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Pericentrin-mediated SAS-6 recruitment promotes centriole assembly.

ABSTRACT: The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.

SUBMITTER: Ito D

PROVIDER: S-EPMC6559791 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Tuning SAS-6 architecture with monobodies impairs distinct steps of centriole assembly.

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Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation.

Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) alone, to block the formation of multiciliate cells, or with Dexamethasone inducible Multicilin (MCI-HGR) to induce the formation of ectopic multiciliate cells. MCI-HGR plus ICD injected samples were compared to ICD alone to identify candidate genes whose expression changes when MCI is active. Epithelial cells projecting hundreds of motile cilia are prominent in the respiratory system, brain ependyma and female reproductive tract where they generate a vigorous fluid flow along luminal surfaces. Despite their importance for organ function, how multiciliate cells arise developmentally in diverse epithelia is poorly understood. We identified a novel coiled-coil protein, we have termed multicilin (MCI), whose expression is Notch regulated and restricted to epithelia where multiciliate cells form in Xenopus embryos. In order to identify early downstream targets of multicilin we blocked the formation of endogenous multiciliate cells by injecting ICD mRNA. In a subset of samples we then misexpressed a dexamethasone inducible form of MCI (MCI-HGR) to identify specific targets of MCI in explants of Xenopus epithelium. In explants expressing MCI-HGR genes specifically expressed in ciliated cells such as FoxJ1 and alpha-tubulin were highly upregulated along with various genes known to be required for ciliogenesis, and a large number of genes likely to be part of the ciliome based on homology of potential structural motifs. 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD or ICD along with MCI-HGR. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 11.5 dexamethasone was added to all conditions to induce MCI. Explants were harvested 5 hours after adding dexamethasone (approximately stage14) and used as the starting material for arrays.

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Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation.

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The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies.

Project description:Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

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Kinetic and structural roles for the surface in guiding SAS-6 self-assembly to direct centriole architecture

Project description:Discovering mechanisms governing organelle assembly is a fundamental pursuit in biology. The centriole is an evolutionarily conserved organelle with a signature 9-fold symmetrical chiral arrangement of microtubules imparted onto the cilium it templates. The first structure in nascent centrioles is a cartwheel, which comprises stacked 9-fold symmetrical SAS-6 ring polymers emerging orthogonal to a surface surrounding each resident centriole. The mechanisms through which SAS-6 polymerization ensures centriole organelle architecture remain elusive. We deploy photothermally-actuated off-resonance tapping high-speed atomic force microscopy to decipher surface SAS-6 self-assembly mechanisms. We show that the surface shifts the reaction equilibrium by ~104 compared to solution. Moreover, coarse-grained molecular dynamics and atomic force microscopy reveal that the surface converts the inherent helical propensity of SAS-6 polymers into 9-fold rings with residual asymmetry, which may guide ring stacking and impart chiral features to centrioles and cilia. Overall, our work reveals fundamental design principles governing centriole assembly. The centriole exhibits an evolutionarily conserved 9-fold radial symmetry that stems from a cartwheel containing vertically stacked ring polymers that harbor 9 homodimers of the protein SAS-6. Here the authors show how dual properties inherent to surface-guided SAS-6 self-assembly possess spatial information that dictates correct scaffolding of centriole architecture.

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