Abstract
Centrosomes are required for faithful chromosome segregation during mitosis. They are composed of a centriole pair that recruits and organizes the microtubule-nucleating pericentriolar material. Centriole duplication is tightly controlled in vivo and aberrations in this process are associated with several human diseases, including cancer and microcephaly. Although factors essential for centriole assembly, such as STIL and PLK4, have been identified, the underlying molecular mechanisms that drive this process are incompletely understood. Combining protein proximity mapping with high-resolution structural methods, we identify CEP85 as a centriole duplication factor that directly interacts with STIL through a highly conserved interaction interface involving a previously uncharacterised domain of STIL. Structure-guided mutational analyses in vivo demonstrate that this interaction is essential for efficient centriolar targeting of STIL, PLK4 activation and faithful daughter centriole assembly. Taken together, our results illuminate a molecular mechanism underpinning the spatiotemporal regulation of the early stages of centriole duplication.
Highlights
Centrosomes are required for faithful chromosome segregation during mitosis
To identify factors implicated in centriole biogenesis, we originally sought to map protein interactions of centriole components using proximity-dependent biotin identification (BioID), including the upstream centriole duplication factors STIL, SASS6, CEP152 and CEP6348
Our analysis of the proximity interaction landscape of these five components revealed that CEP85, a centrosomal protein so far only implicated in regulating centrosome disjunction through its interaction with NEK249, displayed a prominent proximity signature with a number of centriole duplication factors (Supplementary Fig. 1a)
Summary
Centrosomes are required for faithful chromosome segregation during mitosis. They are composed of a centriole pair that recruits and organizes the microtubule-nucleating pericentriolar material. PLK4 activity is regulated through autophosphorylation of its activation loop which in turn promotes trans-autophosphorylation of regulatory sites creating a phosphodegron that is subsequently bound by the SCFSlimb/βTrCP ubiquitin ligase leading to PLK4 ubiquitylation and destruction[34,35,36,37,38] Through this autoinhibitory mechanism PLK4 kinase activity is tightly controlled to ensure a single round of centriole duplication per cell cycle. Consistent with this role, as for PLK4, depletion of STIL prevents centrioles formation, whereas its overexpression triggers centriole overduplication[41, 42] Further underpinning this important function, STIL levels in cells are tightly controlled, with a cell cycle dependent, initial centriolar accumulation at the onset of centriole duplication (at the G1/S transition) and its subsequent degradation during late mitosis in a APC/C dependent manner[41, 43]
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