Nathalie Delgehyr

Multiciliated cells, through the coordinated beating of motile cilia, are vital for moving fluids in the brain and mucus in the airways and in the reproductive organs. Their dysfunction leads to diseases, associated with chronic airway infections, subfertility and ventricular enlargement. A group of transcription factors and a cell cycle variant enable the differentiation of multiciliated cells. However, how the multiciliated differentiation programme is initiated, timed and spatially regulated within developing tissues remains unclear. In this review, we combine findings from airway, brain ventricles and Xenopus epidermal systems to reveal how mechanical cues may interact with transcriptional networks and cell cycle regulators during multiciliogenesis. We suggest a model where mechanical forces serve as contextual regulators that enable, time and control the execution of the multiciliated differentiation programme.

2 A complex and conserved regulatory network drives the cell cycle. Individual components of this network are sometimes used in differentiated cells, i.e. to control organelle destruction in mammalian lens cells or light response in land plants. Some differentiated cells co-opt cell-cycle regulators more largely, to increase their ploidy using a cell cycle variant named endoreplication. Using single-cell RNA-seq profiling and functional assays in differentiating multiciliated cells, we identified a novel type of cell cycle variant that supports cytoplasmic organelle, rather than nuclear content amplification. This variant operates in post-mitotic, centriole-amplifying differentiating multiciliated cells and is characterized by (i) a circular trajectory of the transcriptome, (ii) sequential expression of more than 70% of the genes involved in S, G2 and M-like progression along this trajectory, and (iii) successive waves of cyclins. This cell cycle variant is tailored by the expression of the non-canonical cyclins O and A1 – which replace the transcriptionally silent cyclins E2 and A2 – and by the silencing of the APC/C inhibitor Emi1, two switches also detected in male meiosis, another variant of the canonical cell cycle where centriole and DNA replications are uncoupled. Re-expressing Cyclin E2, cyclin A2 or Emi1 is sufficient to induce partial replication and mitosis, suggesting that change in the regulation of expression of a few cell cycle key players drives a qualitative and quantitative tuning of Cdk activity, allowing the diversion of the cell cycle in the multiciliation variant. We also propose that this new cell cycle variant relies on the existence of a cytoplasmic – or centriolar – Cdk threshold, lower than the S-phase threshold, which affects only the cytoplasmic reorganization. One-Sentence Summary: MCC progenitors undergo a final, tailored iteration of the cell cycle during differentiation, to drive centriole amplification without DNA replication or mitosis.

Multiciliated cells (MCC) ensure proper fluid circulation in various organs in metazoans. Their differentiation is marked by the massive ampliication of cilia-nucleating centrioles and is known to be controlled by various cell cycle components. Tn a companion study, we show that the differentiation of MCC is driven by a genuine cell-cycle variant characterized by sequential and wave-like expression of canonical and non-canonical cyclins such as Cyclin O (CCNO). Patients with CCNO mutations exhibit a subtype of Primary Ciliary Dyskinesia (PCD) designated as Reduced Generation of Multiple Motile Cilia (RGMC), yet the role of CCNO during MCC differentiation remains unclear. Here, using mice and human cellular models, single cell transcriptomics and functional studies, we show that Cena is activated during a strategic temporal window at the crossroads between the onset of MCC differentiation, the entry into the MCC cell cycle variant, and the activation of the centriole biogenesis program. We ind that the absence of Cena leads to a block of MCC progenitor differentiation at the G1/S-like transition, just before the beginning of centriole formation. This leads to a complete lack of centrioles and cilia in mouse brain and human airway MCC. Altogether, our study identifies CCNO as a core regulator of entry into the MCC cell cycle variant and shows that the coupling of centriole biogenesis to an S-like phase, maintained in MCC, is dependent on CCNO. One sentence summary: Cyclin O is necessary for multiciliated cells to enter their differentiation cell cycle variant and allows the massive amplification of centrioles, which serve as basal bodies for cilia nucleation.

Multiciliated cell (MCC) differentiation is a calibrated version of the canonical cell cycle. The MCC cell cycle variant sustains amplification of centrioles for the nucleation of dozens of motile cilia, while avoiding cell division. In this study, we show that the MCC cell cycle variant is an accelerated version of the canonical cell cycle, which superposes two cycles of centriole biogenesis, in order to obtain multiple mature centrioles within a single -instead of double- cell cycle iteration. We further show that the precocious maturation of amplified procentrioles is even determinant for their spatial self-organization, disengagement and apical migration for cilia nucleation. Our findings collectively suggest that the decomposition of centriole biogenesis over two cycle iterations in dividing cells may have been adopted to ensure the growth of a solitary primary cilium, and exemplify how minimal deviations of the canonical cell cycle allow MCC progenitors to both amplify, and accelerate, centriole biogenesis for vital motile ciliogenesis.

SUMMARY: Ependymal cells (ECs) are multi-ciliated cells in the brain that contribute to cerebrospinal fluid flow. ECs are specified at embryonic stages but differentiate later in development. Differentiation depends on genes such as GEMC1 and MCIDAS with E2F4/5, but also on cell cycle related factors. In the mouse brain, we observe that nuclear deformation accompanies EC differentiation. Tampering with these deformations either by decreasing F-actin levels or by severing the link between the nucleus and the actin cytoskeleton blocks differentiation. Conversely, increasing F-actin by knocking out the Arp2/3 complex inhibitor Arpin or artificially deforming the nucleus activates differentiation. These data are consistent with actin polymerization triggering nuclear deformation and jump-starting the signaling that produces ECs. A player in this process is the retinoblastoma1 (RB1) protein whose phosphorylation prompts MCIDAS activation. Overall, this study reveals an important role for actin-based mechanical inputs to the nucleus as controlling factors in cell differentiation.

