Malaria is caused by unicellularPlasmodiumparasites.Plasmodiumrelies 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 acrossPlasmodiumstages 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 inPlasmodiumdespite 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 inPlasmodiumspecies.

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.

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.

Centrioles play key roles in mitotic spindle assembly. Once assembled, centrioles exhibit long-term stability, but how stability is achieved and how it is regulated are not completely understood. In this study we show that SSNA-1, the Caenorhabditis elegans ortholog of Sjogren’s Syndrome Nuclear Antigen 1, is a constituent of centrioles and centriole satellite-like structures. A deletion of ssna-1 results in the formation of extra centrioles. We show that SSNA-1 genetically interacts with the centriole stability factor SAS-1 and is required post assembly for centriole structural integrity. In SSNA-1’s absence, centrioles assemble but fracture leading to extra spindle poles. However, if the efficiency of cartwheel assembly is reduced, the absence of SSNA-1 results in daughter centriole loss and monopolar spindles, indicating that the cartwheel and SSNA-1 cooperate to stabilize centrioles during assembly. Our work thus shows that SSNA-1 contributes to centriole stability during and after assembly, thereby ensuring proper centriole number.

Centromeres are essential for chromosome segregation in eukaryotes, yet their specification is unexpectedly diverse among species and can involve major transitions such as those from localized to chromosome-wide centromeres between monocentric and holocentric species. How this diversity evolves remains elusive. We discovered within-cell variation in the recruitment of the major centromere protein CenH3, reminiscent of variation typically observed among species. While CenH3-containing nucleosomes are distributed in a monocentric fashion on autosomes and bind tandem repeat sequences specific to individual or groups of chromosomes, they show a longitudinal distribution and broad intergenic binding on the X chromosome, which partially recapitulates phenotypes known from holocentric species. Despite this variable CenH3 distribution among chromosomes, all chromosomes are functionally monocentric, marking the first instance of a monocentric species with chromosome-wide CenH3 deposition. Together, our findings illustrate a potential transitional state between mono- and holocentricity or toward CenH3-independent centromere determination and help to understand the rapid centromere sequence divergence between species.

Centrioles are evolutionarily conserved barrel-shaped organelles playing crucial roles in cell division and ciliogenesis. These functions are underpinned by specific structural sub-elements whose functions have been under investigation since many years. The A-C linker structure, connecting adjacent microtubule triplets in the proximal region, has remained unexplored due to its unknown composition. Here, using ultrastructure expansion microscopy, we characterized two recently identified A-C linker proteins, CCDC77 and WDR67, and discovered MIIP as an additional A-C linker protein. Our findings reveal that these proteins localize between microtubule triplets at the A-C linker, forming a complex. Depletion of A-C linker components disrupt microtubule triplet cohesion, leading to breakage at the proximal end. Co-removal of the A-C linker and the inner scaffold demonstrates their joint role in maintaining centriole architecture. Moreover, we uncover an unexpected function of the A-C linker in centriole duplication through torus regulation, underscoring the interplay between these protein modules.