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Introduction: Distal femoral cartilage lesions can be treated using osteochondral autograft transfer (OAT). When impacting plugs into a recipient site, the cartilage may fracture. This study aimed to analyze OAT donor regions and impaction energies to identify characteristics that lead to fracture., Methods: Fifteen cadaver femurs were used with OAT plugs harvested from the following regions: lateral and medial trochlea (LT and MT), lateral and medial intercondylar notch (LIN and MIN). Plugs were impacted into a bone surrogate block using a custom anvil-type system with pre-determined impact heights; 30, 50, 70, and 90 mm. Each plug's cartilage was examined and determined to be intact or fractured. Chi-square was used to compare the rate of chondral fracture for each region., Results: In all, 221 plugs were included. The overall rate of chondral fracture was 45.7%. There was a significant difference in the rate of fracture between regions, with LIN, MIN, LT, and MT, having a fracture rate of 46.6%, 62.7%, 25.0%, and 51.9%, respectively ( P = 0.001). An impact height of 30 mm resulted in a fracture rate of 17.7%. Increasing the impact height from 30 to 50 mm resulted in significantly increased chondral fracture risk ( P = 0.001)., Conclusion: Different donor regions have varying rates of chondral fracture during OAT plug impaction, with the lateral and medial trochlea being the most resistant to chondral fracture at lower forces. Increased impact energy increases risk of chondral fracture. Surgeons should maintain caution and utilize lower impact energy when inserting OAT plugs., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. more...

Tumor-derived extracellular vesicles (TEVs) induce the epithelial-to-mesenchymal transition (EMT) in nonmalignant cells to promote invasion and cancer metastasis, representing a novel therapeutic target in a field severely lacking in efficacious antimetastasis treatments. However, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are absent. Here, the development of a functional phenotypic screening platform based on organic electrochemical transistors (OECTs) for real-time, noninvasive monitoring of TEV-induced EMT and screening of antimetastatic drugs is reported. TEVs derived from the triple-negative breast cancer cell line MDA-MB-231 induce EMT in nonmalignant breast epithelial cells (MCF10A) over a nine-day period, recapitulating a model of invasive ductal carcinoma metastasis. Immunoblot analysis and immunofluorescence imaging confirm the EMT status of TEV-treated cells, while dual optical and electrical readouts of cell phenotype are obtained using OECTs. Further, heparin, a competitive inhibitor of cell surface receptors, is identified as an effective blocker of TEV-induced EMT. Together, these results demonstrate the utility of the platform for TEV-targeted drug discovery, allowing for facile modeling of the transient drug response using electrical measurements, and provide proof of concept that inhibitors of TEV function have potential as antimetastatic drug candidates., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.) more...

Amplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the catalytic subunit of PP6, have amplified Aurora A activity, and as we show here, enlarged mitotic spindles which fail to hold chromosomes tightly together in anaphase, causing defective nuclear structure. Using functional genomics to shed light on the processes underpinning these changes, we discover synthetic lethality between PPP6C and the kinetochore protein NDC80. We find that NDC80 is phosphorylated on multiple N-terminal sites during spindle formation by Aurora A-TPX2, exclusively at checkpoint-silenced, microtubule-attached kinetochores. NDC80 phosphorylation persists until spindle disassembly in telophase, is increased in PPP6C knockout cells, and is Aurora B-independent. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C knockout cells. In regulating NDC80 phosphorylation by Aurora A-TPX2, PP6 plays an important role in mitotic spindle formation and size control and thus the fidelity of cell division., (© 2023 Sobajima et al.) more...

