Biology subjects

Mercey, O.

Publications and source records attributed to Mercey, O..

13 recordsLinked to original sources

bioRxiv2026

DCX enables branching of subpellicular microtubules in Plasmodium falciparum gametocytes and is required for mosquito colonisation

Plasmodium falciparum, the causative agent of malaria, relies on specialised tubulin-based cytoskeletal structures to support its parasitic lifestyle. These include the conoid required for parasite motility and host-cell invasion, as well as subpellicular microtubules (SPMTs) that support parasite shape and rigidity. Here, we investigate the function of the doublecortin-domain protein DCX, a microtubule-binding protein previously associated with the Plasmodium conoid. We first show that, in P. falciparum, DCX is not expressed in the merozoite stage and is not required for the invasion of human erythrocytes. By contrast, DCX is expressed in ookinetes, the motile stage responsible for infecting the mosquito vector, where it associates with conoid tubulin fibres, consistent with a role in stabilising the conoid architecture. Unexpectedly, we find that DCX is required for P. falciparum transmission to the mosquito independently of conoid function. We further link this requirement to the distinctive organisation of SPMTs in P. falciparum gametocytes, which display an unusual branching architecture comprising multiple microtubules of 15 to 18 protofilaments. Deletion of DCX leads to a reduction in SPMT branching and is associated with higher protofilament numbers, revealing a previously unrecognised role for DCX in shaping the ultrastructure of SPMTs in P. falciparum gametocytes. Altogether, our findings uncover the repurposing of DCX across distinct microtubule systems in transmission stages and identify DCX as a key factor mediating microtubule branching and stabilisation in SPMTs required for efficient mosquito transmission.

microbiology↗

bioRxiv2026

Gene Supplementation of MYO7A or activation of Myo7b for treatment of Usher syndrome 1B

Mutations in MYO7A result in the most severe subtype of Usher syndrome, the leading genetic cause of deafblindness. The large size of MYO7A requires dual adeno-associated virus (AAV) vectors for gene transfer or alternative methods to treat retinal defects. Here, we evaluated two treatment approaches: i) Supplementation of the human MYO7A gene via dual mRNA trans-splicing AAVs, and ii) CRISPR/Cas-mediated activation of the related murine Myo7b gene. Upon MYO7A supplementation, the transgenic MYO7A transcript and protein were expressed and correctly localized in retinal pigment epithelial (RPE) and photoreceptors of mice, pigs, and human retinal organoids. In RPE-and photoreceptor-specific Myo7a knockout mice, we could restore MYO7A expression and localization of melanosomes in RPE cells to wild-type levels. Myo7b activation led to partial restoration of melanosome localization, and the localization of MYO7B protein was largely comparable to MYO7A. These findings indicate that both approaches are in principle suitable for the therapy of Usher syndrome.

molecular biology↗

bioRxiv2026

Molecular architecture of the ciliary base in mammalian multiciliated cells

Multiciliated epithelial cells (MCCs) generate tens to hundreds of motile cilia to drive fluid flow in diverse physiological contexts. While the axonemal structure of motile cilia has been described extensively in recent years, the molecular architecture of the transition zone, basal body, and surrounding ciliary environment of MCCs remain more elusive. Here, we use cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to obtain in situ 3D views of the ciliary base within intact MCCs from mammalian trachea, complemented by in situ cross-linking mass spectrometry (XL/MS) and ultrastructure expansion microscopy (U-ExM) for molecular identification. Our data reveal spatially-defined modifications of microtubule architecture from the proximal centriole to the early axoneme, including transition zone-specific features such as an A-B linker bridging microtubule doublets and a helical assembly of microtubule inner proteins (MIPs). We show that the ciliary necklace, a feature observed in many motile cilia, is spatially aligned with the transition zone and quantify its regular organization within the membrane. Our in situ data capture rarely observed events, including intraflagellar transport (IFT) trains connecting to ciliary vesicles tethered to undocked centrioles. The surrounding ciliary environment contains intermediate filaments that encircle the basal bodies and bundled actin filaments that elaborate microvilli structures between the cilia. Integration of XL/MS and U-ExM identified novel microtubule associated proteins (MAPs), MIPs, and membrane-associated proteins localized to these distinct subdomains. This work provides a molecular and structural map of the mammalian MCC ciliary base, revealing architectural principles that underlie its assembly, organization, and function.

cell biology↗

bioRxiv2026

The Joubert gene TMEM67 is required for the correct establishment of spinal dorsal identities in human organoids.

