Neurodevelopmental disorders (NDDs) are often associated with mutations in mitotic genes rather than in genes specifying neuronal

functions. Several mitotic regulators are functionally repurposed to new roles during neurodifferentiation. CENP-F, a kinetochore- and microtubule- interacting protein with well-characterised mitotic functions, is being growingly identified for harbouring mutations in primary microcephaly and in the

Strømme syndrome, an NDD classified as a ciliopathy. The mechanism through which mutant CENP-F contributes to these NDDs remains elusive.

To gain insight into the implication of CENP-F in NDDs, we have optimised imaging methods and developed a comprehensive analysis of CENP-F in post- mitotic cellular systems. We first examined CENP-F during the formation of the primary cilium by optimising quantitative immunofluorescence and single- cell analysis of spinning-disk confocal images. CENP-F and some of its interacting partners were characterised in a time-course analysis of ciliogenesis in

human retinal RPE-1 cells. CENP-F localises at the basal body of the cilium and is absolutely required for proper ciliogenesis. We also studied the requirement for CENP-F during neurodifferentiation by time-lapse videorecording and confocal microscopy. We have developed two AI-based algorithms to analyse high-content images of videorecorded cell cultures: that enabled us to extract information on the dynamics with which phenotypic changes occur during neurodifferentiation and identify steps that are sensitive to and/or impaired by CENP-F loss-of-function. In summary, these imaging protocols identify critical temporal windows during which the lack of CENP-F impacts neurodifferentiation and ciliogenesis, thus providing new tools to dissect roles of CENP-F in NDDs.

Centromere protein F (CENP-F) is a multidomain protein with an established role in regulating microtubule (MT) functions during mitosis. CENP-F mutations are identified in neurodevelopmental disorders (NDDs) associated with primary microcephaly (MCPH), suggesting that CENP-F may also play roles during commitment to the neurogenic lineage. The molecular mechanism(s) through which mutant CENP-F acts in NDDs however remains elusive. Here we have developed a comprehensive analysis of CENP-F in several cellular systems aiming to understand its roles in neurogenesis. First, we investigated CENP-F C-terminal region, which harbours MCPH mutations. In that region we have characterised a nuclear localisation signal (NLS) flanking a KEN box for ubiquitin-dependent proteasomal degradation. We find that the nuclear import receptor importin beta, which binds the NLS, cooperates with MTs in regulating both CENP-F mitotic localisation and the timing of regulated degradation at mitotic exit. Second, we studied CENP-F in cells that exit the cell cycle and enter quiescence, at which stage they nucleate the primary cilium, a MT-based structure with architectural roles during the organisation of brain cortical layers. We find that CENP-F is required for the axoneme formation and for positioning of the cilium basal body. Third, we characterised CENP-F in neurodifferentiating cellular systems. We identified a CENP-F fraction that mobilises from the nucleus to MT-based structures required for neurite formation and branching. These findings illustrate that CENP-F may act at multiple steps during neurodifferentiation: affecting the establishment of the neuronal precursor pool, the process of ciliogenesis, and the formation of neuronal networks. These results suggest possible pathways disrupted by MCPH pathogenetic mutations in CENP-F.