Biology subjects

Fortun, D.

Publications and source records attributed to Fortun, D..

4 recordsLinked to original sources

bioRxiv2025

Deciphering the dynamic proteome of multiciliated cells

Multiciliated cells (MCCs) are essential for generating directional fluid flow across specialized epithelia in various vertebrate organs. MCC differentiation involves a unique, tightly regulated program characterized by massive centriole amplification, independently of DNA replication. Although much is known about the transcriptional control of MCC development, insights into proteome dynamics have been limited due to the lack of suitable models. In this study, we report the generation of a stable inducible MCC line, derived from Xenopus laevis A6 kidney epithelial cells. Upon induction of the MCC master regulator Multicilin (MCI), most A6-MCI cells synchronously differentiate into mature MCCs in 48 hours. Using this novel resource, custom antibodies and super-resolution imaging, we characterized Xenopus deuterosomes, the platforms that allow massive centriole synthesis in vertebrate MCCs. We performed a detailed proteomic profiling throughout MCC differentiation, and uncovered previously uncharacterized regulators. Notably, we highlight a critical role for CDK7 in MCC differentiation in both Xenopus and human systems. Our work provides a valuable resource for mechanistic studies of MCC biology and opens avenues to identify novel therapeutic targets for motile ciliopathies.

cell biology↗

bioRxiv2020

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Super-resolution microscopies, which allow features below the diffraction limit to be resolved, have become an established tool in biological research. However, imaging throughput remains a major bottleneck in using them for quantitative biology, which requires large datasets to overcome the noise of the imaging itself and to capture the variability inherent to biological processes. Here, we develop a multi-focal flat illumination for field independent imaging (mfFIFI) module, and integrate it into an instant structured illumination microscope (iSIM). Our instrument extends the field of view (FOV) to >100x100 {micro}m2 without compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching multiple adjacent images together to perform fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at an effective resolution of [~]35 nm. We apply classification and particle averaging to these large datasets, allowing us to map the 3D organization of post-translational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

biophysics↗

bioRxiv2018

Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM)

For decades, electron microscopy (EM) was the only method able to reveal the ultrastructure of cellular organelles and molecular complexes because of the diffraction limit of optical microscopy. In recent past, the emergence of superresolution fluorescence microscopy enabled the visualization of cellular structures with so far unmatched spatial resolution approaching virtually molecular dimensions. Despite these technological advances, currently super-resolution microscopy does not permit the same resolution level as provided by electron microscopy, impeding the attribution of a protein to an ultrastructural element. Here, we report a novel method of near-native expansion microscopy (UltraExM), enabling the visualization of preserved ultrastructures of macromolecular assemblies with subdiffraction-resolution by standard optical microscopy. UltraExM revealed for the first time the ultrastructural localization of tubulin glutamylation in centrioles. Combined with super-resolution microscopy, UltraExM unveiled the centriolar chirality, an ultrastructural signature, which was only visualizable by electron microscopy.

cell biology↗

bioRxiv2018

Imaging neural activity in the ventral nerve cord of behaving adult Drosophila

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because the portion of the nervous system that contains limb premotor and motor circuits is largely inaccessible to large-scale recording techniques in intact, moving animals - a constraint that is true for both vertebrate and invertebrate models. Here, we introduce a method for 2-photon functional imaging from the ventral nerve cord of behaving adult Drosophila melanogaster. We use this method to reveal patterns of activity across nerve cord populations during grooming and walking and to uncover the functional encoding of moonwalker ascending neurons (MANs), moonwalker descending neurons (MDNs), and a novel class of locomotion-associated descending neurons. This new approach enables the direct investigation of circuits associated with complex limb movements.

neuroscience↗