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Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to the cell equator before segregation in anaphase. Microtubules sprout from nucleation sites such as centrosomes, but kinetochores can also promote microtubule formation. It is unclear, however, how kinetochore-derived microtubules are generated and what their role is in chromosome segregation. Here, we show that the transient outer-kinetochore meshwork known as the fibrous corona serves as an autonomous microtubule nucleation platform. The fibrous corona is essential for the nucleation of kinetochore-derived microtubules, and when dissociated from the core kinetochore, it retains microtubule nucleation capacity. Nucleation relies on a fibrous-corona-bound pool of the LIC1 subunit of the dynein motor complex, which interacts with the γ-tubulin-tethering protein pericentrin (PCNT). PCNT is essential for microtubule nucleation from fibrous coronas, and in centrosome-depleted cells, where nearly all mitotic nucleation occurs at fibrous coronas, chromosome congression is fully dependent on PCNT. We further show that chromosomes in bovine oocytes, which naturally lack centrosomes, have highly expanded fibrous coronas that drive chromosome-derived microtubule nucleation. Preventing fibrous corona expansion in these cells impairs chromosome congression and causes spindle assembly defects. Our results show that fibrous coronas are autonomous microtubule-organizing centers that are important for spindle assembly, which may be especially relevant in acentrosomal cells such as oocytes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1-3]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3, 4]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity. By placing MPS1sen at different subcellular locations, we show that MPS1 activity initiates in the nucleus ∼9-12 min prior to nuclear envelope breakdown (NEB) in a kinetochore-dependent manner and reaches the cytoplasm at the start of NEB. Soon after initiation, MPS1 activity increases with switch-like kinetics, peaking at completion of NEB. We further show that timing and extent of pre-NEB MPS1 activity is regulated by Aurora B and PP2A-B56. MPS1sen phosphorylation declines in prometaphase as a result of formation of kinetochore-microtubule attachments, reaching low but still detectable levels at metaphase. Finally, leveraging the sensitivity and dynamic range of MPS1sen, we show deregulated MPS1 signaling dynamics in colorectal cancer cell lines and tumor organoids with diverse genomic instability phenotypes., Competing Interests: Declaration of Interests A patent application has been filed by Stichting Oncode Institute for the technology disclosed in this publication., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microtubules are essential for polarized transport in neurons, but how their organization guides motor proteins to axons or dendrites is unclear. Because different motors recognize distinct microtubule properties, we used optical nanoscopy to examine the relationship between microtubule orientations, stability, and modifications. Nanometric tracking of motors to super-resolve microtubules and determine their polarity revealed that in dendrites, stable and acetylated microtubules are mostly oriented minus-end out, while dynamic and tyrosinated microtubules are oriented oppositely. In addition, microtubules with similar orientations and modifications form bundles that bias transport. Importantly, because the plus-end-directed Kinesin-1 selectively interacts with acetylated microtubules, this organization guides this motor out of dendrites and into axons. In contrast, Kinesin-3 prefers tyrosinated microtubules and can enter both axons and dendrites. This separation of distinct microtubule subsets into oppositely oriented bundles constitutes a key architectural principle of the neuronal microtubule cytoskeleton that enables polarized sorting by different motor proteins., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)