Biology subjects

Caspy, I.

Publications and source records attributed to Caspy, I..

7 recordsLinked to original sources

bioRxiv2026

Molecular architecture of colossal surface layers from hyperthermophilic archaea

Surface layers (S-layers) are paracrystalline protein lattices that form the outermost layer of the cell envelope in most archaea, providing structural support, protecting against external insults, and co-ordinating interactions with their environment. Despite their widespread occurrence, the molecular and structural details of S-layer architecture in hyperthermophilic archaea remain largely unknown. Here, we report the structure and cellular architecture of the S-layer from the hyperthermophilic archaeon Pyrobaculum arsenaticum by combining in situ electron cryotomography with single-particle electron cryomicroscopy, AlphaFold modelling, and peptide-fingerprinting mass spectrometry. We show that the S-layer is formed by an uncharacterised 292-kDa S-layer protein (SLP) extending 37 nm from the cytoplasmic membrane, making it, to our knowledge, the largest SLP structurally characterised to date. This SLP has a remarkable multidomain architecture comprising 19 immunoglobulin-like domains, 14 canonical and five non-canonical, organised into a lattice-forming core, a stalk, and a unique crown domain that stabilise the S-layer. Comparative genomic analyses unearthed homologous colossal SLP candidates across Thermoproteota, indicating that this distinctive architecture is conserved across diverse archaeal lineages. Together, our findings provide a structural framework for understanding the cell-surface organisation in hyperthermophilic archaea and suggest that these colossal S-layers represent a specialised adaptation to life at high temperatures.

microbiology↗

bioRxiv2025

The mechanics of a continuous self-assembling surface-layer aids cell division in an archaeon

The surface layer or "S-layer" is a planar lattice of glycosylated proteins that coats a wide range of archaea and bacteria instead of a classical cell wall or capsular polysaccharides, insulating them from the extracellular space and providing the cell membrane with physical support. Although the S-layers role as a mechanical support for the membrane might be expected to hinder cell division, we show that in rapidly dividing Sulfolobus acidocaldarius cells, the S-layer protein SlaA self-assembles into flexible lattice that helps flattens the cytokinetic furrow to accelerate ESCRT-III dependent cell division - a role that is especially important under conditions of mechanical stress. Taken together, these results generated using mutational analysis, live and fixed cellular imaging, along with electron cryomicroscopy, define the rules governing S-layer self-assembly and show how the mechanical properties of flexible lattice coats can enhance membrane functions to both physically support a cell and help to drive ESCRT-III dependent cell division. Significance statementThe bounding membrane of a cell must be protected from environmental insults. In many bacteria and archaea that lack a cell wall, this is achieved by an enveloping S-layer. By virtue of its rigid, planar structure, the S-layer is also expected to act as a barrier to the cell shape changes required for cytokinesis. In this study of the S-layer lattice in the archaeon Sulfolobus acidocaldarius however, we demonstrate a role for this self-assembling mechanical support in re-shaping the cytokinetic furrow as an aid to ESCRT-III-mediated division. In showing how mechanically active and passive structures can work together to give rise to mechanically stable cells that can divide, this reconciles what seems like a trade-off between resilience and flexibility.

molecular biology↗

bioRxiv2024

Tissue-Like Multicellular Development Triggered by Mechanical Compression in Archaea

The advent of clonal multicellularity is a critical evolutionary milestone, seen often in eukaryotes, rarely in bacteria, and only once observed in archaea. We show that uniaxial compression induces clonal multicellularity in haloarchaea, forming tissue-like structures. These archaeal tissues are mechanically and molecularly distinct from their unicellular lifestyle, mimicking several eukaryotic features. Archaeal tissues undergo a multinucleate stage followed by tubulin-independent cellularization, orchestrated by active membrane tension at a critical cell size. After cellularization, tissue junction elasticity become akin to animal tissues and give rise to two cell types--radial peribasal and central apicobasal cells-- with distinct actin and protein glycosylation polarity patterns. Our findings highlight the potential convergent evolution of a biophysical mechanism in the emergence of multicellular systems across domains of life.