Primary cilia are essential signaling organelles that mediate key developmental pathways, including Sonic Hedgehog (SHH) and WNT, and are crucial for tissue patterning and homeostasis. Ciliary dysfunction underlies a spectrum of human ciliopathies--such as Joubert syndrome (JBTS) and Meckel syndrome (MKS)--which present with profound neurodevelopmental abnormalities. Although the role of cilia in SHH-dependent ventral spinal cord patterning is well established, their contribution to dorsal spinal cord development, particularly in human systems, remains poorly defined. To address this gap, we utilized human spinal organoids to investigate the function of a ciliopathy-associated protein in dorsal neural tube patterning downstream of BMP4, independent of exogenous SHH. Using TMEM67 knockout human iPSC-derived dorsal spinal organoids, we demonstrate that loss of TMEM67 disrupts the specification of dorsal interneurons, most notably within the dI1 lineage, while concomitantly expanding intermediate dorsal progenitor populations (dI4-dI6). These patterning defects are associated with defective roof plate induction and attenuated BMP4 signaling. Mechanistically, TMEM67 deficiency alters ciliary morphology, decreases cilia number, and impairs recruitment of BMPR2 to the ciliary base, suggesting a direct role for cilia in modulating BMP-induced dorsal spinal patterning. Together, these findings provide new mechanistic insights into the pathogenesis of ciliopathies and underscore the value of human organoid models for elucidating human-specific aspects of neurodevelopmental disorders.

developmental biology↗

bioRxiv2025

HAK-actin, U-ExM-compatible probe to image the actin cytoskeleton

Expansion microscopy (ExM) enables super-resolution imaging by physically enlarging biological samples. While ExM has been successfully applied to study the intracellular microtubule cytoskeleton, reliable probes for visualizing actin fibers remain limited. Here, we present HAK-actin, an engineered actin probe compatible with post-labeling Ultrastructure Expansion Microscopy (U-ExM). We show that HAK-actin delivers robust and uniform actin staining across diverse systems, including human cells, microbial eukaryotes, and mouse retinal tissue. This tool provides a simple, versatile, and reproducible solution for actin cytoskeleton visualization, addressing a critical need in cell biology.

cell biology↗

bioRxiv2025

The Luminal Ring Protein C2CD3 Acts as a Radial In-to-Out Organizer of the Distal Centriole and Appendages

Centrioles are polarized microtubule-based structures with appendages at their distal end that are essential for cilia formation and function. The protein C2CD3 is critical for distal appendage assembly, with mutations linked to orofaciodigital syndrome and other ciliopathies. However, its precise molecular role in appendage recruitment remains unclear. Using Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, and in situ cryo-electron tomography (cryo-ET), we reveal that C2CD3 adopts a radially symmetric 9-fold organization within the centrioles distal lumen. We show that the C-terminal region of C2CD3 localizes close to a [~]100 nm luminal ring structure consisting of [~]27 nodes, while its N-terminal region localizes close to a hook-like structure that attaches to the A-microtubule as it extends from the centriole interior to exterior. This hook structure is adjacent to the DISCO complex (MNR/CEP90/OFD1), which marks future appendage sites. C2CD3 depletion disrupts not only the recruitment of the DISCO complex via direct interaction with MNR but also destabilizes the luminal ring network composed of C2CD3/SFI1/centrin-2/CEP135/NA14, as well as the distal microtubule tip protein CEP162. This reveals an intricate "in-to-out" molecular hub connecting the centriolar lumen, distal microtubule cap, and appendages. Although C2CD3 loss results in shorter centrioles and appendage defects, key structural elements remain intact, permitting continued centriole duplication. We propose that C2CD3 forms the luminal ring structure and extends radially to the space between triplet microtubules, functioning as an architectural hub that scaffolds the distal end of the centriole, orchestrating its assembly and directing appendage formation.

cell biology↗

bioRxiv2025

Unveiling the Molecular Architecture of T Cells and Immune Synapses with Cryo-Expansion Microscopy

Cellular communication is critical for anti-cancer immunity, with tumor cell killing occurring at immunological synapses (IS) formed between effector immune cells and target tumor cells. While optical super-resolution microscopy (SRM) has enlightened the spatial organization of the IS mostly in regular immune cells, visualizing the nanoscale architectural features of IS in its native state, including 3D receptor distribution and the ultrastructural details of the lytic granule release remains challenging. Using cryo-expansion microscopy (cryo-ExM), we unravel the cellular architecture of activated T cells and T cell-target cell pairs. Our approach visualizes actin and microtubule networks during synapse formation, membrane topography, and the distribution of signaling molecules and lytic granules of different types, offering novel insights into IS organization. Finally, we apply U-ExM to glioblastoma tissue, visualizing T cells and their lytic content in situ, highlighting its potential for pre-clinical immunotherapy studies.