In eukaryotes, the spindle assembly checkpoint protects genome stability in mitosis by preventing chromosome segregation until incorrect microtubule-kinetochore attachment geometries have been eliminated and chromosome biorientation has been completed. These error correction and checkpoint processes are linked by the conserved Aurora B and MPS1 Ser/Thr kinases. 1 , 2 MPS1-dependent checkpoint signaling is believed to be initiated by kinetochores without end-on microtubule attachments, 3 , 4 including those generated by Aurora B-mediated error correction. The current model posits that MPS1 competes with microtubules for binding sites at the kinetochore. 3 , 4 MPS1 is thought to first recognize kinetochores not blocked by microtubules and then initiate checkpoint signaling. However, MPS1 is also required for chromosome biorientation and correction of microtubule-kinetochore attachment errors. 5 , 6 , 7 , 8 , 9 This latter function, which must require direct interaction with microtubule-attached kinetochores, is not readily explained within the constraints of the current model. Here, we show that MPS1 transiently localizes to end-on attached kinetochores and that this recruitment depends on the relative activities of Aurora B and its counteracting phosphatase PP2A-B56 rather than microtubule-attachment state per se. MPS1 autophosphorylation also regulates MPS1 kinetochore levels but does not determine the response to microtubule attachment. At end-on attached kinetochores, MPS1 actively promotes microtubule release together with Aurora B. Furthermore, in live cells, MPS1 is detected at attached kinetochores before the removal of microtubules. During chromosome alignment, MPS1, therefore, coordinates both the resolution of incorrect microtubule-kinetochore attachments and the initiation of spindle checkpoint signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.) more...

Nuclear architecture and functions depend on dynamic interactions between nuclear components (such as chromatin) and inner nuclear membrane (INM) proteins. Mutations in INM proteins interfering with these interactions result in disease. However, mechanisms controlling the levels and turnover of INM proteins remain unknown. Here, we describe a mechanism of regulated degradation of the INM SUN domain-containing protein 2 (SUN2). We show that Casein Kinase 2 and the C-terminal domain Nuclear Envelope Phosphatase 1 (CTDNEP1) have opposing effects on SUN2 levels by regulating SUN2 binding to the ubiquitin ligase Skp/Cullin1/F-Box βTrCP (SCF βTrCP ). Upon binding to phosphorylated SUN2, SCF βTrCP promotes its ubiquitination. Ubiquitinated SUN2 is membrane extracted by the AAA ATPase p97 and delivered to the proteasome for degradation. Importantly, accumulation of non-degradable SUN2 results in aberrant nuclear architecture, vulnerability to DNA damage and increased lagging chromosomes in mitosis. These findings uncover a central role of proteolysis in INM protein homeostasis., Competing Interests: LK, WS, MV, DH, EG, UG, PC No competing interests declared, (© 2022, Krshnan et al.) more...

Background: Effective surveillance strategies are required for patients diagnosed with oesophageal squamous cell carcinoma (OSCC) or adenocarcinoma (OAC) for whom chemoradiotherapy (CRT) is used as a potentially-curative, organ-sparing, alternative to surgery. In this study, we evaluated the safety, acceptability and tolerability of a non-endoscopic immunocytological device (the Cytosponge™) to assess treatment response following CRT., Methods: This multicentre, single-arm feasibility trial took place in 10 tertiary cancer centres in the UK. Patients aged at least 16 years diagnosed with OSCC or OAC, and who were within 4-16 weeks of completing definitive or neo-adjuvant CRT, were included. Participants were required to have a Mellow-Pinkas dysphagia score of 0-2 and be able to swallow tablets. All patients underwent a single Cytosponge™ assessment in addition to standard of care (which included post-treatment endoscopic evaluation with biopsy for patients undergoing definitive CRT; surgery for those who received neo-adjuvant CRT). The primary outcome was the proportion of consented, evaluable patients who successfully underwent Cytosponge™ assessment. Secondary and tertiary outcomes included safety, study consent rate, acceptance rate, the suitability of obtained samples for biomarker analysis, and the comparative efficacy of Cytosponge™ to standard histology (endoscopy and biopsy or post-resection specimen) in assessing for residual disease. The trial is registered with ClinicalTrials.gov, NCT03529669., Findings: Between 18 th April 2018 and 16 th January 2020, 41 (42.7%; 95% confidence interval (CI) 32.7-53.2) of 96 potentially eligible patients consented to participate. Thirty-nine (95.1%, 95% CI 83.5-99.4) successfully carried out the Cytosponge™ procedure. Of these, 37 (95%) would be prepared to repeat the procedure. There were only two grade 1 adverse events attributed to use of the Cytosponge™. Thirty-five (90%) of the completed Cytosponge™ samples were suitable for biomarker analysis; 29 (83%) of these were concordant with endoscopic biopsies, three (9%) had findings suggestive of residual cancer on Cytosponge™ not found on endoscopic biopsies, and three (9%) had residual cancer on endoscopic biopsies not detected by Cytosponge™., Interpretation: Use of the Cytosponge TM is safe, tolerable, and acceptable for the assessment of treatment response following CRT in OAC and OSCC. Further evaluation of Cytosponge™ in this setting is warranted., Funding: Cancer Research UK, National Institute for Health Research, Medical Research Council., Competing Interests: The Cytosponge™ technology including the device and TFF3 biomarker has been licensed by the MRC to Covidien (now Medtronic). R.C.F. and M.O.D. are named on patents related to this test. R.C.F. and M.O.D. are shareholders and consultants for Cyted, an early detection company. All other authors declare no competing interests., (© 2022 Published by Elsevier Ltd.) more...

