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

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

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

Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface

Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface

Guided by Light : Optical Control of Microtubule Gliding Assays

Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays in which surface-immobilized motor proteins drive microtubule propulsion are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, biocomputation, and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ∼5-fold enrichment within 6 s upon illumination. Subsequently, proteins are released with a half-life of 13 s when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of nonuniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications

Guided by light: Optical control of microtubule gliding assays

Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays in which surface-immobilized motor proteins drive microtubule propulsion are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, biocomputation, and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a â5-fold enrichment within 6 s upon illumination. Subsequently, proteins are released with a half-life of 13 s when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of nonuniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.BN/Marileen Dogterom LabBN/Bionanoscienc