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Bipolar elongation of filaments of the bacterial actin homolog ParM drives movement of newly replicated plasmid DNA to opposite poles of a bacterial cell. We used a combination of vitreous sectioning and electron cryotomography to study this DNA partitioning system directly in native, frozen cells. The diffraction patterns from overexpressed ParM bundles in electron cryotomographic reconstructions were used to unambiguously identify ParM filaments in Escherichia coli cells. Using a low-copy number plasmid encoding components required for partitioning, we observed small bundles of three to five intracellular ParM filaments that were situated close to the edge of the nucleoid. We propose that this may indicate the capture of plasmid DNA within the periphery of this loosely defined, chromosome-containing region.

The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor–rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters.

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila , is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry. Aerolysin is a secreted bacterial pore forming toxin that inserts into the host plasma membrane, potentially leading to cell death. Here the authors present Cryo-EM structures of aerolysin arrested at different stages of the pore formation process that provide insight into the conformational changes that allow pore formation.

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane β-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal β-signals. Thus in trypanosomes the highly conserved β-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.

Hemolysin β-pore-forming toxins (βPFTs) are key virulence factors of Clostridium perfringens , associated with severe diseases in humans and animals. Yet, the mechanisms by which Clostridium βPFTs recognize and engage specific target cells remain poorly understood. Here, we identify the cellular receptor for C. perfringens necrotizing enteritis toxin F (NetF), a recently discovered toxin implicated in severe enteritis in dogs and foals. We show that NetF binds to the same receptor as anthrax toxin, namely ANTXR2. Using cryo-electron microscopy, we determined the structure of the oligomeric NetF pre-pore as well as the transmembrane pore, both alone and in complex with the extracellular domain of ANTXR2. Unlike anthrax toxin, which binds to the apical MIDAS motif of ANTXR2 – as does the natural ANTXR2 ligand collagen type VI – NetF engages the receptor laterally, spanning both the von Willebrand A and the Ig-like domains. This interaction positions the toxin near the membrane, facilitating contact with membrane lipids and promoting transmembrane pore formation. Our findings uncover key principles of hemolysin βPFT-receptor recognition and advance our understanding of how pathogenic bacteria use these toxins to breach host defenses. This study identifies ANTXR2 as the cellular receptor for Clostridium perfringens toxin NetF and determines its structure bound to the toxin using cryo-EM, revealing a distinct lateral binding mechanism that facilitates membrane pore formation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.

Cryo-electron microscopy can be used to image cells and tissue at high resolution. To ensure electron transparency, the sample thickness must not exceed 500 nm. Focused-ion-beam (FIB) milling has become the standard method for preparing thin samples (lamellae); however, the material removed by the milling process is lost, the imageable area is usually limited to a few square micrometres and the surface layers sustain damage from the ion beam. We have examined c ryo- e lectron m icroscopy o f vi treous s ections (CEMOVIS), a technique based on cutting thin sections with a knife, as an alternative to FIB milling. Vitreous sections also sustain damage, including compression, shearing and cracks. However, samples can be sectioned in series, producing many orders of magnitude more imageable area compared to lamellae, making CEMOVIS an alternative to FIB milling with distinct advantages. Using two-dimensional template matching on images of vitreous sections of Saccharomyces cerevisiae cells, we reconstructed the 60S ribosomal subunit at near-atomic resolution, demonstrating that, in many regions of the sections, the molecular structure of these subunits is largely intact, comparable to FIB-milled lamellae.

Intussusceptive angiogenesis (IA) is a complementary method to sprouting angiogenesis (SA). The hallmark of IA is formation of trans-capillary tissue pillars, their fusion and remodeling of the vascular plexus. In this study, we investigate the formation of the zebrafish caudal vein plexus (CVP) in Tg(fli1a:eGFP) y7 and the synergistic interaction of IA and SA in crafting the archetypical angio-architecture of the CVP. Dynamic in vivo observations and quantitative analyses revealed that the primitive CVP during development was initiated through SA. Further vascular growth and remodeling occurred by IA. Intussusception contributed to the expansion of the CVP by formation of new pillars. Those pillars arose in front of the already existing ones; and in a subsequent step the serried pillars elongated and fused together. This resulted in segregation of larger vascular segments and remodelling of the disorganized vascular meshwork into hierarchical tree-like arrangement. Blood flow was the main driving force for IA, particularly shear stress geometry at the site of pillar formation and fusion. Computational simulations based on hemodynamics showed drop in shear stress levels at locations of new pillar formation, pillar elongation and fusion. Correlative 3D serial block face scanning electron microscopy confirmed the morphological substrate of the phenomena of the pillar formation observed in vivo . The data obtained demonstrates that after the sprouting phase and formation of the primitive capillary meshwork, the hemodynamic conditions enhance intussusceptive segregation of hierarchical vascular tree i.e. intussusceptive arborization resulting in complex vascular structures with specific angio-architecture.