As the primary genetic determinant of immune recognition of self and non-self, the hyperpolymorphic HLA genes play key roles in disease association and transplantation. The large, variably sized HLA class II genes have historically been less well characterized than the shorter HLA class I genes. Here, we have used Pacific Biosciences Single Molecule Real-Time (SMRT®) DNA sequencing to perform four-field resolution HLA typing of HLA-DRB1/3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 from a panel of 181 B-lymphoblastoid cell lines from the International HLA and Immunogenetics Workshops. By interrogating all exons, introns, and the untranslated regions of these important reference cells, we have improved their HLA typing resolution on the IPD-IMGT/HLA database. We observed widespread non-coding polymorphism, with over twice as many unique genomic sequences identified compared with coding sequences (CDS). We submitted 263 unique sequences to the IPD-IMGT/HLA Database, often from multiple cell lines, including 114 confirmations of existing alleles, of which 30 were also extensions to full-length genomic sequences where only CDS was available previously. A total of 149 novel alleles were identified, largely differing from their closest reference allele sequences by a single nucleotide polymorphism (SNP). However, some highly divergent alleles were deemed to be recombinants, only detectable by full-length sequencing with long, phased reads. The fourth-field variation we observed allowed fine mapping of linkage disequilibrium patterns and haplotypes to particular ancestries. This study has highlighted the under-appreciated non-coding diversity in HLA class II genes, with potential implications for population genetic and clinical studies., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.) more...

During cell division, misaligned chromosomes are captured and aligned by motors before their segregation. The CENP-E motor is recruited to polar unattached kinetochores to facilitate chromosome alignment. The spindle checkpoint protein BubR1 (also known as BUB1B) has been reported as a CENP-E interacting partner, but the extent to which BubR1 contributes to CENP-E localization at kinetochores has remained controversial. Here we define the molecular determinants that specify the interaction between BubR1 and CENP-E. The basic C-terminal helix of BubR1 is necessary but not sufficient for CENP-E interaction, and a minimal key acidic patch on the kinetochore-targeting domain of CENP-E is also essential. We then demonstrate that BubR1 is required for the recruitment of CENP-E to kinetochores to facilitate chromosome alignment. This BubR1-CENP-E axis is critical for alignment of chromosomes that have failed to congress through other pathways and recapitulates the major known function of CENP-E. Overall, our studies define the molecular basis and the function for CENP-E recruitment to BubR1 at kinetochores during mammalian mitosis.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.) more...

During mitosis, the formation of microtubule-kinetochore attachments is monitored by the serine/threonine kinase monopolar spindle 1 (MPS1). MPS1 is recruited to unattached kinetochores where it phosphorylates KNL1, BUB1, and MAD1 to initiate the spindle assembly checkpoint. This arrests the cell cycle until all kinetochores have been stably captured by microtubules. MPS1 also contributes to the error correction process rectifying incorrect kinetochore attachments. MPS1 activity at kinetochores requires autophosphorylation at multiple sites including threonine 676 in the activation segment or "T-loop." We now demonstrate that the BUBR1-bound pool of PP2A-B56 regulates MPS1 T-loop autophosphorylation and hence activation status in mammalian cells. Overriding this regulation using phosphomimetic mutations in the MPS1 T-loop to generate a constitutively active kinase results in a prolonged mitotic arrest with continuous turnover of microtubule-kinetochore attachments. Dynamic regulation of MPS1 catalytic activity by kinetochore-localized PP2A-B56 is thus critical for controlled MPS1 activity and timely cell cycle progression., (© 2019 Hayward et al.) more...

In mitosis, the spindle assembly checkpoint (SAC) monitors the formation of microtubule-kinetochore attachments during capture of chromosomes by the mitotic spindle. Spindle assembly is complete once there are no longer any unattached kinetochores. Here, we will discuss the mechanism and key components of spindle checkpoint signalling. Unattached kinetochores bind the principal spindle checkpoint kinase monopolar spindle 1 (MPS1). MPS1 triggers the recruitment of other spindle checkpoint proteins and the formation of a soluble inhibitor of anaphase, thus preventing exit from mitosis. On microtubule attachment, kinetochores become checkpoint silent due to the actions of PP2A-B56 and PP1. This SAC responsive period has to be coordinated with mitotic spindle formation to ensure timely mitotic exit and accurate chromosome segregation. We focus on the molecular mechanisms by which the SAC permissive state is created, describing a central role for CDK1-cyclin B1 and its counteracting phosphatase PP2A-B55. Furthermore, we discuss how CDK1-cyclin B1, through its interaction with MAD1, acts as an integral component of the SAC, and actively orchestrates checkpoint signalling and thus contributes to the faithful execution of mitosis., (© 2019 Federation of European Biochemical Societies.) more...

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1 S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1 S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis., (© 2019 Hayward et al.) more...

Cyclin B-dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis., (© 2019 Alfonso-Pérez et al.) more...

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells., Competing Interests: CP, EB, FR, DH, AP, JC, SB, JW, MG, MS No competing interests declared, (© 2018, Pellacani et al.) more...

Purpose: The purpose of this study was to assess the diagnostic accuracy of 18F-fluorodeoxyglucose with positron emission tomography and computed tomography (FDG-PET-CT) to predict nodal metastases in patients with bladder cancer (BC) scheduled to undergo radical cystectomy (RC)., Methods: We retrospectively reviewed records of patients diagnosed with BC and scheduled to undergo RC at our center from January 2011 through February 2015, who also underwent FDG-PET-CT at the time of diagnosis. All patients underwent RC and an extended pelvic lymph node dissection as the reference standard. The primary endpoints were the sensitivity, specificity and overall accuracy of FDG-PET-CT in detecting lymph node metastasis. We also examined its accuracy in identifying distant metastasis. In addition, we conducted a protocol-driven systematic review and meta-analysis of accuracy of FDG-PET-CT for preoperative staging of BC, as compared to CT alone, as reported in individual studies. To assess the methodological quality of eligible studies, we used the QUADAS-2 tool (a revised tool for the Quality Assessment of Diagnostic Accuracy Studies) and pooled diagnostic accuracy measures using Meta-DiSc statistical software., Results: For detecting nodal metastases in 78 patients, the sensitivity of FDG-PET-CT was 0.56 (95 % CI 0.29-0.80) and the specificity, 0.98 (95 % CI 0.91-1.00). Pooled sensitivity and specificity for detecting lymph node metastasis were 0.57 and 0.95, respectively. Positive likelihood ratio was 9.02. All lesions that were suspicious for distant metastasis were found to be positive on biopsy., Conclusion: FDG-PET-CT was more accurate than CT alone in staging BC in patients undergoing surgery. Standardization of FDG-PET-CT protocol and cost-effectiveness analysis are required before widespread implementation of this technology. more...

