Virginie Hamel

Ciliary microtubule doublets, consisting of an incomplete B-tubule on the surface of a complete A-microtubule, form the fundamental structural framework of motile and sensory cilia/flagella. Ciliary proteins PACRG and FAP20, which are essential for flagellar stability and function, localize to the junction between the A- and the B-tubule. However, their precise role in microtubule doublet assembly and dynamics remains unknown. Here, using a cell-free assay, TIRF-microscopy, and cryo-electron tomography, we demonstrate that in combination PACRG and FAP20 stabilize B-tubule dynamics and microtubule doublet architecture. We show that together these proteins localize to the B-tubules in distinct, high-density patches, which locally stabilize B-tubule by decreasing its depolymerization velocity and increasing its rescue frequency. Cryo-tomography of in vitro reconstructed microtubule doublets in presence of PACRG and FAP20 reveals reduced B-tubule curvature fluctuations, promoting a more rigid and aligned conformation. Altogether, our findings suggest that PACRG and FAP20 function synergistically to reinforce microtubule doublet stability, ensuring ciliary integrity and function.

The ciliary transition zone (TZ) regulates ciliary proteome composition, yet its molecular architecture, protein content, and contribution to motile ciliopathies remain poorly defined. We applied in situ cryo–electron tomography and subtomogram averaging to human multiciliated epithelial cells. This approach resolved TZ-specific doublet microtubules at subnanometer resolution and identified nine constituent proteins. We found that epithelial cell transforming 2 like (ECT2L) and double zinc ribbon and ankyrin repeat-containing protein 1 (DZANK1) form the major linker complexes between adjacent TZ doublet microtubules. Biallelic loss-of-function variants in either gene cause primary ciliary dyskinesia. ECT2L and DZANK1 deficiency disrupted TZ architecture, caused microtubular abnormalities and abnormal bulbous ciliary tips, and impaired mucociliary clearance. These findings establish a direct genetic link between TZ defects and human motile ciliopathy and illustrate how in situ structural biology can uncover mechanisms of human disease.

Expansion microscopy (ExM) enables nanoscale imaging using conventional fluorescence microscopes and has rapidly evolved through advances in hydrogel chemistries and sample-processing strategies. However, most biological applications remain limited to expansion factors below ~20× due to fluorescence dilution and poor hydrogel stability. Here, we introduce BiG-ExM (Boosted Iterative Gel-Expansion Microscopy), an iterative expansion approach that achieves 40–50× linear expansion while preserving high fluorescence signal and sample integrity. BiG-ExM combines an initial ~4× expansion hydrogel, a simple signal-amplification strategy termed FluoBoost, and a newly developed AMPS-based polymer that enables a second ~14× expansion with enhanced mechanical robustness. We validate BiG-ExM across diverse specimens, including nuclear pore complexes, bacteriophage T4 particles, cellular microtubules, and mitochondria visualized by boosted pan-staining. The combination of large expansion and efficient signal retention enables super-resolution light-sheet microscopy and rapid volumetric imaging. BiG-ExM extends the resolution and scalability of optical microscopy for a broad range of biological applications.

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.

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.

Cilia and flagella contribute to cell morphogenesis in multiple organisms. In the parasite Trypanosoma brucei , the flagellum is attached along the length of the cell body and acts as a guide for cell division, while its motility function is required for the completion of cytokinesis. To tease apart the contributions of flagellum length and motility to trypanosome morphogenesis, we investigated the coiled-coil containing domain 40 (CCDC40 or FAP172) protein. Iterative Ultrastructure Expansion Microscopy (iU-ExM) revealed that CCDC40 is associated to the 96-nm repeats of the trypanosome axoneme. CCDC40 depletion by RNAi leads to loss of components from the dynein regulatory complex, inner dynein arms and radial spokes, resulting in disconnected microtubule doublets and disorganised axoneme structure, abrogating motility and resulting in flagella and cell bodies 2-3 times shorter than normal. We show for the first time that this short flagellum phenotype is associated to slower tubulin incorporation and premature acquisition of the maturation marker FLAM8 but not of the locking protein CEP164C. Surprisingly, these short and immotile trypanosomes grow and divide normally. We discuss the significance of these observations for trypanosome morphogenesis and division.

Given the critical role of CD4 T cells in anti-tumor immunity, strategies to harness these cells for cancer immunotherapy are gaining increasing interest. Historically overshadowed by CD8 T cells, cytotoxic CD4 T cells can directly kill MHC class II-expressing tumor cells. However, the defining molecular signature and the mechanisms underlying their cytolytic activity remain poorly understood, particularly in cancer patients. Here, using ex vivo single-cell transcriptomic and spatial analyses of CD4 T cells from paired blood and tumor samples of melanoma patients, we identified Killer Cell Lectin-Like Receptor G1 (KLRG1) as a defining surface marker of cytotoxic CD4 T cells. The CD4 ⁺ KLRG1 ⁺ T cell subset was notably enriched among circulating cells compared with tumor-infiltrating populations, which were instead enriched in T follicular helper (Tfh) states. Functionally, KLRG1 ⁺ CD4 T cells expressed elevated levels of cytotoxic genes and exhibited superior tumor-killing capacity compared with their KLRG1 ^- counterparts. We demonstrated that their cytotoxicity is granulysin-dependent, as confirmed by CRISPR/Cas9-mediated gene deletion. Mechanistically, CD4 T cells spared MHC class II ⁺ cells lacking the KLRG1 ligands CD324 and CD325, such as professional antigen-presenting cells (APCs), indicating that cytotoxicity was selectively directed towards tumor cells while preserving immune cells. Finally, by investigating how the tumor microenvironment may impair CD4 T cell cytotoxicity, we showed that tumor-derived factors, including interleukin-6 (IL-6), are key drivers promoting the transition of cytotoxic CD4 T cells toward a Tfh phenotype. In summary, our findings define KLRG1 as a defining cell surface marker of cytotoxic CD4 T cells in cancer patients, as well as a key regulator that protects MHC class II ⁺ APCs. Moreover, targeting the IL-6 signalling pathway may enhance CD4 T cell anti-tumor cytotoxicity, offering new avenues for cancer immunotherapy.

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.