Dissecting the molecular mechanisms of corona assembly and CENP-E kinetochore recruitment

During mitosis, accurate chromosome segregation is ensured by chromosome biorientation on the mitotic spindle. This process is facilitated by kinetochores, specialized multi-protein structures that assemble directly on the centromeric chromatin. Kinetochores mediate the physical attachment of chromosomes to spindle microtubules and monitor biorientation of sister chromatids, thereby safeguarding mitotic fidelity. In metazoans, unattached kinetochores are further characterized by the assembly of the fibrous corona, an outermost layer that facilitates microtubule attachment. The corona forms during prometaphase and dissolves once chromosome biorientation is achieved. Despite its discovery over five decades ago, the fibrous crescent structure remains poorly understood due to its transient and complex nature. In this doctoral work, we sought to investigate the corona using a multidisciplinary approach combining cell biology, biochemistry, and structural biology. We resolved a high-resolution structure of the ROD-Zwilch-ZW10 (RZZ) complex, which serves as the core building block of the corona. Additionally, we established a novel biochemical assay to study how the mitotic dynein adaptor Spindly and the kinase MPS1 promote corona polymerization in vitro. Further investigations enabled us to propose a recruitment model for CENP-E (kinesin-7) that relies on interactions with BUBR1 and the RZZS complex. Building on this work, we demonstrated that RZZS and CENP-E, which are co-dependent for their localization at kinetochores, form a platform for dynein recruitment. Notably, we observed that when corona assembly is inhibited through inhibition of MPS1 kinase, CENP-E becomes essential for retaining RZZS at kinetochores. Additionally, we found that an RZZS mutant mimicking its phosphorylation bypasses this requirement, indicating a second, unknown receptor for polymeric RZZS. Further investigations allowed us to develop a model for RZZ recruitment based on its interactions with CENP-E and the spindle assembly checkpoint (SAC) protein BUB1. Interestingly, the binding site of RZZ on BUB1 partially overlaps with the binding site of another central SAC component, MAD1, highlighting the close relation between the corona and SAC signalling.