Errorless chromosome segregation requires load-bearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Both the Ndc80-tail, which we reveal to be essential for microtubule end-tracking, and Ndc80-bound Ska stabilize microtubule ends in a stalled conformation. Modulation of force-coupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.

The NF-κB essential modulator NEMO is the core regulatory component of the inhibitor of κB kinase complex, which is a critical checkpoint in canonical NF-κB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1β, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-1β. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional relevance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is defective in mediating IL-1β–induced NF-κB activation.

Errorless chromosome segregation requires loadbearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Under force, Ska stabilizes end-on microtubule attachments in parallel with the Ndc80-tail, which we reveal to be essential for end-tracking by Ndc80 multimers. Modulation of forcecoupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.

Identification and analysis of small molecule bioactivity in target-agnostic cellular assays and monitoring changes in phenotype followed by identification of the biological target are a powerful approach for the identification of novel bioactive chemical matter in particular when the monitored phenotype is disease-related and physiologically relevant. Profiling methods that enable the unbiased analysis of compound-perturbed states can suggest mechanisms of action or even targets for bioactive small molecules and may yield novel insights into biology. Here we report the enantioselective synthesis of natural-product-inspired 8-oxotetrahydroprotoberberines and the identification of Picoberin, a low picomolar inhibitor of Hedgehog (Hh)-induced osteoblast differentiation. Global transcriptome and proteome profiling revealed the aryl hydrocarbon receptor (AhR) as the molecular target of this compound and identified a cross talk between Hh and AhR signaling during osteoblast differentiation.

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