Publications by authors named "Patrick Meraldi"

57 Publications

Spindle-Length-Dependent HURP Localization Allows Centrosomes to Control Kinetochore-Fiber Plus-End Dynamics.

Curr Biol 2019 11 24;29(21):3563-3578.e6. Epub 2019 Oct 24.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland; Translational Research Centre in Onco-hematology, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland. Electronic address:

During mitosis, centrosomes affect the length of kinetochore fibers (k-fibers) and the stability of kinetochore-microtubule attachments, implying that they regulate k-fiber dynamics. However, the exact cellular and molecular mechanisms of this regulation remain unknown. Here, we created human cells with only one centrosome to investigate these mechanisms. Such cells formed asymmetric bipolar spindles that resulted in asymmetric cell divisions. K-fibers in the acentrosomal half-spindles were shorter, more stable, and had a reduced poleward microtubule flux at minus ends and more frequent pausing events at their plus ends. This indicates that centrosomes regulate k-fiber dynamics both locally at minus ends and far away at plus ends. At the molecular level, we find that the microtubule-stabilizing protein HURP is enriched on the k-fiber plus ends in the acentrosomal half-spindles of cells with only one centrosome. HURP depletion rebalances k-fiber stability and plus-end dynamics in such cells and improves spindle and cell division symmetry. Our data from 3 different cell lines indicate that HURP accumulates on k-fibers inversely proportionally to half-spindle length. We therefore propose that centrosomes regulate k-fiber plus ends indirectly via length-dependent accumulation of HURP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2019.08.061DOI Listing
November 2019

Identification of a Synergistic Multi-Drug Combination Active in Cancer Cells via the Prevention of Spindle Pole Clustering.

Cancers (Basel) 2019 Oct 22;11(10). Epub 2019 Oct 22.

Institute of Pharmaceutical Sciences of Western Switzerland, Faculty of Sciences, University of Geneva, 1 Rue Michel-Servet, CMU, 1211 Geneva 4, Switzerland.

A major limitation of clinically used cancer drugs is the lack of specificity resulting in toxicity. To address this, we performed a phenotypically-driven screen to identify optimal multidrug combinations acting with high efficacy and selectivity in clear cell renal cell carcinoma (ccRCC). The search was performed using the Therapeutically Guided Multidrug Optimization (TGMO) method in ccRCC cells (786-O) and nonmalignant renal cells and identified a synergistic low-dose four-drug combination () with high efficacy and negligible toxicity. We discovered that inhibits multipolar spindle pole clustering, a survival mechanism employed by cancer cells with spindle abnormalities. This phenotype was also observed in 786-O cells resistant to sunitinib, the first line ccRCC treatment, as well as in melanoma cells with distinct percentages of supernumerary centrosomes. We conclude that -treatment shows a high efficacy in cells prone to form multipolar spindles. Our data suggest a highly effective and selective treatment strategy for malignant and drug-resistant cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11101612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826636PMC
October 2019

Cell polarity-dependent centrosome separation in the embryo.

J Cell Biol 2019 12 23;218(12):4112-4126. Epub 2019 Oct 23.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland

In animal cells, faithful chromosome segregation depends on the assembly of a bipolar spindle driven by the timely separation of the two centrosomes. Here we took advantage of the highly stereotypical cell divisions in embryos to identify new regulators of centrosome separation. We find that at the two-cell stage, the somatic AB cell initiates centrosome separation later than the germline P1 cell. This difference is strongly exacerbated by the depletion of the kinesin-13 KLP-7/MCAK, resulting in incomplete centrosome separation at NEBD in AB but not P1. Our genetic and cell biology data indicate that this phenotype depends on cell polarity via the enrichment in AB of the mitotic kinase PLK-1, which itself limits the cortical localization of the dynein-binding NuMA orthologue LIN-5. We postulate that the timely separation of centrosomes is regulated in a cell type-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201902109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891102PMC
December 2019

Mild replication stress causes chromosome mis-segregation via premature centriole disengagement.

Nat Commun 2019 08 8;10(1):3585. Epub 2019 Aug 8.

Department of Cell Physiology and Metabolism, University of Geneva, 1211, Geneva 4, Switzerland.

