Microtubule-dependent orchestration of centriole amplification in brain multiciliated cells

Reviewer #1 (Public Review):

The manuscript by Boudjema et al. describes the cellular events underlying centriole amplification and apical migration to allow the assembly of hundreds of motile cilia in multi-ciliated cells. For this, they use cell culture models in combination with fixed and live cell imaging using antibody staining and fluorescence from endogenously tagged centriole and deuterostome markers, respectively. The work is largely descriptive and functional analyses are restricted to treatment with the microtubule depolymerizing drug nocodazole. The imaging is state-of-the-art including confocal microscopy, live imaging with optical sectioning and high optical and temporal resolution, as well as super-resolution imaging by ultra-expansion microscopy.

The study does a good job of providing a very detailed description of the dynamics of centrioles and deuterostomes that lead to centriole amplification and apical migration in multiciliated cells. This detailed view was missing in previous work. It also reveals the involvement of microtubules at multiple steps: the formation of a cloud of deuterostome precursors, the nuclear envelope tethering of newly formed centrioles, their separation, and their migration to the apical surface.

It would have been useful to expand the analysis of the role of microtubules by including analyses of the requirement for specific microtubule motors, for a better understanding and additional evidence that microtubule-based transport is involved. A weak point is that there is no visualization of microtubules together with deuterosomes and centrioles at the different steps of centriole amplification and migration, to directly address how these structures may interact with and move along microtubules.

Overall, apart from experimental aspects and since this is largely a descriptive study, the manuscript would benefit from more precise language and a better description of the complex events underlying centriole amplification and movements.

Reviewer #2 (Public Review):

This important work will be of interest to centriole and cilia cell biologists. It describes in detail how microtubules control multiple aspects of centriole amplification in brain multiciliated cells. This study provides a greater time-resolved and molecular proteomic mapping of the different steps involved, with or without microtubule disruption. Boudjema et al. show that microtubules are important throughout the centriole amplification process, from the early stages, where the procentrioles emerge from a pericentriolar "nest", through the growth stage where microtubules maintain the perinuclear localisation, to the detachment stage, where microtubules assist in perinuclear disengagement and apical migration. The results are generally well supported by the evidence, but the manuscript would benefit significantly from some heavy editing to introduce more niche terms, standardize abbreviations in text, and labels on figures to help bring the readers, especially non-specialists, along with them - increasing the accessibility of their work.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Boudjerna and Balagé et al. aim to elucidate the spatial origin of centriole amplification and the mechanisms behind the formation of an apical-basal body patch in multiciliated cells (MCCs). To this end, they focused on the role of microtubules and developed new tools for spatiotemporal and high-resolution analysis of different stages of centriole amplification, including the centrosome stages, A-stage, G-stage, and MCC-stage. Among these tools, the MEF-MCC cells grown on micropatterns stands out for its versatility as it is not tissue-specific and does not require epithelial cell-to-cell contact for differentiation. Additionally, the Cen2-GFP; mRuby-Deup1 knock-in mouse model was used to study different stages of centriole amplification in physiological brain MCCs. This model offers an advantage over the previously described Cen2-GFP model by enabling the resolution of early events in centriole amplification through the visualization of Deup1-positive structures and their dynamics. Finally, the authors leveraged powerful imaging techniques, including super-resolution microscopy, the U-ExM, and high-resolution live cell imaging in order to detect and track centriole amplification, elongation, disengagement, and migration.

By combining the MEF-MCC and knock-in mouse model with spatiotemporal imaging in control and nocodazole-treated cells(treated acutely or chronically), the authors define the sequence of events during centriole amplification, revealing the critical roles of microtubules for the first time. Initially, the centrosome-mediated microtubule network forms, organizing a pericentrosomal nest from which procentrioles and deuterosomes emerge. Their findings indicate the importance of microtubules in recruiting and maintaining pericentriolar material clouds that contain DEUP1, PCNT, SAS6, PLK1, PLK4, and tubulins. Following the amplification stage, the procentrioles mature, leading to cells displaying numerous MTOCs, as demonstrated by regrowth experiments. Mature centrioles then disengage from deuterosomes, attach to the nuclear envelope, and migrate to the apical surface facilitated by microtubules.

Strengths:

The manuscript provides new insights into the regulatory function of microtubules in centriole amplification. Addressing the role of microtubules during different stages of centriole amplification required the development of new tools to study brain MCCs, which will be useful in future studies of MCCs. A notable strength of this manuscript is the authors' thorough and quantitative analysis of highly dynamic processes in MCCs. The precision and detail in describing these dynamic events are impressive. This comprehensive analysis advances our understanding of MCC biology.

Weaknesses:

The role of microtubules and other molecular players during different stages of centriole amplification in brain MCCs can be further studied and strengthened using the tools developed in the manuscript. A more quantitative description of some of the analysis performed in the manuscript is required to strengthen the conclusions.