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The accuracy of cell division requires precise regulation of the cellular machinery governing DNA/genome duplication, ensuring its equal distribution among the daughter cells. The control of the centrosome cycle is crucial for the formation of a bipolar spindle, ensuring error-free segregation of the genome. The cell and centrosome cycles operate in close synchrony along similar principles. Both require a single duplication round in every cell cycle, and both are controlled by the activity of key protein kinases. Nevertheless, our comprehension of the precise cellular mechanisms and critical regulators synchronizing these two cycles remains poorly defined. Here, we present our hypothesis that the spatiotemporal regulation of a dynamic equilibrium of mitotic kinases activities forms a molecular clock that governs the synchronous progression of both the cell and the centrosome cycles.

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Centrosomes are the main microtubule-organizing centers in animal cells. Due to the semiconservative nature of centrosome duplication, the two centrosomes differ in age. In asymmetric stem cell divisions, centrosome age can induce an asymmetry in half-spindle lengths. However, whether centrosome age affects the symmetry of the two half-spindles in tissue culture cells thought to divide symmetrically is unknown. Here, we show that in human epithelial and fibroblastic cell lines centrosome age imposes a mild spindle asymmetry that leads to asymmetric cell daughter sizes. At the mechanistic level, we show that this asymmetry depends on a cenexin-bound pool of the mitotic kinase Plk1, which favors the preferential accumulation on old centrosomes of the microtubule nucleation-organizing proteins pericentrin, γ-tubulin, and Cdk5Rap2, and microtubule regulators TPX2 and ch-TOG. Consistently, we find that old centrosomes have a higher microtubule nucleation capacity. We postulate that centrosome age breaks spindle size symmetry via microtubule nucleation even in cells thought to divide symmetrically.

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A tight synchrony between the DNA and centrosome cycle is essential for genomic integrity. Centriole disengagement, which licenses centrosomes for duplication, occurs normally during mitotic exit. We recently demonstrated that mild DNA replication stress typically seen in cancer cells causes premature centriole disengagement in untransformed mitotic human cells, leading to transient multipolar spindles that favour chromosome missegregation. How mild replication stress accelerates the centrosome cycle at the molecular level remained, however, unclear. Using ultrastructure expansion microscopy, we show that mild replication stress induces premature centriole disengagement already in G2 via the ATR-Chk1 axis of the DNA damage repair pathway. This results in a sub-critical Plk1 kinase activity that primes the pericentriolar matrix for Separase-dependent disassembly but is insufficient for rapid mitotic entry, causing premature centriole disengagement in G2. We postulate that the differential requirement of Plk1 activity for the DNA and centrosome cycles explains how mild replication stress disrupts the synchrony between both processes and contributes to genomic instability.

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Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing "HURP-gap". The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps.

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During mitosis, chromosomes must bind spindle microtubules via kinetochores in a stable yet dynamic manner to ensure rapid frictionless movements. A recent study identifies the first complex that specifically reduces friction in the kinetochore-microtubule interface to ensure efficient chromosome segregation.

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Centrioles are central structural elements of centrosomes and cilia. In human cells, daughter centrioles are assembled adjacent to existing centrioles in S-phase and reach their full functionality with the formation of distal and subdistal appendages one-and-a-half cell cycles later, as they exit their second mitosis. Current models postulate that the centriolar protein centrobin acts as placeholder for distal appendage proteins that must be removed to complete distal appendage formation. Here, we investigated, in non-transformed human epithelial RPE1 cells, the mechanisms controlling centrobin removal and its effect on distal appendage formation. Our data are consistent with a speculative model in which centrobin is removed from older centrioles due to a higher affinity for the newly born daughter centrioles, under the control of the centrosomal kinase PLK1. This removal also depends on the presence of subdistal appendage proteins on the oldest centriole. Removing centrobin, however, is not required for the recruitment of distal appendage proteins, even though this process is equally dependent on PLK1. We conclude that PLK1 kinase regulates centrobin removal and distal appendage formation during centriole maturation via separate pathways.

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Mutations in the WDR62 gene cause primary microcephaly, a pathological condition often associated with defective cell division that results in severe brain developmental defects. The precise function and localization of WDR62 within the mitotic spindle is, however, still under debate, as it has been proposed to act either at centrosomes or on the mitotic spindle. Here we explored the cellular functions of WDR62 in human epithelial cell lines using both short-term siRNA protein depletions and long-term CRISPR/Cas9 gene knockouts. We demonstrate that WDR62 localizes at spindle poles, promoting the recruitment of the microtubule-severing enzyme katanin. Depletion or loss of WDR62 stabilizes spindle microtubules due to insufficient microtubule minus-end depolymerization but does not affect plus-end microtubule dynamics. During chromosome segregation, WDR62 and katanin promote efficient poleward microtubule flux and favor the synchronicity of poleward movements in anaphase to prevent lagging chromosomes. We speculate that these lagging chromosomes might be linked to developmental defects in primary microcephaly.

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Mitosis, under the control of the microtubule-based mitotic spindle, is an attractive target for anti-cancer treatments, as cancer cells undergo frequent and uncontrolled cell divisions. Microtubule targeting agents that disrupt mitosis or single molecule inhibitors of mitotic kinases or microtubule motors kill cancer cells with a high efficacy. These treatments have, nevertheless, severe disadvantages: they also target frequently dividing healthy tissues, such as the haematopoietic system, and they often lose their efficacy due to primary or acquired resistance mechanisms. An alternative target that has emerged in dividing cancer cells is their ability to "cluster" the poles of the mitotic spindle into a bipolar configuration. This mechanism is necessary for the specific survival of cancer cells that tend to form multipolar spindles due to the frequent presence of abnormal centrosome numbers or other spindle defects. Here we discuss the recent development of combinatorial treatments targeting spindle pole clustering that specifically target cancer cells bearing aberrant centrosome numbers and that have the potential to avoid resistance mechanism due their combinatorial nature.

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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.

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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 (C2) with high efficacy and negligible toxicity. We discovered that C2 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 C2-treatment shows a high efficacy in cells prone to form multipolar spindles. Our data suggest a highly effective and selective C2 treatment strategy for malignant and drug-resistant cancers.

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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 Caenorhabditis elegans 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.

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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.

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BACKGROUND: Crenolanib is a tyrosine kinase inhibitor targeting PDGFR-α, PDGFR-ß 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.

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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.

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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.

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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.

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Purpose: 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 BRCA2 gene affects the clinical outcome of ovarian cancer patients.Experimental Design: A study cohort of 353 women with ovarian cancer who underwent genetic germline testing for BRCA1 and BRCA2 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 (n = 316) was used as a validation cohort.Results: In the study cohort, 78 patients were carriers of germline mutations of BRCA2 After adjustment for FIGO stage and macroscopic residual disease, BRCA2 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; P = 0.001] and prolonged PFI (29.7 vs. 15.5 months, P = 0.011), compared with noncarriers. BRCA2 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; P = 0.094) or PFI (19 vs. 15.5 months, P = 0.146). In the TCGA cohort, only BRCA2 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; P = 0.004) and 5-year OS (78%; adjusted HR, 0.09; 95% CI, 0.02-0.38; P = 0.001).Conclusions: Among ovarian cancer patients, BRCA2 carriers with mutations located in the RAD51-BD (exon 11) have prolonged PFS, PFI, and OS. Clin Cancer Res; 24(2); 326-33. ©2017 AACR.

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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.

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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.

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