Replication stress, a hallmark of cancerous and pre-cancerous lesions, is linked to structural chromosomal aberrations. Recent studies demonstrated that it could also lead to numerical chromosomal instability (CIN). The mechanism, however, remains elusive. Here, we show that inducing replication stress in non-cancerous cells stabilizes spindle microtubules and favours premature centriole disengagement, causing transient multipolar spindles that lead to lagging chromosomes and micronuclei. Premature centriole disengagement depends on the G2 activity of the Cdk, Plk1 and ATR kinases, implying a DNA-damage induced deregulation of the centrosome cycle. Premature centriole disengagement also occurs spontaneously in some CIN+ cancer cell lines and can be suppressed by attenuating replication stress. Finally, we show that replication stress potentiates the effect of the chemotherapeutic agent taxol, by increasing the incidence of multipolar cell divisions. We postulate that replication stress in cancer cells induces numerical CIN via transient multipolar spindles caused by premature centriole disengagement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11584-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687892PMC
August 2019

Anti-angiogenic effects of crenolanib are mediated by mitotic modulation independently of PDGFR expression.

Br J Cancer 2019 07 25;121(2):139-149. Epub 2019 Jun 25.

Molecular Pharmacology Group, School of Pharmaceutical Sciences, University of Lausanne and University of Geneva, Rue Michel-Servet, 1211, Geneva, Switzerland.

Background: Crenolanib is a tyrosine kinase inhibitor targeting PDGFR-α, PDGFR-β and Fms related tyrosine kinase-3 (FLT3) that is currently evaluated in several clinical trials. Although platelet-derived growth factor receptor (PDGFR) signalling pathway is believed to play an important role in angiogenesis and maintenance of functional vasculature, we here demonstrate a direct angiostatic activity of crenolanib independently of PDGFR signalling.

Methods: The activity of crenolanib on cell viability, migration, sprouting, apoptosis and mitosis was assessed in endothelial cells, tumour cells and fibroblasts. Alterations in cell morphology were determined by immunofluorescence experiments. Flow-cytometry analysis and mRNA expression profiles were used to investigate cell differentiation. In vivo efficacy was investigated in human ovarian carcinoma implanted on the chicken chorioallantoic membrane (CAM).

Results: Crenolanib was found to inhibit endothelial cell viability, migration and sprout length, and induced apoptosis independently of PDGFR expression. Treated cells  showed altered actin arrangement and nuclear aberrations. Mitosis was affected at several levels including mitosis entry and centrosome clustering. Crenolanib suppressed human ovarian carcinoma tumour growth and angiogenesis in the CAM model.

Conclusions: The PDGFR/FLT3 inhibitor crenolanib targets angiogenesis and inhibits tumour growth in vivo unrelated to PDGFR expression. Based on our findings, we suggest a broad mechanism of action of crenolanib.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41416-019-0498-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738084PMC
July 2019

Bub1-the zombie protein that CRISPR cannot kill.

Authors:
Patrick Meraldi

EMBO J 2019 04 8;38(7). Epub 2019 Mar 8.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019101912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443206PMC
April 2019

Mitotic live-cell imaging at different timescales.

Methods Cell Biol 2018 ;145:1-27

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland. Electronic address:

Mitosis is a highly dynamic and choreographed process in which chromosomes are captured by the mitotic spindle and physically segregated into the two daughter cells to ensure faithful transmission of the genetic material. Live-cell fluorescence microscopy enables these dynamics to be analyzed over diverse temporal scales. Here we present the methodologies to study chromosome segregation at three timescales: we first show how automated tracking of kinetochores enables investigation of mitotic spindle and chromosome dynamics in the seconds-to-minutes timescale; next we highlight how new DNA live dyes allow the study of chromosome segregation over a period of several hours in any cell line; finally, we demonstrate how image sequences acquired over several days can reveal the fate of whole cell populations over several consecutive cell divisions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.mcb.2018.03.009DOI Listing
December 2018

Complete microtubule-kinetochore occupancy favours the segregation of merotelic attachments.

Nat Commun 2018 05 23;9(1):2042. Epub 2018 May 23.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211, Geneva 4, Switzerland.

Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20 microtubules, even though single microtubules can generate sufficient force to move chromosomes. Here, we show that high microtubule occupancy at kinetochores ensures robust chromosome segregation by providing a strong mechanical force that favours segregation of merotelic attachments during anaphase. Using low doses of the microtubules-targeting agent BAL27862 we reduce microtubule occupancy and observe that spindle morphology is unaffected and bi-oriented kinetochores can still oscillate with normal intra-kinetochore distances. Inter-kinetochore stretching is, however, dramatically reduced. The reduction in microtubule occupancy and inter-kinetochore stretching does not delay satisfaction of the spindle assembly checkpoint or induce microtubule detachment via Aurora-B kinase, which was so far thought to release microtubules from kinetochores under low stretching. Rather, partial microtubule occupancy slows down anaphase A and increases incidences of lagging chromosomes due to merotelically attached kinetochores.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-04427-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966435PMC
May 2018

p37/UBXN2B regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man.

J Cell Biol 2018 02 8;217(2):483-493. Epub 2017 Dec 8.

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland

Spindle orientation determines the axis of division and is crucial for cell fate, tissue morphogenesis, and the development of an organism. In animal cells, spindle orientation is regulated by the conserved Gαi-LGN-NuMA complex, which targets the force generator dynein-dynactin to the cortex. In this study, we show that p37/UBXN2B, a cofactor of the p97 AAA ATPase, regulates spindle orientation in mammalian cells by limiting the levels of cortical NuMA. p37 controls cortical NuMA levels via the phosphatase PP1 and its regulatory subunit Repo-Man, but it acts independently of Gαi, the kinase Aurora A, and the phosphatase PP2A. Our data show that in anaphase, when the spindle elongates, PP1/Repo-Man promotes the accumulation of NuMA at the cortex. In metaphase, p37 negatively regulates this function of PP1, resulting in lower cortical NuMA levels and correct spindle orientation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201707050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800812PMC
February 2018

Location of Mutation in Gene and Survival in Patients with Ovarian Cancer.

Clin Cancer Res 2018 01 30;24(2):326-333. Epub 2017 Oct 30.

Department of Medical Oncology, Centre Léon Bérard, Lyon, France.

BRCA2 plays a central role in homologous recombination by loading RAD51 on DNA breaks. The objective of this study is to determine whether the location of mutations in the RAD51-binding domain (RAD51-BD; exon 11) of gene affects the clinical outcome of ovarian cancer patients. A study cohort of 353 women with ovarian cancer who underwent genetic germline testing for and genes was identified. Progression-free survival (PFS), platinum-free interval (PFI), and overall survival (OS) were analyzed. The Cancer Genome Atlas (TCGA) cohort of ovarian cancer ( = 316) was used as a validation cohort. In the study cohort, 78 patients were carriers of germline mutations of After adjustment for FIGO stage and macroscopic residual disease, carriers with truncating mutations in the RAD51-BD have significantly prolonged 5-year PFS [58%; adjusted HR, 0.36; 95% confidence interval (CI), 0.20-0.64; = 0.001] and prolonged PFI (29.7 vs. 15.5 months, = 0.011), compared with noncarriers. carriers with mutations located in other domains of the gene do not have prolonged 5-year PFS (28%, adjusted HR, 0.67; 95% CI, 0.42-1.07; = 0.094) or PFI (19 vs. 15.5 months, = 0.146). In the TCGA cohort, only carriers harboring germline or somatic mutations in the RAD51-BD have prolonged 5-year PFS (46%; adjusted HR, 0.30; 95% CI, 0.13-0.68; = 0.004) and 5-year OS (78%; adjusted HR, 0.09; 95% CI, 0.02-0.38; = 0.001). Among ovarian cancer patients, carriers with mutations located in the RAD51-BD (exon 11) have prolonged PFS, PFI, and OS. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-17-2136DOI Listing
January 2018

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

Cancer Cell 2017 10;32(4):444-459.e7

Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin 10126, Italy. Electronic address:

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2017.09.002DOI Listing
October 2017

The Elephant in the Room: The Role of Microtubules in Cancer.

Adv Exp Med Biol 2017 ;1002:93-124

Department of Cell Physiology and Metabolism, Faculty of Medicine, Geneva University, 1211, Geneva, Switzerland.

