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.

iU-ExM: nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy.

Expansion microscopy (ExM) is a highly effective technique for super-resolution fluorescence microscopy that enables imaging of biological samples beyond the diffraction limit with conventional fluorescence microscopes. Despite the development of several enhanced protocols, ExM has not yet demonstrated the ability to achieve the precision of nanoscopy techniques such as Single Molecule Localization Microscopy (SMLM). Here, to address this limitation, we have developed an iterative ultrastructure expansion microscopy (iU-ExM) approach that achieves SMLM-level resolution. With iU-ExM, it is now possible to visualize the molecular architecture of gold-standard samples, such as the eight-fold symmetry of nuclear pores or the molecular organization of the conoid in Apicomplexa. With its wide-ranging applications, from isolated organelles to cells and tissue, iU-ExM opens new super-resolution avenues for scientists studying biological structures and functions.

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.