie S9 Reversible inhibition of CENP-E motor activity prevents loss of spindle pole integrity in the absence of CLASPs. CLASPs RNAi depleted HeLa cells stably expressing H2B-Histone-GFP/α-tubulin-mRFP were treated with GSK-932295 CENP-E inhibitor as described for Movie S8. Most of the cells remained bipolar until the drug was washed out 2 h after the NEB (26/32 cells). Following CENP-E inhibitor wash out, cells often became multipolar in less than 1 h (12/24).

Movie S10 Loss of spindle pole integrity in the absence of CLASPs is irreversible and once occurred cannot be rescued by CENP-E inhibition. CLASPs RNAi depleted HeLa cells stably expressing H2B-Histone-GFP/α-tubulin-mRFP were treated with GSK-932295 CENP-E inhibitor as described for Movie S14. Cells (5/5 cells) that were already multipolar when the drug was added, remained multipolar during the 2 h incubation period with the drug and after the drug washout.