The attribution of a protein to an ultrastructural element by optical microscopy represents a major challenge in biology. Here, we report a method of near-native expansion microscopy (U-ExM), enabling the visualization of preserved ultrastructures of macromolecules by optical microscopy. Combined with super-resolution, U-ExM unveiled the centriolar chirality, only visualizable by electron microscopy. We demonstrate the general applicability of U-ExM by imaging different cellular structures including microtubules and mitochondria in cellulo .

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo–electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

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.

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.

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with singlemolecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo–electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas . We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five-to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight-or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six-to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight-and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.The formation of centrosomes in animal cells, as well as that of cilia and flagella in eukaryotic cells, critically depends on the evolutionarily conserved microtubule-based cylindrical centriole. In most species, newly formed centrioles are organized around a nine-fold radially symmetric 'cartwheel' structure. This cartwheel comprises a central hub ∼25 nm in diameter from which nine spokes emanate and connect through a pinhead structure to nine microtubule triplets, which are linked together and constitute a peripheral 'microtubule wall' (reviewed in refs 1,2). A generally acknowledged model posits that the cartwheel acts as a molecular scaffold for the formation of the nine-fold symmetric architecture of centrioles (reviewed in refs 1-7). In its strictest sense, this 'scaffold model' of centriole formation postulates that the nine-fold symmetric cartwheel is formed first and then dictates the assembly of a nine-fold symmetric microtubule wall.A key prediction of the scaffold model is that changes in cartwheel symmetry should dictate likewise changes in the architecture of the centriole. A central component of the cartwheel is SAS-6, a protein that is essential for cartwheel formation across eukaryotes (reviewed in refs 1,2). In Chlamydomonas, however, despite the absence of cartwheels, ∼20% of cells bearing a deletion of the sole SAS-6 gene nevertheless assemble centrioles that comprise a circular microtubule wall with a nine-fold symmetry in ∼70% of cases, but with seven-, eight-, ten-or eleven-fold symmetries in the remaining cases 8 . These observations suggest that the cartwheel is critical for proper assembly of the microtubule wall in Chlamydomonas, but also that in this species the centriolar microtubules have an inherent ability to assemble into near-nine-fold symmetrical structures. In contrast, knockdown of SAS-6 proteins in other systems, including human cells 9,10 , results in a failure...