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The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryotomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner Junctions, underscoring its precise spatial regulation.

Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. The full localization of the inner scaffold protein Fam161A requires Poc1. As ciliary forces are increased, Fam161A is reduced, indicative of a force-dependent molecular remodeling of the inner scaffold. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces., (© 2024 Ruehle et al.)

Excess centrosomes cause defects in mitosis, cell-signaling, and cell migration, and therefore their assembly is tightly regulated. Plk4 controls centriole duplication at the heart of centrosome assembly, and elevation of Plk4 promotes centrosome amplification (CA), a founding event of tumorigenesis. Here, we investigate the transcriptional consequences of elevated Plk4 and find Unkempt, a gene encoding an RNA binding protein with roles in translational regulation, to be one of only two upregulated mRNAs. Unk protein localizes to centrosomes and Cep131-positive centriolar satellites and is required for Plk4-induced centriole overduplication in an RNA-binding dependent manner. Translation is enriched at centrosomes and centriolar satellites with Unk and Cep131 promoting this localized translation. A transient centrosomal downregulation of translation occurs early in Plk4-induced CA. CNOT9, an Unk interactor and component of the translational inhibitory CCR4-NOT complex, localizes to centrosomes at this time. In summary, centriolar satellites and Unk promote local translation as part of a translational program that ensures centriole duplication., Competing Interests: The authors declare no competing financial interests.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC + mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma., (© 2024. The Author(s).)

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila . A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

RNAs encoding some centrosomal components are trafficked to the organelle during mitosis. Some RNAs, including ASPM , localize to the centrosome co-translationally. However, the relative position of these RNAs and their protein after trafficking to centrosomes remained unclear. We find that mislocalization of ASPM RNA from the centrosome does not affect the localization of ASPM protein. Further, ASPM RNA and ASPM protein reside in two physically close yet distinct subcellular spaces, with ASPM RNA on the astral side of the centrosome and ASPM protein on the spindle side. This suggests subtly distinct locations of ASPM RNA translation and ASPM protein function., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)