Long-range migration of centrioles to the apical surface of the olfactory epithelium

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Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50 to 100 μm from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 μm/min. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium prior to other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.

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  1. Evaluation Summary:

    Analyzing the long-distance migration of centrioles to the dendrite tip of multicilated olfactory neurons in mice, Ching et al. use expansion microscopy to show that centrioles migrate as clusters, which mature as they reach the apical surface. The super-resolution data are impressive and the claims are generally supported by the data. Although the manuscript is largely descriptive, it is an important addition to the field, and will be of broad interest to cell biologists.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    Ching et al investigate the long-range migration and maturation of cilia-forming centrioles in multiciliated OSNs of the adult mouse. They develop techniques to perform expansion microscopy of the tissue, as well as explant culture and live imaging. Images obtained from expansion microscopy in this tissue are just spectacular, and provide suborganelle details of centriole organization as they traverse the OSN dendritic projections and eventually dock and form cilia. Although the study is predominantly descriptive and observational, the increased resolution gained by expansion microscopy allow them to do things like count the exact number of centrioles in progenitors and mature OSNs, and describe the sub-organelle organization of centrioles as they migrate, mature and build cilia.

    Key findings include the observation that centrioles migrate in clusters, though it is unclear what molecularly connects the clusters. Using makers of centriole maturation they show that immature centrioles migrate and subsequently lose those markers when they reach the apical surface where ciliogenesis will occur. PCM factors associated with MT nucleation remain associated with centrioles throughout the migration and maturation process where they presumably retain their MT nucleation capacity. Distal and subdistal appendages are present only in a signal centriole in immature OSNs, presumably the parent centriole, and that these markers only associate with the rest of the centrioles once they have docked at the apical surface. Stabilizing MTs using taxol in an ALI culture system leads to decreased migration of OSN centrioles suggesting a role for dynamic microtubules in their movement. Overall the data are very high quality. The study raises more questions than answers - how do centrioles cluster, how do they migrate, what controls their maturation in both space and time. The paper lays the groundwork for tackling these questions and provides some intriguing hypotheses to guide those future studies.

  3. Reviewer #2 (Public Review):

    Olfactory sensory neurons (OSN) are a unique type of multi-ciliated cell that have a distinct and dynamic morphology. These cells have numerous centrioles that will generate their numerous cilia that project off the end of their dendrite. The Ching et al. manuscript addresses the question of where these centrioles are generated and how they are transported to the dendritic knobs. The authors use expansion microscopy to quantify centriole number and to characterize centriole maturation including localization of a wide range of centriolar proteins. They develop both an ex vivo culture system and an explant based ALI culture system to visualize centriole migration towards the dendritic knob. Finally they show that microtubule (MT) stabilization with Taxol leads to a defect in the centriole movements.

    Overall, there is a lot of beautiful data in this paper and the claims are generally supported by the data. While OSNs have been fairly well characterized at the EM level, it is certainly a worthwhile endeavor to revisit some of this description with newer technologies and with live imaging. To that end this manuscript is largely descriptive and while that is often used negatively, here I think it is an important addition to the field. In particular the development of novel culture systems promises to bring new insights to the field. However, certain aspects of these culture systems require further validation / description. Additionally, while the authors attempt to bring some mechanism into their analysis via MT manipulation, the results are preliminary and in my opinion would require deeper analysis to support their claims.

  4. Reviewer #3 (Public Review):

    Analyzing the long-distance migration of centrioles at the dendrite tip of olfactory neurons in adult mice, Ching et al. first show that centrioles migrate along with dendrite extension, forming multiple clusters of centrioles distributed along a single dendrite. Interestingly, the maturation of centrioles occurs during their migration. Indeed, some of the migrating centrioles still contain the STIL and SAS-6 markers, while all centrioles in mature OSNs are devoid of these proteins. The authors show that immature OSNs carry only one cilium, organized from the mother centriole, the only one that has appendages at this stage. Maturation of the other centrioles, which allows them to acquire distal appendages, therefore occurs near the apical membrane. The mother centriole also has subdistal appendages, while the other centrioles acquire a single basal foot, as in multiciliated cells bearing motile cilia. The authors then show that treatment with paclitaxel, a drug that stabilizes microtubules, leads to an increase in the number of centrioles in the subapical region of the epithelium, suggesting a defect in centriole migration. Overall, this is an interesting work that sheds light on a cell type whose ciliogenesis is poorly characterized. Various protocols are developed for this study, including a protocol for explant culture. However, this study would be strengthened by further analysis of the different stages of centriole assembly and maturation, as well as the mechanisms underlying centriole migration.