cell biology↗

bioRxiv2024

Structure of the Pseudomonas aeruginosa PAO1 Type IV pilus

Type IV pili (T4Ps), which are abundant in many bacterial and archaeal species, have been shown to play important roles in both surface sensing and twitching motility, with implications for adhesion, biofilm formation and pathogenicity. While Type IV pilus (T4P) structures from other organisms have been previously solved, a high-resolution structure of the native, fully assembled T4P of Pseudomonas aeruginosa, one of the major human pathogens, is not available. Here, we report a 3.2 [A]-resolution structure of the P. aeruginosa PAO1 T4P determined by electron cryomicroscopy (cryo-EM). PilA subunits constituting the T4P exhibit a classical pilin fold featuring an extended N-terminal -helix linked to a C-terminal globular {beta}-sheet-containing domain, which are packed tightly along the pilus. The N-terminal helices constitute the pilus core where they stabilise the tubular assembly via hydrophobic interactions. The -helical core of the pilus is surrounded by the C-terminal globular domain of PilA that coats the outer surface of the pilus, mediating interactions with the surrounding environment. Comparison of the P. aeruginosa T4P with T4P structures from other organisms, both at the level of the pilin subunits and the fully assembled pili, allows us to enumerate key differences, and detect common architectural principles in this abundant class of prokaryotic filaments. This study provides a structural framework for understanding the molecular and cell biology of these important cellular appendages mediating interaction of prokaryotes to surfaces.

microbiology↗

bioRxiv2023

CryoEM PSII structure reveals adaptation mechanisms to environmental stress in Chlorella ohadii

Performing photosynthesis in the desert is a challenging task since it requires a fast adaptation to extreme illumination and temperature changes. To understand adaptive mechanisms, we purified Photosystem II (PSII) from Chlorella ohadii, a green alga from the desert soil surface, and identified structural elements that might enable the photosystem functioning under harsh conditions. The 2.72 [A] cryogenic electron-microscopy (cryoEM) structure of PSII exhibited 64 subunits, encompassing 386 chlorophylls, 86 carotenoids, four plastoquinones, and several structural lipids. At the luminal side of PSII, the oxygen evolving complex was protected by a unique subunit arrangement - PsbO (OEE1), PsbP (OEE2), CP47, and PsbU (plant OEE3 homolog). PsbU interacted with PsbO, CP43, and PsbP, thus stabilising the oxygen evolving shield. Substantial changes were observed on the stromal electron acceptor side - PsbY was identified as a transmembrane helix situated alongside PsbF and PsbE enclosing cytochrome b559, supported by the adjacent C-terminal helix of Psb10. These four transmembrane helices bundled jointly, shielding cytochrome b559 from the solvent. The bulk of Psb10 formed a cap protecting the quinone site and probably contributed to the PSII stacking. So far, the C. ohadii PSII structure is the most complete description of the complex, suggesting numerous future experiments. A protective mechanism that prevented QB from rendering itself fully reduced is proposed.

plant biology↗

bioRxiv2021

Structure of Dunaliella Photosystem II reveals conformational flexibility of stacked and unstacked supercomplexes

Photosystem II (PSII) generates an oxidant whose redox potential is high enough to enable water oxidation1,2, a substrate so abundant that it assures a practically unlimited electron source for life on earth3. Our knowledge on the mechanism of water photooxidation was greatly advanced by high-resolution structures of prokaryotic PSII4-6. Here we show high-resolution structures of eukaryotic PSII from the green algae Dunaliella salina at two distinct conformations. The conformers are also present in stacked PSII, exhibiting flexibility that is relevant to the grana formation in chloroplasts of the green lineage. CP29, one of PSII associated light harvesting antennae, plays a major role in distinguishing the two conformations of the supercomplex. We also show that the stacked PSII dimer, a form suggested to support the organization of thylakoid membranes7,8, can appear in many different orientations providing a flexible stacking mechanism for the arrangement of grana stacks in thylakoids. Our findings provide a structural basis for the heterogenous nature of the eukaryotic PSII on multiple levels.

plant biology↗

bioRxiv2021

Structure of plant PSI-plastocyanin complex reveals strong hydrophobic interactions.

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 [A] resolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB / Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared to reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover. One Sentence SummaryGenetic engineering, kinetics and cryo-EM structural data reveal a mechanism in a major step of oxygenic photosynthesis

biochemistry↗