Self‐assembling Long Coiled‐coil Proteins Driving the Formation of a Nanoscale Cylindrical Architecture at Human Centrosomes

Abstract

The centrosome, a unique membrane‐less organelle that serves as the main microtubule (MT)‐organizing center in animal cells, plays a pivotal role in the orderly progression of the cell cycle. Improper function of the centrosome results in abnormal cell division and proliferation that can lead the development of various human disorders. Thus, elucidating the molecular mechanisms underlying how centrosomal scaffold proteins orchestrate to generate a micrometer‐scale architecture in the three‐dimensional intracellular space is likely a key step to determining the etiology of centrosome‐associated human diseases. We found that two long coiled‐coil proteins, Cep63 and Cep152, interact with each other to form a heterotetrameric complex that self‐assembles into a higher‐order cylindrical architecture around a centriole, a barrel‐shaped organelle buried inside the centrosome. Mechanisms underlying how Cep63 and Cep152 reach their threshold concentrations in the vast cytosolic space of a cell to trigger the self‐assembly process remains not known. Using purified recombinant proteins, here we demonstrated that Cep152 and Cep63 possess an intrinsic activity of co‐phase‐separating into matrix‐like condensates and form a nanoscale cylindrical architecture. By combining sedimentation equilibrium ultracentrifugation with interferometric scattering mass spectrometry, we further showed that the heterotetrameric building block generates octameric and hexadecameric complexes in a concentration‐dependent manner in vitro, suggesting that the cylindrical self‐assembly is formed through stepwise processes. Subsequent analyses led to the discovery of a short uncharacterized region, named self‐assembly motif (SAM) from each Cep63 and Cep152, that exhibits a unique ability to undergo density transition and cooperatively generate the cylindrical self‐assembly in vitro. Fluorescent recovery after photobleaching revealed that the self‐assembly is capable of undergoing internal rearrangement within the assembly, while dynamically exchanging its components with those in the surroundings. Thus, the SAM motifs possess a highly flexible physicochemical nature that drives the Cep63‐Cep152 heterotetrameric complex to generate biomolecular condensates and self‐organize into a higher‐order cylindrical architecture likely through the repetitive cycles of controlled interactions among its constituent components. Introduction of single missense SAM mutations found in human cancer databases crippled proper recruitment and organization of Cep63 and Cep152 in vivo, thus consequently altering the function of its downstream effector, Polo‐like kinase 4, a key regulator of centriole biogenesis. Because the architecture and function of the centrosome is evolutionarily conserved, this work may serve a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms. Furthermore, given that abnormalities in the pericentriolar architecture are tightly associated with various human disorders, such as cancers, microcephaly, and dwa...