Microtubules are the backbone of all eukaryotic cells cytoskeleton. Their dynamic behaviour constitutes the basis for many biological processes such as cellular motility, cytoplasmic transport and cell division. Some the most effective chemotherapeutics, such as the taxanes, are microtubule interfering drugs. Moreover, many studies suggest that microtubule dynamics are altered in cancer cell divisions and linked to chromosomal instability, aneuploidy and development of drug resistances. The elephant in the room, however, is that despite all these evidences, the exact role of microtubules in malignancies remains elusive, partially due to the lack of clear genetic alterations linking microtubules to cancer. This review will discuss the molecular mechanisms that might alter microtubule dynamics in cancer cells, the pro and cons of the different theories linking these alterations to cancer progression, and the possible directions to address future key questions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-57127-0_5DOI Listing
September 2017

Symmetry Does not Come for Free: Cellular Mechanisms to Achieve a Symmetric Cell Division.

Results Probl Cell Differ 2017 ;61:301-321

Medical Faculty, Department of Physiology and Metabolism, University of Geneva, 1211, Geneva 4, Switzerland.

During mitosis cells can divide symmetrically to proliferate or asymmetrically to generate tissue diversity. While the mechanisms that ensure asymmetric cell division have been extensively studied, it is often assumed that a symmetric cell division is the default outcome of mitosis. Recent studies, however, imply that the symmetric nature of cell division is actively controlled, as they reveal numerous mechanisms that ensure the formation of equal-sized daughter cells as cells progress through cell division. Here we review our current knowledge of these mechanisms and highlight possible key questions in the field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-53150-2_14DOI Listing
July 2017

Combination of ruthenium(II)-arene complex [Ru(η-p-cymene)Cl(pta)] (RAPTA-C) and the epidermal growth factor receptor inhibitor erlotinib results in efficient angiostatic and antitumor activity.

Sci Rep 2017 02 22;7:43005. Epub 2017 Feb 22.

School of Pharmaceutical Sciences, University of Geneva (UNIGE), Geneva, Switzerland.

Ruthenium-based compounds show strong potential as anti-cancer drugs and are being investigated as alternatives to other well-established metal-based chemotherapeutics. The organometallic compound [Ru(η-p-cymene)Cl(pta)], where pta = 1,3,5-triaza-7-phosphaadamantane (RAPTA-C) exhibits broad acting anti-tumor efficacy with intrinsic angiostatic activity. In the search for an optimal anti-angiogenesis drug combination, we identified synergistic potential between RAPTA-C and the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. This drug combination results in strong synergistic inhibition of cell viability in human endothelial (ECRF24 and HUVEC) and human ovarian carcinoma (A2780 and A2780cisR) cells. Additionally, erlotinib significantly enhances the cellular uptake of RAPTA-C relative to treatment with RAPTA-C alone in human ovarian carcinoma cells, but not endothelial cells. Drug combinations induce the formation of chromosome bridges that persist after mitotic exit and delay abscission in A2780 and A2780cisR, therefore suggesting initiation of cellular senescence. The therapeutic potential of these compounds and their combination is further validated in vivo on A2780 tumors grown on the chicken chorioallantoic membrane (CAM) model, and in a preclinical model in nude mice. Immunohistochemical analysis confirms effective anti-angiogenic and anti-proliferative activity in vivo, based on a significant reduction of microvascular density and a decrease in proliferating cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep43005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320450PMC
February 2017

The Ska complex promotes Aurora B activity to ensure chromosome biorientation.

J Cell Biol 2016 Oct 3;215(1):77-93. Epub 2016 Oct 3.