Michel BornensMichel Bornens was born in Paris in 1938 and died there in 2022. After studying at the ‘Agro’, a thesis at the CNRS in Villejuif and a post-doc in the USA, he worked at the Centre de Cytologie Expérimentale in Ivry sur Seine and then at the Centre de Génétique Moléculaire in Gif-sur-Yvette, before taking part in the setting up of the cell biology department at the Institut Curie in Paris in 1995.It is 5pm when Michel Bornens returns to the lab. He has gone out to buy Le Monde, which has just arrived at the kiosk next door. He clears off his desk, pulls down the curtains slightly, puts on the music, opens the newspaper wide and starts reading. This image says a lot about Michel. The importance that politics had in his life. His strong commitment to the values of solidarity, sharing and freedom, and to the defence of the common good. And his curiosity about the whole world. It also says that he felt at home in his office.He did not experience the boundary, so often claimed today, between the private sphere and that of work in the laboratory. Science was an integral part of his life. Above all, he liked to think. He liked to think and imagine the world. Of course, he wanted to understand first and foremost, but his desire to observe and admire was already immense. He was a thinker and a contemplator.He was curious about everything around him, from the farms of the Jura to the ports of Brittany. He was as enthusiastic about the making of pelardon [a type of cheese] as he was about the building of boats. He was fed by music and painting. And he loved above all to learn the stories. The stories of the craftsmen who make the instruments, of the directors who make the movies or of the farmers who work the land.His insatiable curiosity led him to be interested in all living organisms. He saw in them, in different forms, the manifestation of the same great principles of life. Michel believed that these principles were at work in every single cell. In the early 1980s, for many researchers, the cell was a bag of enzymes whose targets and function had to be identified. For Michel the cell was a compartmentalised, structured, organised and spatially oriented entity (Fig. 1). The centrosome, by organising the microtubules, had to be somehow linked to the entire intracellular space as well as to its limits, the plasma and nuclear membranes (Fig. 2). The microtubule network could therefore measure and integrate spatial information, concentrate and titrate biochemical information, and then combine all these processes in the regulation of cell behaviour. This is how he contributed to the emergence of cell biology in France.The researchers who worked with him all had fond memories of it. We were both excited to feel that we were part of a team of adventurous explorers and slightly worried that we were quite far from the common frontiers of knowledge. Fortunately, Michel's stature and his ability to share his enthusiasm for the great mysteries of biology were there to reassure us. He encouraged us not only to consider new hypotheses, but also to try out of the ordinary experiments. Thus, Fréderic Tournier injected centrosomes from human lymphocytes into frog oocytes to trigger their parthenogenesis and produce frogs without fathers. Matthieu Piel covered phantoms of enucleated cells with frog egg extracts to study the respective capacities of the two centrioles to organise microtubules. It must be said that Michel guided us by example, having himself had a calf slaughtered by surprise to find out whether the stress of the slaughterhouse was the cause of the alignment of centrioles in the thymus... Fundamental experiments, even if they didn't always look like it. Indeed, from one thing to another, and from unfinished experiments to technological twists and turns, Michel and his team managed to describe nothing less than – the structure and composition of the centrosome as well as a number of key rules on how it regulates the organisation of the microtubule network, the progression of the cell cycle, the orientation of the mitotic spindle, the final stage of daughter cell abscission, the differentiation of the myotubes and the polarisation of the cells in response to the geometry of their adhesive environment.Michel's thinking went far beyond what could be experimentally tested. He was trying to conceptualise how the cell senses, acts, optimises and progresses in time. As an autonomous entity, an organism in its own right, it had to be at all times the product of its history and the preparation of its future in order to function in the present. At any given moment, the structure of the cell and its physiological state – reflected in the expression profiles of proteins and their phosphorylation states – are the consequence of its history. And they predetermine its capacity to react. The adapting cell must be able to take its time and behave differently according to its past experiences. It therefore necessarily has memory and calculation capacities. These functions are key for cells to learn. Little is known about this today. It may seem worrying that there are such gaps in our understanding, and one might be afraid to venture into such obscure areas. But for Michel, it was necessary to confront the limits of our knowledge rather than ignore them – an indispensable condition for setting up the experimental models that would allow us to ask the right questions under the right conditions.Michel was aware that cellular functions could not be understood by adding up the role of each protein. The number and combinatorial nature of possible interactions quickly became unmanageable. And even if supercomputers made them possible, the classic experiments of gene silencing or overexpression would probably not allow the redundancies and compensation mechanisms that ensure the essential functions of the cell to be taken into account. The...

Multiciliated ependymal cells line all brain cavities. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centrioles, persist once formed and withstand the forces produced by the external fluid flow and by their own cilia beating. Here, we show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. Moreover, disruption of the actin network, or specifically of the apical actin network, causes motile cilia and their centrioles to detach from the apical surface of ependymal cell. In conclusion, cilia beating controls the apical actin network around centrioles; the mechanical resistance of this actin network contributes, in turn, to centriole stability.