Biology subjects

Van der Stappen, P.

Publications and source records attributed to Van der Stappen, P..

6 recordsLinked to original sources

bioRxiv2026

Molecular organization of the Chlorella sorokiniana pyrenoid

To overcome the enzymatic limitations of Rubisco, algae operate CO2-concentrating mechanisms (CCMs) that deliver concentrated CO2 to Rubisco tightly packaged in a specialized microcompartment called a pyrenoid. Pyrenoids are globally important biomolecular condensates, but their convergent evolution means that their molecular composition and emergent architecture cannot be inferred across clades. Here we characterize the pyrenoid of the Trebouxiophyceae alga, Chlorella sorokiniana. Using cryo-electron tomography, we provide an architectural overview of the pyrenoid and visualize pyrenoid-specific protein complexes. Quantitative proteomics and Rubisco co-immunoprecipitation followed by mass spectrometry demonstrate that inorganic carbon delivery machinery is conserved across green algae but the pyrenoid structural components are not. In vitro reconstitution supports the role of two previously undescribed proteins, one in assembly of pyrenoid traversing thylakoids (putative matrix thylakoid tether; PMTT) and another in starch tethering to the Rubisco matrix (putative matrix starch tether; PMST). In Nicotiana benthamiana, PMTT localized to the thylakoid stromal lamellae and PMST to chloroplast starch granules. Our findings provide insights into the molecular logic of pyrenoid assembly; how proteins mediate condensate-membrane and condensate-starch interactions; and expands the pyrenoid plant engineering toolkit, setting the stage for engineering a Chlorella pyrenoid into plants.

plant biology↗

bioRxiv2026

Molecular architecture of the ciliary base in mammalian multiciliated cells

Multiciliated epithelial cells (MCCs) generate tens to hundreds of motile cilia to drive fluid flow in diverse physiological contexts. While the axonemal structure of motile cilia has been described extensively in recent years, the molecular architecture of the transition zone, basal body, and surrounding ciliary environment of MCCs remain more elusive. Here, we use cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to obtain in situ 3D views of the ciliary base within intact MCCs from mammalian trachea, complemented by in situ cross-linking mass spectrometry (XL/MS) and ultrastructure expansion microscopy (U-ExM) for molecular identification. Our data reveal spatially-defined modifications of microtubule architecture from the proximal centriole to the early axoneme, including transition zone-specific features such as an A-B linker bridging microtubule doublets and a helical assembly of microtubule inner proteins (MIPs). We show that the ciliary necklace, a feature observed in many motile cilia, is spatially aligned with the transition zone and quantify its regular organization within the membrane. Our in situ data capture rarely observed events, including intraflagellar transport (IFT) trains connecting to ciliary vesicles tethered to undocked centrioles. The surrounding ciliary environment contains intermediate filaments that encircle the basal bodies and bundled actin filaments that elaborate microvilli structures between the cilia. Integration of XL/MS and U-ExM identified novel microtubule associated proteins (MAPs), MIPs, and membrane-associated proteins localized to these distinct subdomains. This work provides a molecular and structural map of the mammalian MCC ciliary base, revealing architectural principles that underlie its assembly, organization, and function.

cell biology↗

bioRxiv2026

Chloroplast-encoded small subunit extensions reshape the Chlamydomonas chlororibosome

Chloroplast ribosomes (chlororibosomes) synthesize the core protein components of the photosynthetic apparatus, yet their structural diversity outside flowering plants remains largely unexplored. Here, we combine in situ cryo-electron tomography (cryo-ET) with single-particle cryo-electron microscopy (cryo-EM) to determine the structure of the chlororibosome from the unicellular green alga Chlamydomonas reinhardtii. Subtomogram averaging of chlororibosomes in their native environment, resolved to [~]5 [A] resolution and in distinct translational states, reveals particles both free in the stroma and loosely tethered to thylakoid membranes. These in situ reconstructions uncover an additional "arm" domain on the small subunit. High-resolution single-particle reconstruction of isolated chlororibosomes to [~]2.5 [A], in states bound either to the inhibitory translation factor pY or to a nascent chain-linked P-site tRNA, reveals that this domain is built primarily from extensive chloroplast-encoded insertions and extensions of conserved small subunit proteins, supported by chlororibosome-specific ribosomal proteins. The arm domain is located around the mRNA entry and exit channels, suggesting a role in stabilizing the mRNA trajectory through the small subunit and organizing chloroplast polysomes. Together, these data reveal unexpected structural variation of algal chlororibosomes and suggest that chloroplast translation has diversified substantially even among relatively closely related photosynthetic lineages.

