Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: CENP-F, a microtubule regulator in cell division and neurodevelopmental disorders

This thesis focuses on Centromere protein F (CENP-F), a large multidomain protein that interacts with kinetochores, microtubules and motor proteins during mitosis. While CENP-F is aberrantly expressed in many cancer types, mutations are being growingly identified in neurodevelopmental disorders (NDDs) associated with primary microcephaly (MCPH), a systemic condition associated with impaired brain growth, suggesting that CENP-F may be functionally repurposed in cells that exit mitosis and undergo neurodifferentiation. The mechanism through which mutant CENP-F may contribute to NDDs remains elusive. In my project I have characterised three neurogenesis-relevant aspects in which CENP-F is implicated. First, I studied CENP-F during the cell cycle and in mitosis, focusing on its C-terminal region, within which viable MCPH mutations fall. That region contains a nuclear localisation signal (NLS) flanking a KEN box that drives ubiquitin-dependent proteasomal degradation at mitotic exit. Using importin beta overexpression in newly engineered, inducible cell lines, I have found that importin beta regulates both CENP-F localisation during mitosis, and the timing of degradation at mitotic exit. Errors in regulation of these processes can impair cell division and, if occurring during neural stem cell expansion, can result in insufficient production of neuronal precursors. Second, I followed up and characterised CENP-F during neurogenic differentiation in several cellular models, including primary neurons explanted from the cortex: I have identified a CENP-F fraction localising at microtubule-based structures and required for neurite formation and branching. Third, I examined CENP-F in quiescent cells that assemble the primary cilium, a crucial structure with architectural roles in the organisation of the brain cortical layers. I found that CENP-F is required for the formation of a functional axoneme and for proper positioning of the cilium basal body at the level of the nuclear envelope. Together, these findings suggest that CENP-F acts at multiple steps during neurodifferentiation, potentially affecting the proper generation of the neuronal precursor pool, the process of ciliogenesis, and the formation of neuronal networks. These results begin to shed some light on processes through which CENP-F mutations can yield neurodevelopmental disorders.