Cilia assembly and development

For the general public

Cilia and flagella are small cellular protrusions at the cell surface that play various roles: they can be motile and propel cells (spermatozoa) and fluids (respiratory mucus), or they can be immotile and serve as cellular antennae capturing extracellular signals, allowing cells to respond and adapt to their environment. Abnormalities in cilia formation or function are responsible for many rare and often severe hereditary diseases, grouped under the term ciliopathies. More, cilia and centrioles on which they are built, are also important players in the regulation of cancerous processes because of their role in the control of cell division and cell signaling. Our team aims to understand how cilia are assembled from centrioles and what are the mechanisms underlying their diversity of function. We are developing functional genetic, biochemical and high-resolution imaging approaches to identify and understand the function of new genes involved in cilia assembly. We mainly use the Drosophila model and mouse or human cell models. Our work has implications for the understanding of diseases associated with dysfunctions of these organelles.

Join the team!

Characterizing the function of proteins associated with ciliopathies

The PhD project aims to understand the precise function of two proteins whose mutations in humans are associated with retinal ciliopathy. In this project, the PhD candidate will combine the power of functional genetic approaches in Drosophila (RNAi and/or CrispR-Cas9 genome editing) with cutting-edge imaging strategies (Expansion microscopy coupled with spinning-confocal and STED microscopies) and biochemical strategies to determine the contribution of each protein to cilia assembly and maintenance.

Complete job offer

• Bénédicte DURAND — ORCID — Professor, UCBL, HDR – benedicte.durand@univ-lyon1.fr – 04 78 77 28 13
• Marine LAPORTE — ORCID — Researcher, INSERM – marine.laporte2@univ-lyon1.fr – 04 78 77 28 65
• Véronique MOREL — ORCID — Researcher, CNRS – veronique.morel@univ-lyon1.fr – 04 26 68 82 99
• Joëlle THOMAS — ORCID — Associate Professor, UCBL – joelle.thomas@univ-lyon1.fr – 04 78 77 28 65
• Marine BRUNET — PhD student, UCBL – marine.brunet@etu.univ-lyon1.fr – 04 26 68 82 99
• Julien PERRICHET — Research Assistant, UCBL – julien.perrichet@univ-lyon1.fr – 04 78 77 28 65
• Jennifer VIEILLARD — Research Assistant, UCBL - jennifer.vieillard@univ-lyon1.fr – 04 78 77 28 65
• Sarah DE FREITAS — M2 Research Internship, UCBL – sarah.de-freitas@etu.univ-lyon1.fr – 04 78 77 28 65
• Emilie JOUX — M2 Research Internship, UCBL – mailto:emilie.joux@etu.univ-lyon1.fr – 04 78 77 28 65

Alumni

• Amélie BILLON — PhD student
• Alison CARRET — PhD student
• Emilie FONTAINE — PhD student
• Hajer MAARCHA — M2 Research Intern
• Jean-André LAPART — PhD student
• Céline AUGIERE — PhD student
• Jennifer VIEILLARD — PhD student
• Dominique BAAS — Associate Professor
• Marie PASCHAKI — Associate Professor
• Elisabeth CORTIER — Research Assistant
• Jean-Luc DUTEYRAT — Research Assistant

1. Cep131-Cep162 and Cby-Fam92 complexes cooperatively maintain Cep290 at the basal body and contribute to ciliogenesis initiation.
   Wu Z, Chen H, Zhang Y, Wang Y, Wang Q, Augière C, Hou Y, Fu Y, Peng Y, Durand B, Wei Q.
   PLoS Biology (2024) — Show abstract