Growth and Development, Biozentrum, University of Basel, 4056 Basel, Switzerland Cell Cycle and Cancer, Group of Biomedical Research in Gynecology, Vall d'Hebron Research Institute (VHIR)-UAB, 08035 Barcelona, Spain

Chromosome biorientation and accurate segregation rely on the plasticity of kinetochore-microtubule (KT-MT) attachments. Aurora B facilitates KT-MT dynamics by phosphorylating kinetochore proteins that are critical for KT-MT interactions. Among the substrates whose microtubule and kinetochore binding is curtailed by Aurora B is the spindle and kinetochore-associated (Ska) complex, a key factor for KT-MT stability. Here, we show that Ska is not only a substrate of Aurora B, but is also required for Aurora B activity. Ska-deficient cells fail to biorient and display chromosome segregation errors underlying suppressed KT-MT turnover. These defects coincide with KNL1-Mis12-Ndc80 network hypophosphorylation, reduced mitotic centromere-associated kinesin localization, and Aurora B T-loop phosphorylation at kinetochores. We further show that Ska requires its microtubule-binding capability to promote Aurora B activity in cells and stimulates Aurora B catalytic activity in vitro. Finally, we show that protein phosphatase 1 counteracts Aurora B activity to enable Ska kinetochore accumulation once biorientation is achieved. We propose that Ska promotes Aurora B activity to limit its own microtubule and kinetochore association and to ensure that KT-MT dynamics and stability fall within an optimal balance for biorientation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201603019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5057281PMC
October 2016

Two Ways to Get Mad at Kinetochores.

Authors:
Patrick Meraldi

Dev Cell 2015 Dec;35(5):535-536

Cell Physiology and Metabolism Department, University of Geneva, Geneva CH-1211, Switzerland. Electronic address:

The spindle assembly checkpoint ensures that mitotic cells only segregate their sister chromatids once all chromosomes are attached via kinetochores by microtubules of the mitotic spindle. Reporting in Developmental Cell, Silió et al. (2015) show that in human cells the signaling cascade controlling the checkpoint operates through two separate branches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2015.11.021DOI Listing
December 2015

Centrosomes in spindle organization and chromosome segregation: a mechanistic view.

Authors:
Patrick Meraldi

Chromosome Res 2016 Jan;24(1):19-34

Cell Physiology and Metabolism Department, University of Geneva, Geneva, Switzerland.

Centrosomes are complex structures, which are embedded into the opposite poles of the mitotic spindle of most animals, acting as microtubule organizing centres. Surprisingly, in several biological systems, such as flies, chicken, or human cells, centrosomes are not essential for cell division. Nonetheless, they ensure faithful chromosome segregation. Moreover, mis-functioning centrosomes can act in a dominant-negative manner, resulting in erroneous mitotic progression. Here, I review the mechanisms by which centrosomes contribute to proper spindle organization and faithful chromosome segregation under physiological conditions and discuss how errors in centrosome function impair transmission of the genomic material in a pathological setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10577-015-9508-2DOI Listing
January 2016

Centrosome age regulates kinetochore-microtubule stability and biases chromosome mis-segregation.

Elife 2015 Aug 19;4. Epub 2015 Aug 19.

Department of Cellular Physiology and Metabolism, University of Geneva, Geneva, Switzerland.

The poles of the mitotic spindle contain one old and one young centrosome. In asymmetric stem cell divisions, the age of centrosomes affects their behaviour and their probability to remain in the stem cell. In contrast, in symmetric divisions, old and young centrosomes are thought to behave equally. This hypothesis is, however, untested. In this study, we show in symmetrically dividing human cells that kinetochore-microtubules associated to old centrosomes are more stable than those associated to young centrosomes, and that this difference favours the accumulation of premature end-on attachments that delay the alignment of polar chromosomes at old centrosomes. This differential microtubule stability depends on cenexin, a protein enriched on old centrosomes. It persists throughout mitosis, biasing chromosome segregation in anaphase by causing daughter cells with old centrosomes to retain non-disjoint chromosomes 85% of the time. We conclude that centrosome age imposes via cenexin a functional asymmetry on all mitotic spindles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.07909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4579388PMC
August 2015

The equatorial position of the metaphase plate ensures symmetric cell divisions.

Elife 2015 Jul 18;4. Epub 2015 Jul 18.

Department of Physiology and Metabolism, Medical Faculty, University of Geneva, Geneva, Switzerland.

Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. When we offset the metaphase plate position by creating an asymmetric centriole distribution on each pole, we find that metaphase plates relocate to the middle of the spindle before anaphase. The spindle assembly checkpoint enables this centering mechanism by providing cells enough time to correct metaphase plate position. The checkpoint responds to unstable kinetochore-microtubule attachments resulting from an imbalance in microtubule stability between the two half-spindles in cells with an asymmetric centriole distribution. Inactivation of the checkpoint prior to metaphase plate centering leads to asymmetric cell divisions and daughter cells of unequal size; in contrast, if the checkpoint is inactivated after the metaphase plate has centered its position, symmetric cell divisions ensue. This indicates that the equatorial position of the metaphase plate is essential for symmetric cell divisions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.05124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536468PMC
July 2015

CRL4(RBBP7) is required for efficient CENP-A deposition at centromeres.

J Cell Sci 2015 May 20;128(9):1732-45. Epub 2015 Mar 20.

Department of Biology, Institute of Biochemistry, Swiss Federal Institute of Technology, 8093 Zurich, Switzerland

The mitotic spindle drives chromosome movement during mitosis and attaches to chromosomes at dedicated genomic loci named centromeres. Centromeres are epigenetically specified by their histone composition, namely the presence of the histone H3 variant CENP-A, which is regulated during the cell cycle by its dynamic expression and localization. Here, we combined biochemical methods and quantitative imaging approaches to investigate a new function of CUL4-RING E3 ubiquitin ligases (CRL4) in regulating CENP-A dynamics. We found that the core components CUL4 and DDB1 are required for centromeric loading of CENP-A, but do not influence CENP-A maintenance or pre-nucleosomal CENP-A levels. Interestingly, we identified RBBP7 as a substrate-specific CRL4 adaptor required for this process, in addition to its role in binding and stabilizing soluble CENP-A. Our data thus suggest that the CRL4 complex containing RBBP7 (CRL4(RBBP7)) might regulate mitosis by promoting ubiquitin-dependent loading of newly synthesized CENP-A during the G1 phase of the cell cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.162305DOI Listing
May 2015

TRAIP is a regulator of the spindle assembly checkpoint.

J Cell Sci 2014 Dec 21;127(Pt 24):5149-56. Epub 2014 Oct 21.

Service of Dermatology, Lausanne University Hospital, CHUV, 1011 Lausanne, Switzerland

Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.152579DOI Listing
December 2014

Modulation of the chromatin phosphoproteome by the Haspin protein kinase.

Mol Cell Proteomics 2014 07 14;13(7):1724-40. Epub 2014 Apr 14.

From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland; Faculty of Science, University of Zurich, Zurich, Switzerland

Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr(3) of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg(2) and Lys(4) adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys(4) of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser(137) of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser(137) resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser(137) might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.M113.034819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083111PMC
July 2014

SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown.

J Cell Biol 2014 Mar 24;204(7):1099-109. Epub 2014 Mar 24.

Institute of Biochemistry, Department of Biology, and 2 Light Microscopy Center, Swiss Federal Institute of Technology Zurich, CH-8093 Zurich, Switzerland.

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN-KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201310116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971743PMC
March 2014

Nonautonomous movement of chromosomes in mitosis.

Dev Cell 2013 Oct;27(1):60-71

Centre for Mechanochemical Cell Biology, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

Kinetochores are the central force-generating machines that move chromosomes during cell division. It is generally assumed that kinetochores move in an autonomous manner. However, we reveal here that movements of neighboring sister-kinetochore pairs in metaphase are correlated in a distance-dependent manner. This correlation increases in the absence of kinetochore oscillations or stable end-on attachments. This suggests that periodic movements of bioriented chromosomes limit the correlated motion of nonsisters. Computer simulations show that these correlated movements can occur when elastic crosslinks are placed between the K-fibers of oscillating kinetochores. Strikingly, inhibition of the microtubule crosslinking motor kinesin-5 Eg5 leads to an increase in nonsister correlation and impairs periodic oscillations. These phenotypes are partially rescued by codepletion of the kinesin-12 Kif15, demonstrating a function for kinesin-5 and kinesin-12 motors in driving chromosome movements, possibly as part of a crosslinking structure that correlates the movements of nonsister kinetochores.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2013.08.004DOI Listing
October 2013

Effect of Cell Shape and Dimensionality on Spindle Orientation and Mitotic Timing.

PLoS One 2013 18;8(6):e66918. Epub 2013 Jun 18.