immunology↗

bioRxiv2024

Glutamylation imbalance leads to photoreceptor degeneration

The stereotypic structure of microtubules, assembled from conserved /{beta}-tubulin dimers is subject to a complex diversity of Post-translational Modifications (PTMs). PTMs are predicted to fine-tune microtubule properties and interactions with other proteins, thus allowing microtubules to perform specific functions. Cilia accumulate several types of tubulin PTMs, such as polyglutamylation, polyglycylation, detyrosination and acetylation, whose functions are not yet fully understood. Recently, mutations of AGBL5, coding for the deglutamylating enzyme CCP5, have been associated to retinitis pigmentosa, suggesting that perturbation of polyglutamylation leads to the degeneration of photoreceptor cells. However, the molecular mechanisms underlying this degeneration remain unknown. Here, using super-resolution Ultrastructure Expansion Microscopy in mouse and human photoreceptor cells, we found that most tubulin PTMs are accumulated at the level of the connecting cilium, a structure linking the outer and inner segments of photoreceptor cells. Using mouse models with increased glutamylation (Ccp5-/- and Ccp1-/-), or loss of tubulin acetylation (Atat1-/-), we demonstrated that aberrant glutamylation, but not loss of acetylation, resulted in perturbed molecular architecture of the outer segment, with the loss of the bulge region and destabilization of the distal axoneme. Concurrently, we observed a substantial impairment in tubulin glycylation and intraflagellar transport. Altogether our results indicate that glutamylation plays a crucial role in the maintenance of the molecular architecture of the outer segment and point to tubulin PTM imbalance as possible culprit in retinal degeneration.

cell biology↗

bioRxiv2023

Fine-tuning FAM161A gene augmentation therapy to restore retinal function

In 15 years, inherited retinal diseases have seen gene therapy as a springboard to hope. Many preclinical investigations focused on vectors with maximal gene expression capabilities. But despite an efficient gene transfer, little physiological improvement was noted for certain ciliopathies. FAM161A is an essential protein for the structure of photoreceptor connecting cilium (CC). In the absence of FAM161A, cilia disorganize resulting in outersegment collapses and vision impairment. Within the human retina, FAM161A produces two isoforms: the long with exon 4, and the short, lacking it. To restore CC in Fam161a-deficient mice, we compared AAV vectors with different promoter activities, doses, and human isoforms injected subretinally in 14-days Fam161atm1b/tm1b mice, shortly after the onset of cilium disorganization. All vectors improved cell survival, but only combining both isoforms using the weak FCBR1-F0.4 promoter allowed precise FAM161A expression in the CC and enhanced retinal function. Our study on FAM161A gene replacement for RP28, a rod-cone-related disease, underscores the critical need for precise therapeutic gene regulation, appropriate vector dosing and delivery of both isoforms. Fine tuning of therapeutic gene expression, tailored to disease traits, is crucial for restoring retinal function. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

neuroscience↗

bioRxiv2023

Probing the sub-cellular mechanisms of LCA5-Leber Congenital Amaurosis and associated gene therapy with expansion microscopy

Leber Congenital Amaurosis (LCA) is a group of Inherited Retinal Diseases (IRDs) characterized by the early onset and rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with Ultrastructure Expansion Microscopy (U-ExM), we revealed the structural and molecular defects underlying LCA type 5 (LCA5) with unprecedented resolution. We showed that LCA5-encoded lebercilin, together with Retinitis Pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localize at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrated that mutant mice deficient for lebercilin exhibit early axonemal defects at the bulge region and the distal OS, accompanied by reduced level of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, we probed the LCA5 gene augmentation therapy strategy using U-ExM by monitoring its subcellular outcome. We found that, expression of LCA5 partially restores the bulge region, preserves OS axoneme structure and membrane disc formation, as well as photoreceptor survival.

molecular biology↗

bioRxiv2022

Nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy (iU-ExM)

Expansion microscopy is an approach of super-resolution fluorescence microscopy that does not yet achieve the precision of nanoscopy techniques such as single-molecule light microscopy (SMLM). Here, we developed an iterative ultrastructure expansion microscopy approach (iU-ExM), which now matches the SMLM resolution as demonstrated using standard references such as the nuclear pores. Applicable to both cells and tissues, iU-ExM allows a broad research community to access high precision super-resolution microscopy.

cell biology↗

bioRxiv2021

The connecting cilium inner scaffold provides a structural foundation to maintain photoreceptor integrity

Retinal degeneration is a leading cause of human blindness due to progressive loss of ciliated photoreceptors cells. While this degradation can be associated with cohesion defects of the microtubule-based connecting cilium (CC) structure, the underlying mechanism is not understood. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC-inner scaffold (CC-IS) containing POC5, CENTRIN and FAM161A. Monitoring CC-IS assembly during photoreceptor development in mouse reveals that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Consistently, Fam161a mutations lead to a specific CC-IS loss and trigger microtubule doublets spreading, prior to outer segment collapse and photoreceptor degeneration, providing a molecular mechanism for retinitis pigmentosa disease. One Sentence SummaryThe connecting cilium inner scaffold acts as a structural zipper granting photoreceptor integrity.

cell biology↗

bioRxiv2017

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells

Multiciliated cells (MCCs) harbour dozens to hundreds of motile cilia, which beat in a synchronized and directional manner, thus generating hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation critically depends on the synthesis and release of numerous centrioles by specialized structures called deuterosomes. Little is known about the composition, organization and regulation of deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome-stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, the host gene of microRNA-449abc. We show that the CDC20B protein associates to deuterosomes and is required for the release of centrioles and the subsequent production of cilia in mouse and Xenopus MCCs. CDC20B interacts with PLK1, which has been shown to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescued centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of a new biological function, deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.

cell biology↗