molecular biology↗

bioRxiv2026

Sticker number modulates pyrenoid condensate assembly to support algal fitness

The valency of intrinsically disordered proteins underpins liquid-liquid phase separation (LLPS), yet how this parameter shapes condensate function and cellular fitness remains poorly understood. Here we exploit the algal pyrenoid-a minimal, two component LLPS system-to directly link condensate properties to physiological performance. Pyrenoid assembly is driven by a disordered, multivalent Linker protein that binds Rubisco at symmetry-related surface sites, with the number of binding motifs ("stickers") varying across species. Using Chlamydomonas reinhardtii, we systematically tuned sticker number from two to nine and examined effects on Rubisco condensation, pyrenoid architecture and CO2 fixation. Three stickers were sufficient for condensation in vitro, but at least four were required for pyrenoid assembly in vivo. Cryo-electron tomography and single-molecule tracking revealed that increasing sticker number enhances Rubisco packing and mobility, while time-resolved imaging and competition assays demonstrated that sticker number governs the kinetics of pyrenoid formation and determines cellular fitness under fluctuating carbon conditions. Our findings establish sticker number as an evolutionary tuning parameter that balances condensate formation, dynamics, and function, providing a quantitative framework for linking the molecular grammar of phase separation to biological fitness.

cell biology↗

bioRxiv2025

The Luminal Ring Protein C2CD3 Acts as a Radial In-to-Out Organizer of the Distal Centriole and Appendages

Centrioles are polarized microtubule-based structures with appendages at their distal end that are essential for cilia formation and function. The protein C2CD3 is critical for distal appendage assembly, with mutations linked to orofaciodigital syndrome and other ciliopathies. However, its precise molecular role in appendage recruitment remains unclear. Using Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, and in situ cryo-electron tomography (cryo-ET), we reveal that C2CD3 adopts a radially symmetric 9-fold organization within the centrioles distal lumen. We show that the C-terminal region of C2CD3 localizes close to a [~]100 nm luminal ring structure consisting of [~]27 nodes, while its N-terminal region localizes close to a hook-like structure that attaches to the A-microtubule as it extends from the centriole interior to exterior. This hook structure is adjacent to the DISCO complex (MNR/CEP90/OFD1), which marks future appendage sites. C2CD3 depletion disrupts not only the recruitment of the DISCO complex via direct interaction with MNR but also destabilizes the luminal ring network composed of C2CD3/SFI1/centrin-2/CEP135/NA14, as well as the distal microtubule tip protein CEP162. This reveals an intricate "in-to-out" molecular hub connecting the centriolar lumen, distal microtubule cap, and appendages. Although C2CD3 loss results in shorter centrioles and appendage defects, key structural elements remain intact, permitting continued centriole duplication. We propose that C2CD3 forms the luminal ring structure and extends radially to the space between triplet microtubules, functioning as an architectural hub that scaffolds the distal end of the centriole, orchestrating its assembly and directing appendage formation.

cell biology↗

bioRxiv2024

Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii

In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures of biomolecules in their native context. However, challenges remain in the efficient production of large-scale cryo-ET datasets, as well as the community sharing of this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling of the green alga Chlamydomonas reinhardtii, a useful model organism for in situ visualization of numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances in cryo-ET data acquisition and processing, we generated a dataset of 1829 reconstructed and annotated tomograms, which we provide as a community resource to drive method development and inspire biological discovery. To assay the quality of this dataset, we performed subtomogram averaging (STA) of both soluble and membrane-bound complexes ranging in size from >3 MDa to [~]200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset, as well as the promise of modern cryo-ET workflows and open data sharing towards visual proteomics.

cell biology↗