Laboratory for Surface Science and Technology, ETH Zurich, Zurich, Switzerland.

The formation and orientation of the mitotic spindle is a critical feature of mitosis. The morphology of the cell and the spatial distribution and composition of the cells' adhesive microenvironment all contribute to dictate the position of the spindle. However, the impact of the dimensionality of the cells' microenvironment has rarely been studied. In this study we present the use of a microwell platform, where the internal surfaces of the individual wells are coated with fibronectin, enabling the three-dimensional presentation of adhesive ligands to single cells cultured within the microwells. This platform was used to assess the effect of dimensionality and cell shape in a controlled microenvironment. Single HeLa cells cultured in circular microwells exhibited greater tilting of the mitotic spindle, in comparison to cells cultured in square microwells. This correlated with an increase in the time required to align the chromosomes at the metaphase plate due to prolonged activation of the spindle checkpoint in an actin dependent process. The comparison to 2D square patterns revealed that the dimensionality of cell adhesions alone affected both mitotic timings and spindle orientation; in particular the role of actin varied according to the dimensionality of the cells' microenvironment. Together, our data revealed that cell shape and the dimensionality of the cells' adhesive environment impacted on both the orientation of the mitotic spindle and progression through mitosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066918PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688943PMC
October 2017

The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A.

J Cell Biol 2013 May 6;201(4):559-75. Epub 2013 May 6.

Department of Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland.

Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2-depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201209107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653362PMC
May 2013

Mitotic spindle (DIS)orientation and DISease: cause or consequence?

J Cell Biol 2012 Dec;199(7):1025-35

Department of Physiology and Metabolism, University of Geneva, 1211 Geneva, Switzerland.

Correct alignment of the mitotic spindle during cell division is crucial for cell fate determination, tissue organization, and development. Mutations causing brain diseases and cancer in humans and mice have been associated with spindle orientation defects. These defects are thought to lead to an imbalance between symmetric and asymmetric divisions, causing reduced or excessive cell proliferation. However, most of these disease-linked genes encode proteins that carry out multiple cellular functions. Here, we discuss whether spindle orientation defects are the direct cause for these diseases, or just a correlative side effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201209015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529530PMC
December 2012

Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex.

PLoS One 2012 18;7(9):e44717. Epub 2012 Sep 18.

Molecular Biology, FLI, Jena, Germany.

Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044717PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445539PMC
March 2013

Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis.

J Cell Biol 2012 Sep;198(5):847-63

Biocenter, Division of Molecular Pathophysiology, Innsbruck Medical University, A-6020 Innsbruck, Austria.

Chromokinesins are microtubule plus end-directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the dynamics of microtubules, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201110060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432768PMC
September 2012

Kinetochores accelerate centrosome separation to ensure faithful chromosome segregation.

J Cell Sci 2012 Feb 7;125(Pt 4):906-18. Epub 2012 Mar 7.

Institute of Biochemistry, ETH Zurich, Schafmattstrasse 18, 8093 Zürich, Switzerland.

At the onset of mitosis, cells need to break down their nuclear envelope, form a bipolar spindle and attach the chromosomes to microtubules via kinetochores. Previous studies have shown that spindle bipolarization can occur either before or after nuclear envelope breakdown. In the latter case, early kinetochore-microtubule attachments generate pushing forces that accelerate centrosome separation. However, until now, the physiological relevance of this prometaphase kinetochore pushing force was unknown. We investigated the depletion phenotype of the kinetochore protein CENP-L, which we find to be essential for the stability of kinetochore microtubules, for a homogenous poleward microtubule flux rate and for the kinetochore pushing force. Loss of this force in prometaphase not only delays centrosome separation by 5-6 minutes, it also causes massive chromosome alignment and segregation defects due to the formation of syntelic and merotelic kinetochore-microtubule attachments. By contrast, CENP-L depletion has no impact on mitotic progression in cells that have already separated their centrosomes at nuclear envelope breakdown. We propose that the kinetochore pushing force is an essential safety mechanism that favors amphitelic attachments by ensuring that spindle bipolarization occurs before the formation of the majority of kinetochore-microtubule attachments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.091967DOI Listing
February 2012