Molecular basis of centriole duplication

Division Of Basic Sciences - Nci

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Abstract

The centrosome, a unique membrane-less multiprotein organelle that serves as the main microtubule-organizing center in animal cells, plays a pivotal role in the orderly progression of the cell cycle. Since faulty assembly and duplication of the centrosome results in abnormal cell division, which then leads to various human disorders, elucidating the molecular mechanisms underlying centrosome assembly and function is likely a key step to understanding the etiology of centrosome-associated human diseases. By combining cell biology with biophysical methods and X-ray crystallography, we demonstrated that two pericentriolar scaffolds, Cep152 and Cep63, possess intrinsic activity of co-phase-separating into condensates and form a heterotetrameric complex that serves as a building block for generating a nanoscale cylindrical self-assembly around a centriole. Remarkably, two short uncharacterized regions named Self-Assembly Motifs (one each from Cep63 and Cep152) cooperatively conferred physicochemical properties that allowed them to undergo density transition and self-assemble into a cylindrical architecture. Interestingly, the Cep152-Cep63 condensates exhibited a rapid turnover, underwent fusion with other assemblies, and carried out a significant degree of internal rearrangement within a condensate. A Cep152-Cep63 cylindrical architecture that self-assembled on a flat substrate displayed a decreased but still detectable level of dynamic turnover. Interestingly, Polo-like kinase 4 (Plk4), a key regulator of centriole biogenesis, also dynamically phase-separated from a Cep152-bound state around a centriole (i.e., ring state) into a dot-like, low-nanoscale spherical condensate (i.e., dot state) upon autophosphorylating its C-terminal cryptic polo-box domain. Additional in vitro and in vivo data suggest that the Plk4 condensate serves as an assembling body at the future procentriole assembly site by amassing downstream procentriole assembly components such as STIL and Sas6 and facilitating Plk4-mediated centriole biogenesis. Thus, the formation of biomolecular condensates appears to be a fundamental step that not only promotes the self-assembly of a pericentriolar architecture but also triggers the process of centriole duplication. Along with this progress, we have been focusing on examining the mechanism underlying pericentriolar material (PCM) organization, self-assembling activity of pericentriolar scaffold proteins, molecular basis of building higher-order PCM architectures. To this end, we performed size-exclusion chromatography, sedimentation equilibrium ultracentrifugation, and interferometric scattering mass spectrometry and showed that the heterotetrameric building block generates octameric and hexadecameric complexes in a concentration-dependent manner, suggesting that the cylindrical self-assembly is formed through stepwise processes. By using MINFLUX nanoscopy, which offers low-nanometer-scale localization precision in a three-dimensional space, we further showed that mutants defective in forming the Cep63-Cep152 heterotetramer exhibited crippled pericentriolar Cep152 organization, consequently failing to promote polo-like kinase 4 (Plk4)'s dynamic relocalization from around the centriole to the future procentriole assembly site as well as Plk4-mediated centriole duplication. Remarkably, the entire self-assembly process could be driven by two short, uncharacterized regions (which we named "self-assembly modules") in Cep63 and Cep152 capable of cophase-separating and generating cylindrical self-assemblies in vitro. Fluorescence recovery after photobleaching revealed that the self-assembled architecture is highly dynamic, undergoing internal rearrangement within the assembly while exchanging its components with those in the surroundings. Dynamic turnover of pericentriolar Cep63 and Cep152 has also been observed in human centrosomes. Taken together, given the evolutionarily conserved organization of PCM, this work could serve as a paradigm for investigating the structure and function of centrosomal scaffolds in other organisms, while offering a new direction for probing organizational defects in PCM-related human diseases.