α-tubulin detyrosination fine-tunes kinetochore-microtubule attachments.

Post-translational cycles of α-tubulin detyrosination and tyrosination generate microtubule diversity, the cellular functions of which remain largely unknown. Here we show that α-tubulin detyrosination regulates kinetochore-microtubule attachments to ensure normal chromosome oscillations and timely anaphase onset during mitosis. Remarkably, detyrosinated α-tubulin levels near kinetochore microtubule plus-ends depend on the direction of chromosome motion during metaphase.

Balancing Plk1 activity levels: The secret of synchrony between the cell and the centrosome cycle.

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.

Mild replication stress causes premature centriole disengagement via a sub-critical Plk1 activity under the control of ATR-Chk1.

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.

Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules.

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.

Cell division: The science friction of chromosome attachment.

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.

PLK1 controls centriole distal appendage formation and centrobin removal via independent pathways.

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.

WDR62 localizes katanin at spindle poles to ensure synchronous chromosome segregation.

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.

Forcing dividing cancer cells to die; low-dose drug combinations to prevent spindle pole clustering.

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.

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

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.

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

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.

Cell polarity-dependent centrosome separation in the embryo.

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.

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

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.

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

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.

Bub1-the zombie protein that CRISPR cannot kill.

The conserved Bub1 kinase was proposed to be non‐essential for the mitotic spindle assembly checkpoint based on recent findings that knocking‐out Bub1 by CRISPR/Cas9 did not impair checkpoint function in human cells. New studies now demonstrate that CRISPR/Cas9 Bub1 knockout cells still express low levels of Bub1 protein and that only removal of this remaining Bub1 pool by RNA interference substantially weakens spindle assembly checkpoint signaling.

Mitotic live-cell imaging at different timescales.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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.

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.

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

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.

Two Ways to Get Mad at Kinetochores.

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.