   Cilia play critical roles in cell signal transduction and organ development. Defects in cilia function result in a variety of genetic disorders. Cep290 is an evolutionarily conserved ciliopathy protein that bridges the ciliary membrane and axoneme at the basal body (BB) and plays critical roles in the initiation of ciliogenesis and TZ assembly. How Cep290 is maintained at BB and whether axonemal and ciliary membrane localized cues converge to determine the localization of Cep290 remain unknown. Here, we report that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module close to the membrane synergistically control the BB localization of Cep290 and the subsequent initiation of ciliogenesis in Drosophila. Concurrent deletion of any protein of the Cep131-Cep162 module and of the Cby-Fam92 module leads to a complete loss of Cep290 from BB and blocks ciliogenesis at its initiation stage. Our results reveal that the first step of ciliogenesis strictly depends on cooperative and retroactive interactions between Cep131-Cep162, Cby-Fam92 and Cep290, which may contribute to the complex pathogenesis of Cep290-related ciliopathies.

2. Evolution: The ancient history of cilia assembly regulation.
   Azimzadeh J, Durand B.
   Current Biology (2023) — Show abstract

   A new study identifies a conserved regulatory mechanism for cilia assembly in the closest unicellular relatives of animals, suggesting that this mechanism was already present in a common unicellular ancestor and was repurposed during the transition to multicellularity.

3. Drosophila transition fibers are essential for IFT-dependent ciliary elongation but not basal body docking and ciliary budding
   Hou Y, Zheng S, Wu Z, Augière C, Morel V, Cortier E, Duteyrat JL, Zhang Y, Chen H, Peng Y, Durand B, Wei Q.
   Current Biology (2023) — Show abstract

   Cilia are highly conserved organelles critical for animal development and perception. Dysfunction of cilia has been linked to a wide spectrum of human genetic diseases, termed ciliopathies.1,2 Transition fibers (TFs) are striking ciliary base structures essential for cilia assembly. Vertebrates' TFs that originate from centriole distal appendages (DAs) mediate basal body docking to ciliary vesicles to initiate ciliogenesis and regulate the entry of ciliary proteins for axoneme assembly via intraflagellar transport (IFT) machinery.3 Although no distal appendages can be observed on Drosophila centrioles,4,5 three key TF proteins, FBF1, CEP164, and CEP89, have obvious homologs in Drosophila. We aimed to compare their functions with their mammalian counterparts in Drosophila ciliogenesis. Here, we show that all three proteins are localized like TF proteins at the ciliary base in both sensory neurons and spermatocytes, the only two types of ciliated cells in flies. Fbf1 and Cep89 are essential for the formation of IFT-dependent neuronal cilia, but Cep164 is dispensable for ciliogenesis in flies. Strikingly, none are required for basal body docking and transition zone (TZ) assembly in IFT-dependent neuronal cilia or IFT-independent spermatocyte cilia. Furthermore, we demonstrate that Unc is essential to recruit all three TF proteins and establish a hierarchical order, with Cep89 acting on Fbf1. Collectively, our results not only demonstrate that TF proteins are required for IFT-dependent ciliogenesis in Drosophila, in agreement with an evolutionarily conserved function of these proteins in regulating ciliary protein entry, but also that the basal body docking function of TFs has diverged during evolution.

4. Drosophila Nesprin-1 Isoforms Differentially Contribute to Muscle Function
   Rey A, Schaeffer L, Durand B, Morel V.
   Cells (2021) — Show abstract

   Nesprin-1 is a large scaffold protein connecting nuclei to the actin cytoskeleton via its KASH and Calponin Homology domains, respectively. Nesprin-1 disconnection from nuclei results in altered muscle function and myonuclei mispositioning. Furthermore, Nesprin-1 mutations are associated with muscular pathologies such as Emery Dreifuss muscular dystrophy and arthrogryposis. Nesprin-1 was thus proposed to mainly contribute to muscle function by controlling nuclei position. However, Nesprin-1's localisation at sarcomere's Z-discs, its involvement in organelles' subcellular localization, as well as the description of numerous isoforms presenting different combinations of Calponin Homology (CH) and KASH domains, suggest that the contribution of Nesprin-1 to muscle functions is more complex. Here, we investigate the roles of Nesprin-1/Msp300 isoforms in muscle function and subcellular organisation using Drosophila larvae as a model. Subsets of Msp300 isoform were down-regulated by muscle-specific RNAi expression and muscle global function and morphology were assessed. We show that nuclei anchoring in mature muscle and global muscle function are disconnected functions associated with different Msp300 isoforms. Our work further uncovers a new and unsuspected role of Msp300 in myofibril registration and nuclei peripheral displacement supported by Msp300 CH containing isoforms, a function performed by Desmin in mammals.

