Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface

Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.Smith Family FoundationMassachusetts Life Sciences CenterKinship Foundation. Searle Scholars ProgramNational Institute of General Medical Sciences (U.S.) (Grant number GM088313

TechEthos D5.5 Complementing the ALLEA European Code of Conduct for Research Integrity

<p>The European Code of Conduct for Research Integrity (hereafter named the "European Code of Conduct"), published by the European Federation of Academies of Sciences and Humanities (ALLEA), serves the European research community as a framework for self-regulation across all scientific and scholarly disciplines and for all research settings. The latest revision of the European Code of Conduct was <a href="https://allea.org/allea-publishes-2023-revised-edition-of-the-european-code-of-conduct-for-research-integrity/">released in June 2023</a> and takes account of the latest social, political, and technological developments, as well as trends emerging in the research landscape. These revisions took place in the context of the EU-funded TechEthos project, with the aim to also identify gaps and necessary additions related to the integration of ethics in research protocols and the possible implications of new technologies and their applications.</p><p>In addition to extensive input from within the ALLEA community, detailed feedback from 31 stakeholder organisations and projects was considered during the revision of the European Code of Conduct. As part of this stakeholder consultation process, the views of the TechEthos consortium partners were collected both in writing and during an online workshop. In addition to input on broader research integrity issues, the unique expertise within the TechEthos consortium allowed for the collection of critical input on the ethical, legal, and societal aspects related to the development and application of new technologies, as well as on the responsible use of new technologies in collecting, analysing, and publishing research results. This deliverable provides a short history of the European Code of Conduct, details the revision process leading to the recently published edition, summarizes the feedback from the TechEthos consortium and implementation thereof, and describes current and ongoing communication and dissemination activities. </p><p>The full version of the European Code of Conduct, as well as a document that summarizes the stakeholder consultation process and its outcomes, can be accessed directly via <a href="https://allea.org/code-of-conduct/">https://allea.org/code-of-conduct/</a>:</p><ul><li>ALLEA (2023) The European Code of Conduct for Research Integrity – Revised Edition 2023. Berlin. DOI 10.26356/ECOC. (<a href="https://allea.org/wp-content/uploads/2023/06/European-Code-of-Conduct-Revised-Edition-2023.pdf">link</a>)</li><li>Summary and Outcomes of the Stakeholder Consultation. (<a href="https://allea.org/wp-content/uploads/2023/06/Feedback-to-Stakeholders-on-2023-ECoC-Revision.pdf">link</a>)</li></ul&gt

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle

Evolution and Function of the Mitotic Checkpoint

The mitotic checkpoint evolved to prevent cell division when chromosomes have not established connections with the chromosome segregation machinery. Many of the fundamental molecular principles that underlie the checkpoint, its spatiotemporal activation, and its timely inactivation have been uncovered. Most of these are conserved in eukaryotes, but important differences between species exist. Here we review current concepts of mitotic checkpoint activation and silencing. Guided by studies in model organisms and our phylogenomics analysis of checkpoint constituents and their functional domains and motifs, we highlight ancient and taxa-specific aspects of the core checkpoint modules in the context of mitotic checkpoint function

Integration of Kinase and Phosphatase Activities by BUBR1 Ensures Formation of Stable Kinetochore-Microtubule Attachments

SummaryMaintenance of chromosomal stability depends on error-free chromosome segregation. The pseudokinase BUBR1 is essential for this, because it is a core component of the mitotic checkpoint and is required for formation of stable kinetochore-microtubule attachments. We have identified a conserved and highly phosphorylated domain (KARD) in BUBR1 that is crucial for formation of kinetochore-microtubule attachments. Deletion of this domain or prevention of its phosphorylation abolishes formation of kinetochore microtubules, which can be reverted by inhibiting Aurora B activity. Phosphorylation of KARD by PLK1 promotes direct interaction of BUBR1 with the PP2A-B56α phosphatase that counters excessive Aurora B activity at kinetochores. As a result, removal of BUBR1 from mitotic cells or inhibition of PLK1 reduces PP2A-B56α kinetochore binding and elevates phosphorylation of Aurora B substrates on the outer kinetochore. We propose that PLK1 and BUBR1 cooperate to stabilize kinetochore-microtubule interactions by regulating PP2A-B56α-mediated dephosphorylation of Aurora B substrates at the kinetochore-microtubule interface

Reconstitution of basic mitotic spindles in cell-like confinement

Bipolar organization of the mitotic spindle is the result of forces generated by dynamic microtubules and associated proteins in interaction with chromosomes and the cell boundary1–4. Biophysical experiments on isolated spindle components have provided important insights into the force-generating properties of different components5–8, but a quantitative understanding of the force balance that results from their concerted action is lacking. Here we present an experimental platform based on water-in-oil emulsion droplets that allows for the bottom-up reconstitution of basic spindles. We find a typical metaphase organization, where two microtubule asters position symmetrically at moderate distance from the mid-zone, is readily obtained even in the absence of chromosomes. Consistent with simulations, we observe an intrinsic repulsive force between two asters that can be counterbalanced alternatively by cortical pulling forces, anti-parallel microtubule crosslinking, or adjustment of microtubule dynamics, emphasizing the robustness of the system. Adding motor proteins that slide anti-parallel microtubules apart drives the asters to maximum separation, as observed in cells during anaphase9,10. Our platform offers a valuable complementary approach to in vivo experiments where essential mitotic components are typically removed, instead of added, one by one.</jats:p

Inner centromere localization of the CPC maintains centromere cohesion and allows mitotic checkpoint silencing

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation

Dissecting the roles of human BUB1 in the spindle assembly checkpoint

Mitotic chromosome segregation is initiated by the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC) when chromosomes have not attached to spindle microtubules. Unattached kinetochores catalyze the formation of a diffusible APC/CCDC20 inhibitor that is composed of BUBR1, BUB3, MAD2 and a second molecule of CDC20. Kinetochore recruitment of these proteins as well as SAC activation rely on the mitotic kinase BUB1, but the molecular mechanism by which BUB1 accomplishes this in human cells is unknown. We show that BUBR1 and BUB3 kinetochore recruitment by BUB1 is dispensable for SAC activation. Unlike its yeast and nematode orthologs, human BUB1 does not associate stably with the MAD2 activator MAD1 and, although required for accelerating loading of MAD1 onto kinetochores, is dispensable for its steady-state levels there. Instead, we identify a 50 amino acid segment harboring the recently reported ABBA motif close to a KEN box as critical for BUB1's role in SAC signaling. The presence of this segment correlates with SAC activity and efficient binding of CDC20 but not MAD1 to kinetochores.</jats:p