Chromatin-associated proteins are essential for the specification and maintenance of cell identity. They exert these functions through modulating and maintaining transcriptional patterns. To elucidate the functions of the Jmjd2 family of H3K9/H3K36 histone demethylases, we generated conditional Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c/Gasc1 single, double and triple knockout mouse embryonic stem cells (ESCs). We report that while individual Jmjd2 family members are dispensable for ESC maintenance and embryogenesis, combined deficiency for specifically Jmjd2a and Jmjd2c leads to early embryonic lethality and impaired ESC self-renewal, with spontaneous differentiation towards primitive endoderm under permissive culture conditions. We further show that Jmjd2a and Jmjd2c both localize to H3K4me3-positive promoters, where they have widespread and redundant roles in preventing accumulation of H3K9me3 and H3K36me3. Jmjd2 catalytic activity is required for ESC maintenance, and increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity., (© 2016 The Authors.) more...

The spindle assembly checkpoint (SAC) ensures proper chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Checkpoint signalling requires the kinetochore localization of the Mad1-Mad2 complex that in more complex eukaryotes depends on the Rod-Zwilch-ZW10 (RZZ) complex. The kinetochore protein Zwint has been proposed to be the kinetochore receptor for RZZ, but here we show that Bub1 and not Zwint is required for RZZ recruitment. We find that the middle region of Bub1 encompassing a domain essential for SAC signalling contributes to RZZ localization. In addition, we show that a distinct region in Bub1 mediates kinetochore localization of BubR1 through direct binding, but surprisingly removal of this region increases checkpoint strength. Our work thus uncovers how Bub1 coordinates checkpoint signalling by distinct domains for RZZ and BubR1 recruitment and suggests that Bub1 localizes antagonistic checkpoint activities. more...

Microinjection is a powerful technique that can be used to study protein function. Early Drosophila embryos are particularly amenable to microinjection due to their large size and their single cell status. Here, we report methods to microinject these embryos with various reagents to study the function of proteins at centrosomes and centrosome function more generally. Although precise details vary between laboratories, many aspects of the process are conserved. We describe the process from setting up a fly cage to imaging the injected embryos on a spinning disk confocal microscope and use specific examples to highlight the potency of this technique., (Copyright © 2015 Elsevier Inc. All rights reserved.) more...

During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation. more...

Upon entry into mitosis, many microtubules are nucleated that coordinately integrate into a stable, yet dynamic, mitotic spindle apparatus. In a recent publication, we examined microtubule-generating pathways within a single model system, the Drosophila syncytial embryo. We found that, following depolymerisation of metaphase spindle microtubules by cold treatment, spindles regenerate predominantly from microtubules nucleated within the vicinity of chromatin. We also showed this chromatin-mediated microtubule nucleation is mediated by the Drosophila homolog of a vertebrate spindle assembly factor (SAF), HURP and is dependent on the conserved microtubule amplifying protein complex, Augmin. Here, we expand our investigation into Drosophila SAFs, providing evidence that, in vitro, both D-HURP and D-TPX2 are able to bind to and stabilize microtubules. We show that GFP-D-HURP purified from embryos interacts with Importin-β and Augmin and, consistent with this, demonstrate that the underlying basis of chromatin-mediated microtubule nucleation in Drosophila syncytial embryos is dependent on Ran-GTP. more...

The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. We have tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. Our work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.) more...

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC. more...

Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, whereas depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. The authors used a radiometric proximity assay in a high-throughput screen to identify small-molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabeled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microplates. Seventy nonaggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualized using pericentrin antibodies confirmed that 2 of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show that pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery. more...

Objectives: To determine the contemporary prevalence of and mortality in patients with coronary artery disease (CAD) not amenable to revascularization., Background: A growing number of patients have severe CAD with ongoing angina despite optimal medical therapy which is not amenable to traditional revascularization. Limited data exist on contemporary prevalence and outcome for these patients., Methods: Clinical and angiographic data were reviewed for 493 consecutive patients undergoing coronary angiography and revascularization if indicated. Patients were categorized into six groups: (1) normal coronary arteries, (2) CAD <70%, (3) CAD >70% with complete revascularization by percutaneous intervention or coronary artery bypass grafting, (4) CAD >70% with partial revascularization, (5) CAD >70% treated medically, and (6) CAD >70% on optimal medical therapy with no revascularization option. All-cause mortality at 3 years was determined., Results: Prevalence for groups 1-6 was 14.8, 19.5, 36.9, 12.8, 9.3, and 6.7%, respectively. Three-year mortality increased with angiographic severity of CAD: 2.7, 6.3, 8.2, 12.7, 17.4, and 15.2%, respectively. Patients with incomplete revascularization (groups 4-6, n = 142) had higher mortality than completely revascularized patients (groups 1-3, n = 351): 14.8 vs. 6.6% (P = 0.004)., Conclusions: In a contemporary series of patients undergoing coronary angiography, 28.8% (142/493) of patients had significant CAD and did not undergo complete revascularization, including 12.8% partially revascularized, 9.3% managed medically, and 6.7% with "no-option." These patients had higher mortality at 3 years (14.8 vs. 6.6%, P = 0.004) when compared with completely revascularized patients., (Copyright 2010 Wiley-Liss, Inc.) more...

Nek2 is a cell cycle-regulated serine/threonine protein kinase that is up-regulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic C termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic, and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A having a weak NLS, and Nek2B having no NLS. Finally, we identified a 28-kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization, enabling it to have both nuclear and cytoplasmic functions. more...

Aneuploidy and chromosome instability are two of the most common abnormalities in cancer cells. They arise through defects in cell division and, specifically, in the unequal segregation of chromosomes between daughter cells during mitosis. A number of cell cycle dependent protein kinases have been identified that control mitotic progression and chromosome segregation. Some of these localize to the centrosome and regulate mitotic spindle formation. One such protein is Nek2, a member of the NIMA-related serine/threonine kinase family. Data are emerging that Nek2 is abnormally expressed in a wide variety of human cancers. In this review, we summarize current knowledge on the expression, regulation and function of Nek2, consider how Nek2 may contribute to chromosome instability, and ask whether it might make an attractive target for chemotherapeutic intervention in human cancer. more...

Aneuploidy and chromosome instability are common abnormalities in human cancer. Loss of control over mitotic progression, multipolar spindle formation, and cytokinesis defects are all likely to contribute to these phenotypes. Nek2 is a cell cycle-regulated protein kinase with maximal activity at the onset of mitosis that localizes to the centrosome. Functional studies have implicated Nek2 in regulation of centrosome separation and spindle formation. Here, we present the first study of the protein expression levels of the Nek2 kinase in human cancer cell lines and primary tumors. Nek2 protein is elevated 2- to 5-fold in cell lines derived from a range of human tumors including those of cervical, ovarian, breast, prostate, and leukemic origin. Most importantly, by immunohistochemistry, we find that Nek2 protein is significantly up-regulated in preinvasive in situ ductal carcinomas of the breast as well as in invasive breast carcinomas. Finally, by ectopic expression of Nek2A in immortalized HBL100 breast epithelial cells, we show that increased Nek2 protein leads to accumulation of multinucleated cells with supernumerary centrosomes. These data highlight the Nek2 kinase as novel potential target for chemotherapeutic intervention in breast cancer. more...