5. Interplay of RFX transcription factors 1, 2 and 3 in motile ciliogenesis.
   Lemeille S, Paschaki M, Baas D, Morlé L, Duteyrat JL, Ait-Lounis A, Barras E, Soulavie F, Jerber J, Thomas J, Zhang Y, Holtzman MJ, Kistler WS, Reith W, Durand B.
   Nucleic Acid Res (2020) — Show abstract

   Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo.

6. Dzip1 and Fam92 form a ciliary transition zone complex with cell type specific roles in Drosophila.
   Lapart JA, Gottardo M, Cortier E, Duteyrat JL, Augière C, Mangé A, Jerber J, Solassol J, Gopalakrishnan J, Thomas J, Durand B
   eLife (2019) — Show abstract

   Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. Here, we show that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. We identify cell type specific roles of this functional module in two different tissues. While it is required for TZ assembly in all Drosophila ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. We therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization.

7. salto/CG13164 is required for sperm head morphogenesis in Drosophila.
   Augière C, Lapart JA, Duteyrat JL, Cortier E, Maire C, Thomas J, Durand B.
   Mol. Biol. Cell. (2019) — Show abstract

   Producing mature spermatozoa is essential for sexual reproduction in metazoans. Spermiogenesis involves dramatic cell morphological changes going from sperm tail elongation and nuclear reshaping to cell membrane remodeling during sperm individualization and release. The sperm manchette plays a critical scaffolding function during nuclear remodeling by linking the nuclear lamina to the cytoskeleton. Here, we describe the role of an uncharacterized protein in Drosophila, salto/CG13164, involved in nuclear shaping and spermatid individualization. Salto has dynamic localization during spermatid differentiation, being progressively relocated from the sperm-nuclear dense body, which is equivalent to the mammalian sperm manchette, to the centriolar adjunct and acrosomal cap during spermiogenesis. salto-null male flies are sterile and exhibit complete spermatid individualization defects. salto-deficient spermatids show coiled spermatid nuclei at late maturation stages and stalled individualization complexes. Our work sheds light on a novel component involved in cytoskeleton-based cell-morphological changes during spermiogenesis.

8. Altered GLI3 and FGF8 signaling underlies Acrocallosal syndrome phenotypes in Kif7 depleted mice.
   Putoux A, Baas D, Paschaki M, Morlé L, Maire C, Attié-Bitach T, Thomas S, Durand B.
   Hum Mol Genet. (2019) — Show abstract

   Acrocallosal syndrome (ACLS) is a rare genetic disorder characterized by agenesis or hypoplasia of corpus callosum (CC), polydactyly, craniofacial dysmorphism and severe intellectual deficiency. We previously identified KIF7, a key ciliary component of the Sonic hedgehog (SHH) pathway, as being a causative gene for this syndrome, thus including ACLS in the group of ciliopathies. In both humans and mice, KIF7 depletion leads to abnormal GLI3 processing and over-activation of SHH target genes. To understand the pathological mechanisms involved in CC defects in this syndrome, we took advantage of a previously described Kif7-/- mouse model to demonstrate that in addition to polydactyly and neural tube closure defects, these mice present CC agenesis with characteristic Probst bundles, thus recapitulating major ACLS features. We show that CC agenesis in these mice is associated with specific patterning defects of the cortical septum boundary leading to altered distribution of guidepost cells required to guide the callosal axons through the midline. Furthermore, by crossing Kif7-/- mice with Gli3Δ699 mice exclusively producing the repressive isoform of GLI3 (GLI3R), we demonstrate that decreased GLI3R signaling is fully responsible for the ACLS features in these mice, as all phenotypes are rescued by increasing GLI3R activity. Moreover, we show that increased FGF8 signaling is responsible in part for CC defects associated to KIF7 depletion, as modulating FGF8 signaling rescued CC formation anteriorly in Kif7-/- mice. Taken together our data demonstrate that ACLS features rely on defective GLI3R and FGF8 signaling.

