UNDERSTANDING THE SPINDLE ASSEMBLY CHECKPOINT AND THE ERROR CORRECTION MACHINERY WITHIN THE KINETOCHORE FRAMEWORK

Kinetochores are large protein assemblies built on chromosomal loci named centromeres. Four distinct modules accomplish the main functions of kinetochores. The first module, in the inner kinetochore, contributes a sturdy interface with centromeric chromatin. The second module, the outer kinetochore, contributes a microtubule-binding interface. The third module, the spindle assembly checkpoint, is a feedback control mechanism that monitors the state of kinetochore-microtubule attachment to control progression of the cell cycle. The fourth module discerns correct from improper attachments, preventing the stabilization of the latter and allowing the selective stabilization of the former. The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome bi-orientation on the mitotic spindle. In this thesis, I report work showing that the small-molecule Reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the RZZ complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using Reversine, I also demonstrated that MPS1 is required for the correction of tensionless chromosome-microtubule attachments. An important conclusion from this work is that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. My studies describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle assembly checkpoint, and suggest that these processes are co-regulated and are likely to involve the same catalytic machinery

The pleiotropic deubiquitinase Ubp3 confers aneuploidy tolerance

Aneuploidy-or an unbalanced karyotype in which whole chromosomes are gained or lost-causes reduced fitness at both the cellular and organismal levels but is also a hallmark of human cancers. Aneuploidy causes a variety of cellular stresses, including genomic instability, proteotoxic and oxidative stresses, and impaired protein trafficking. The deubiquitinase Ubp3, which was identified by a genome-wide screen for gene deletions that impair the fitness of aneuploid yeast, is a key regulator of aneuploid cell homeostasis. We show that deletion of UBP3 exacerbates both karyotype-specific phenotypes and global stresses of aneuploid cells, including oxidative and proteotoxic stress. Indeed, Ubp3 is essential for proper proteasome function in euploid cells, and deletion of this deubiquitinase leads to further proteasome-mediated proteotoxicity in aneuploid yeast. Notably, the importance of UBP3 in aneuploid cells is conserved. Depletion of the human homolog of UBP3, USP10, is detrimental to the fitness of human cells upon chromosome missegregation, and this fitness defect is accompanied by autophagy inhibition. We thus used a genome-wide screen in yeast to identify a guardian of aneuploid cell fitness conserved across species. We propose that interfering with Ubp3/USP10 function could be a productive avenue in the development of novel cancer therapeutics

A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

In mitosis, the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic spindle checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate. The spindle checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened checkpoint signaling system that may contribute to chromosome instability in tumors. Because complete absence of the spindle checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the checkpoint might provide at therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study, we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel

Psychological care, patient education, orthotics, ergonomics and prevention strategies for neck pain: a systematic overview update as part of the ICON project

Objectives: To conduct an overview on psychological interventions, orthoses, patient education, ergonomics, and 1°/2° neck pain prevention for adults with acute-chronic neck pain. Search Strategy: Computerized databases and grey literature were searched (2006-2012). Selection Criteria: Systematic reviews of randomized controlled trials (RCTs) on pain, function/disability, global perceived effect, quality-of-life and patient satisfaction were retrieved. Data Collection & Analysis: Two independent authors selected articles, assessed risk of bias using AMSTAR tool and extracted data. The GRADE tool was used to evaluate the body of evidence and an external panel to provide critical review. Main Results: We retrieved 30 reviews (5-9 AMSTAR score) reporting on 75 RCTs with the following moderate GRADE evidence. For acute whiplash associated disorder (WAD), an education video in emergency rooms (1RCT, 405participants] favoured pain reduction at long-term follow-up thus helping 1 in 23 people [Standard Mean Difference: - 0.44(95%CI: -0.66 to -0.23)). Use of a soft collar (2RCTs, 1278participants) was not beneficial in the long-term. For chronic neck pain, a mind-body intervention (2RCTs, 1 meta-analysis, 191participants) improved short-term pain/function in 1 of 4 or 6 participants. In workers, 2-minutes of daily scapula-thoracic endurance training (1RCT, 127participants) over 10 weeks was beneficial in 1 of 4 participants. A number of psychosocial interventions, workplace interventions, collar use and self-management educational strategies were not beneficial. Reviewers' Conclusions: Moderate evidence exists for quantifying beneficial and non-beneficial effects of a limited number of interventions for acute WAD and chronic neck pain. Larger trials with more rigorous controls need to target promising interventions

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore

Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

To satisfy the mitotic checkpoint and drive chromosome congression, the Mps1 kinase lets go of kinetochores by phosphorylating itself in trans (see also related papers by Maciejowski et al. and Santaguida et al. in this issue)

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis

CONSORT Harms 2022 statement, explanation, and elaboration: updated guideline for the reporting of harms in randomized trials.

Randomized controlled trials remain the reference standard for healthcare research on effects of interventions, and the need to report both benefits and harms is essential. The Consolidated Standards of Reporting Trials (the main CONSORT) statement includes one item on reporting harms (i.e., all important harms or unintended effects in each group). In 2004, the CONSORT group developed the CONSORT Harms extension; however, it has not been consistently applied and needs to be updated. Here, we describe CONSORT Harms 2022, which replaces the CONSORT Harms 2004 checklist, and shows how CONSORT Harms 2022 items could be incorporated into the main CONSORT checklist. Thirteen items from the main CONSORT were modified to improve harms reporting. Three new items were added. In this article, we describe CONSORT Harms 2022 and how it was integrated into the main CONSORT checklist and elaborate on each item relevant to complete reporting of harms in randomized controlled trials. Until future work from the CONSORT group produces an updated checklist, authors, journal reviewers, and editors of randomized controlled trials should use the integrated checklist presented in this paper