Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users

Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

A meta-analysis of genome-wide association studies of epigenetic age acceleration

Funding: Generation Scotland received core support from the Chief Scientist Office of the Scottish Government Health Directorates (CZD/16/6) and the Scottish Funding Council (HR03006). Genotyping and DNA methylation profiling of the GS samples was carried out by the Genetics Core Laboratory at the Wellcome Trust Clinical Research Facility, Edinburgh, Scotland and was funded by the Medical Research Council UK and the Wellcome Trust (Wellcome Trust Strategic Award “STratifying Resilience and Depression Longitudinally” ((STRADL) Reference 104036/Z/14/Z)). Funding details for the cohorts included in the study by Lu et al. (2018) can be found in their publication. HCW is supported by a JMAS SIM fellowship from the Royal College of Physicians of Edinburgh and by an ESAT College Fellowship from the University of Edinburgh. AMM & HCW acknowledge the support of the Dr. Mortimer and Theresa Sackler Foundation. SH acknowledges support from grant 1U01AG060908-01. REM is supported by Alzheimer’s Research UK major project grant ARUK-PG2017B-10. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: Summary statistics from the research reported in the manuscript will be made available immediately following publication on the Edinburgh Data Share portal with a permanent digital object identifier (DOI). According to the terms of consent for Generation Scotland participants, requests for access to the individual-level data must be reviewed by the GS Access Committee ([email protected]). Individual-level data are not immediately available, due to confidentiality considerations and our legal obligation to protect personal information. These data will, however, be made available upon request and after review by the GS access committee, once ethical and data governance concerns regarding personal data have been addressed by the receiving institution through a Data Transfer Agreement.Peer reviewedPublisher PD

On the road to cancer:Aneuploidy and the mitotic checkpoint

Abnormal chromosome content - also known as aneuploidy - is the most common characteristic of human solid tumours. It has therefore been proposed that aneuploidy contributes to, or even drives, tumour development. The mitotic checkpoint guards against chromosome mis-segregation by delaying cell-cycle progression through mitosis until all chromosomes have successfully made spindle-microtubule attachments. Defects in the mitotic checkpoint generate aneuploidy and might facilitate tumorigenesis, but more severe disabling of checkpoint signalling is a possible anticancer strategy

Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness