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Article Reference Article 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 Disclaimer: layout of this document may differ from the published version. 1 / 1 LETTERS 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 NATURE CELL BIOLOGY VOLUME 14 j NUMBER 3 j MARCH 2012 295 © 2012 Macmillan Publishers Limited. All rights reserved. LETTERS 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 1 1 2 Control 2 3 1 2 1 2 3 4 4 Cytochalasin D 1 1 2 Astrin 2 3 3 1 2 1 2 RNAi TOGp 3 3 3 1 1 2 CLASP1/2 2 3 4 4 Figure 1 CLASP1/2 depletion causes the formation of multipolar spindles. siRNAs against astrin, TOGp and CLASPs (mean ± s:d:; n D 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 D 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). 296 NATURE CELL BIOLOGY VOLUME 14 j NUMBER 3 j MARCH 2012 © 2012 Macmillan Publishers Limited. All rights reserved. LETTERS 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
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