9. Genetic specification of left-right asymmetry in the diaphragm muscles and their motor innervation.
   Charoy C, Dinvaut S, Chaix Y, Morlé L, Sanyas I, Bozon M, Kindbeiter K, Durand B, Skidmore JM, De Groef L, Seki M, Moons L, Ruhrberg C, Martin JF, Martin DM, Falk J, Castellani V.
   Elife (2017) — Show abstract

   The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

10. Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells.
    Vieillard J, Paschaki M, Duteyrat JL, Augière C, Cortier E, Lapart JA, Thomas J and Durand B.
    Cell Biology (2016) — Show abstract

    The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. We show that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. Our results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells.

11. Drosophila melanogaster as a model for basal body research.
    Jana SC, Bettencourt-Dias M, Durand B, and Megraw TL.
    Cilia (2016) — Show abstract

    The fruit fly, Drosophila melanogaster, is one of the most extensively studied organisms in biological research and has centrioles/basal bodies and cilia that can be modelled to investigate their functions in animals generally. Centrioles are nine-fold symmetrical microtubule-based cylindrical structures required to form centrosomes and also to nucleate the formation of cilia and flagella. When they function to template cilia, centrioles transition into basal bodies. The fruit fly has various types of basal bodies and cilia, which are needed for sensory neuron and sperm function. Genetics, cell biology and behaviour studies in the fruit fly have unveiled new basal body components and revealed different modes of assembly and functions of basal bodies that are conserved in many other organisms, including human, green algae and plasmodium. Here we describe the various basal bodies of Drosophila, what is known about their composition, structure and function.

12. RFX2 Is a Major Transcriptional Regulator of Spermiogenesis.
    Kistler WS, Baas D, Lemeille S, Paschaki M, Seguin-Estevez Q, Barras E, Ma W, Duteyrat JL, Morlé L, Durand B and Reith W.
    Plos Genetics (2015) — Show abstract

    Spermatogenesis consists broadly of three phases: proliferation of diploid germ cells, meiosis, and finally extensive differentiation of the haploid cells into effective delivery vehicles for the paternal genome. Despite detailed characterization of many haploid developmental steps leading to sperm, only fragmentary information exists on the control of gene expression underlying these processes. Here we report that the RFX2 transcription factor is a master regulator of genes required for the haploid phase. A targeted mutation of Rfx2 was created in mice. Rfx2-/- mice are perfectly viable but show complete male sterility. Spermatogenesis appears to progress unperturbed through meiosis. However, haploid cells undergo a complete arrest in spermatid development just prior to spermatid elongation. Arrested cells show altered Golgi apparatus organization, leading to a deficit in the generation of a spreading acrosomal cap from proacrosomal vesicles. Arrested cells ultimately merge to form giant multinucleated cells released to the epididymis. Spermatids also completely fail to form the flagellar axoneme. RNA-Seq analysis and ChIP-Seq analysis identified 139 genes directly controlled by RFX2 during spermiogenesis. Gene ontology analysis revealed that genes required for cilium function are specifically enriched in down- and upregulated genes showing that RFX2 allows precise temporal expression of ciliary genes. Several genes required for cell adhesion and cytoskeleton remodeling are also downregulated. Comparison of RFX2-regulated genes with those controlled by other major transcriptional regulators of spermiogenesis showed that each controls independent gene sets. Altogether, these observations show that RFX2 plays a major and specific function in spermiogenesis.