Ultrastructure Expansion Microscopy applied to C. elegans embryos.

Visualization of organelles using expansion microscopy has been previously applied to Caenorhadbitis elegans adult gonads or worms. However, its application to embryos has remained a challenge due to the protective eggshell barrier. Here, by combining freeze-cracking and ultrastructure expansion microscopy (U-ExM), we demonstrate a four-time isotropic expansion of C. elegans embryos. As an example structure, we chose the nuclear pore and demonstrate that we achieve sufficient resolution to distinguish them individually. Our work provides proof of principle for U-ExM in C. elegans embryos, which will be applicable for imaging a wide range of cellular structures in this model system.

Time-series reconstruction of the molecular architecture of human centriole assembly.

Centriole biogenesis, as in most organelle assemblies, involves the sequential recruitment of sub-structural elements that will support its function. To uncover this process, we correlated the spatial location of 24 centriolar proteins with structural features using expansion microscopy. A time-series reconstruction of protein distributions throughout human procentriole assembly unveiled the molecular architecture of the centriole biogenesis steps. We found that the process initiates with the formation of a naked cartwheel devoid of microtubules. Next, the bloom phase progresses with microtubule blade assembly, concomitantly with radial separation and rapid cartwheel growth. In the subsequent elongation phase, the tubulin backbone grows linearly with the recruitment of the A-C linker, followed by proteins of the inner scaffold (IS). By following six structural modules, we modeled 4D assembly of the human centriole. Collectively, this work provides a framework to investigate the spatial and temporal assembly of large macromolecules.

Unraveling the Bombus terrestris Hemolymph, an Indicator of the Immune Response to Microbial Infections, through Complementary Mass Spectrometry Approaches.

Pollinators, including Bombus terrestris, are crucial for maintaining biodiversity in ecosystems and for agriculture. Deciphering their immune response under stress conditions is a key issue for protecting these populations. To assess this metric, we analyzed the B. terrestris hemolymph as an indicator of their immune status. Hemolymph analysis was carried out using mass spectrometry, MALDI molecular mass fingerprinting was used for its effectiveness in assessing the immune status, and high-resolution mass spectrometry was used to measure the impact of experimental bacterial infections on the "hemoproteome". By infecting with three different types of bacteria, we observed that B. terrestris reacts in a specific way to bacterial attacks. Indeed, bacteria impact survival and stimulate an immune response in infected individuals, visible through changes in the molecular composition of their hemolymph. The characterization and label-free quantification of proteins involved in specific signaling pathways in bumble bees by bottom-up proteomics revealed differences in protein expression between the non-experimentally infected and the infected bees. Our results highlight the alteration of pathways involved in immune and defense reactions, stress, and energetic metabolism. Lastly, we developed molecular signatures reflecting the health status of B. terrestris to pave the way for diagnosis/prognosis tools in response to environmental stress.

Correction: Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

[This corrects the article DOI: 10.1371/journal.pbio.3001020.].

Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

Essential function of the alveolin network in the subpellicular microtubules and conoid assembly in Toxoplasma gondii.

The coccidian subgroup of Apicomplexa possesses an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR). The APR serves as microtubule-organizing center for the 22 subpellicular microtubules (SPMTs) that are linked to a patchwork of flattened vesicles, via an intricate network composed of alveolins. Here, we capitalize on ultrastructure expansion microscopy (U-ExM) to localize the Toxoplasma gondii Apical Cap protein 9 (AC9) and its partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Parasites conditionally depleted in AC9 or AC10 replicate normally but are defective in microneme secretion and fail to invade and egress from infected cells. Electron microscopy revealed that the mature parasite mutants are conoidless, while U-ExM highlighted the disorganization of the SPMTs which likely results in the catastrophic loss of APR and conoid.