Centromeric chromatin clearings demarcate the site of kinetochore formation.

The centromere is the chromosomal locus that recruits the kinetochore, directing faithful propagation of the genome during cell division. The kinetochore has been interrogated by electron microscopy since the middle of the last century, but with methodologies that compromised fine structure. Using cryo-ET on human mitotic chromosomes, we reveal a distinctive architecture at the centromere: clustered 20-25 nm nucleosome-associated complexes within chromatin clearings that delineate them from surrounding chromatin. Centromere components CENP-C and CENP-N are each required for the integrity of the complexes, while CENP-C is also required to maintain the chromatin clearing. We further visualize the scaffold of the fibrous corona, a structure amplified at unattached kinetochores, revealing crescent-shaped parallel arrays of fibrils that extend >1 µm. Thus, we reveal how the organization of centromeric chromatin creates a clearing at the site of kinetochore formation as well as the nature of kinetochore amplification mediated by corona fibrils.

Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling.

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) allows higher resolution imaging of unfixed cellular samples while preserving architecture, but it requires samples to be vitreous and thin enough for transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue via plunge-freezing and use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid at variable depth inside the tissue. Lamellae generated in Alzheimer's disease brain tissue reveal intact subcellular structures including components of autophagy and potential pathologic tau fibrils. Furthermore, we reveal intact compact myelin and functional cytoplasmic expansions. These images indicate that plasma FIB milling with cryo-ET may be used to elucidate nanoscale structures within the human brain.

Sustained rhoptry docking and discharge requires Toxoplasma gondii intraconoidal microtubule-associated proteins.

In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10-12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity.

Combining per-particle cryo-ET and cryo-EM single particle analysis to elucidate heterogeneous DNA-protein organization.

Cryo-electron microscopy single particle analysis (cryo-EM SPA) and cryo-electron tomography (cryo-ET) have historically been employed as distinct approaches for investigating molecular structures of disparate sample types, focusing on highly purified biological macromolecules and in situ cellular contexts, respectively. However, these techniques offer inherently complementary structural insights that, when combined, provide a more comprehensive understanding of complex biological systems. For example, if both techniques are applied to the same purified biological macromolecules, cryo-ET has the ability to resolve highly flexible yet strong signal features on an individual target molecule which will not be preserved in the high-resolution cryo-EM SPA results. In this review, we highlight recent achievements utilizing such applications to unveil new insights into the chromatin assembly and activities of DNA-protein assemblies. This convergence of cryo-EM SPA and cryo-ET holds great promise for elucidating new structural aspects of these essential molecular processes.

Native ultrastructure of fresh human brain vitrified directly from autopsy revealed by cryo-electron tomography with cryo-plasma focused ion beam milling.

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following chemical fixation, staining, and mechanical sectioning, which limit attainable resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) offers the potential to image unfixed cellular samples at higher resolution while preserving their native structures, but it requires samples to be frozen free from crystalline ice and thin enough to image via transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate the native ultrastructure of unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue directly on cryo-EM grids via plunge-freezing, as opposed to high pressure freezing which is generally used for thick samples. Following vitrification, we use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid. In comparison to gallium FIB, which is commonly used for biological samples, xenon plasma FIB is powerful enough to efficiently mill large volume samples, such as human brain tissue. Additionally, our approach allows for lamellae to be generated at variable depth inside the tissue as opposed to being limited to starting at the surface of the tissue. Lamellae generated in Alzheimer's disease brain tissue and imaged by cryo-ET reveal intact subcellular structures including components of autophagy and potential tau fibrils. Furthermore, we visualize myelin revealing intact compact myelin and functional cytoplasmic expansions such as cytoplasmic channels and the inner tongue. From these images we also measure the dimensions of myelin membranes, providing insight into how myelin basic protein forces out oligodendrocyte cytoplasm to form compact myelin and tightly links intracellular polar head groups of the oligodendrocyte plasma membrane. This approach provides a first view of unfixed, never previously frozen human brain tissue prepared by cryo-plasma FIB milling and imaged at high resolution by cryo-ET.

A hydrophilic polyimidazolium antibiotic targeting the membranes of Gram-negative bacteria.

OBJECTIVES: The rise of MDR Gram-negative bacteria (GNB), especially those resistant to last-resort drugs such as carbapenems and colistin, is a global health risk and calls for increased efforts to discover new antimicrobial compounds. We previously reported that polyimidazolium (PIM) compounds exhibited significant antimicrobial activity and minimal mammalian cytotoxicity. However, their mechanism of action is relatively unknown. We examined the efficacy and mechanism of action of a hydrophilic PIM (PIM5) against colistin- and meropenem-resistant clinical isolates. METHODS: MIC and time-kill testing was performed for drug-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. N-phenyl-1-naphthylamine and propidium iodide dyes were employed to determine membrane permeabilization. Spontaneous resistant mutants and single deletion mutants were generated to understand potential resistance mechanisms to the drug. RESULTS: PIM5 had the same effectiveness against colistin- and meropenem-resistant strains as susceptible strains of GNB. PIM5 exhibited a rapid bactericidal effect independent of bacterial growth phase and was especially effective in water. The polymer disrupts both the outer and cytoplasmic membranes. PIM5 binds and intercalates into bacterial genomic DNA upon entry of cells. GNB do not develop high resistance to PIM5. However, the susceptibility and uptake of the polymer is moderately affected by mutations in the two-component histidine kinase sensor BaeS. PIM5 has negligible cytotoxicity on human cells at bacterial-killing concentrations, comparable to the commercial antibiotics polymyxin B and colistin. CONCLUSIONS: PIM5 is a potent broad-spectrum antibiotic targeting GNB resistant to last-resort antibiotics.

Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography.

The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

Nanoscale details of mitochondrial constriction revealed by cryoelectron tomography.

Mitochondria adapt to changing cellular environments, stress stimuli, and metabolic demands through dramatic morphological remodeling of their shape, and thus function. Such mitochondrial dynamics is often dependent on cytoskeletal filament interactions. However, the precise organization of these filamentous assemblies remains speculative. Here, we apply cryogenic electron tomography to directly image the nanoscale architecture of the cytoskeletal-membrane interactions involved in mitochondrial dynamics in response to damage. We induced mitochondrial damage via membrane depolarization, a cellular stress associated with mitochondrial fragmentation and mitophagy. We find that, in response to acute membrane depolarization, mammalian mitochondria predominantly organize into tubular morphology that abundantly displays constrictions. We observe long bundles of both unbranched actin and septin filaments enriched at these constrictions. We also observed septin-microtubule interactions at these sites and elsewhere, suggesting that these two filaments guide each other in the cytosolic space. Together, our results provide empirical parameters for the architecture of mitochondrial constriction factors to validate/refine existing models and inform the development of new ones.

Origin and arrangement of actin filaments for gliding motility in apicomplexan parasites revealed by cryo-electron tomography.

The phylum Apicomplexa comprises important eukaryotic parasites that invade host tissues and cells using a unique mechanism of gliding motility. Gliding is powered by actomyosin motors that translocate host-attached surface adhesins along the parasite cell body. Actin filaments (F-actin) generated by Formin1 play a central role in this critical parasitic activity. However, their subcellular origin, path and ultrastructural arrangement are poorly understood. Here we used cryo-electron tomography to image motile Cryptosporidium parvum sporozoites and reveal the cellular architecture of F-actin at nanometer-scale resolution. We demonstrate that F-actin nucleates at the apically positioned preconoidal rings and is channeled into the pellicular space between the parasite plasma membrane and the inner membrane complex in a conoid extrusion-dependent manner. Within the pellicular space, filaments on the inner membrane complex surface appear to guide the apico-basal flux of F-actin. F-actin concordantly accumulates at the basal end of the parasite. Finally, analyzing a Formin1-depleted Toxoplasma gondii mutant pinpoints the upper preconoidal ring as the conserved nucleation hub for F-actin in Cryptosporidium and Toxoplasma. Together, we provide an ultrastructural model for the life cycle of F-actin for apicomplexan gliding motility.

Inflammasomes primarily restrict cytosolic Salmonella replication within human macrophages.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that utilizes its type III secretion systems (T3SSs) to inject virulence factors into the host cell and colonize the host. In turn, a subset of cytosolic immune receptors respond to T3SS ligands by forming multimeric signaling complexes called inflammasomes, which activate caspases that induce interleukin-1 (IL-1) family cytokine release and an inflammatory form of cell death called pyroptosis. Human macrophages mount a multifaceted inflammasome response to Salmonella infection that ultimately restricts intracellular bacterial replication. However, how inflammasomes restrict Salmonella replication remains unknown. We find that caspase-1 is essential for mediating inflammasome responses to Salmonella and subsequent restriction of bacterial replication within human macrophages, with caspase-4 contributing as well. We also demonstrate that the downstream pore-forming protein gasdermin D (GSDMD) and ninjurin-1 (NINJ1), a mediator of terminal cell lysis, play a role in controlling Salmonella replication in human macrophages. Notably, in the absence of inflammasome responses, we observed hyperreplication of Salmonella within the cytosol of infected cells, and we also observed increased bacterial replication within vacuoles, suggesting that inflammasomes control Salmonella replication primarily within the cytosol and also within vacuoles. These findings reveal that inflammatory caspases and pyroptotic factors mediate inflammasome responses that restrict the subcellular localization of intracellular Salmonella replication within human macrophages.

Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.

Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometre-scale resolution. With high-resolution images of predation in a native (hydrated, unstained) state, we discover several surprising features of the process, including macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following growth and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid.

High-throughput cryo-ET structural pattern mining by unsupervised deep iterative subtomogram clustering.

Cryoelectron tomography directly visualizes heterogeneous macromolecular structures in their native and complex cellular environments. However, existing computer-assisted structure sorting approaches are low throughput or inherently limited due to their dependency on available templates and manual labels. Here, we introduce a high-throughput template-and-label-free deep learning approach, Deep Iterative Subtomogram Clustering Approach (DISCA), that automatically detects subsets of homogeneous structures by learning and modeling 3D structural features and their distributions. Evaluation on five experimental cryo-ET datasets shows that an unsupervised deep learning based method can detect diverse structures with a wide range of molecular sizes. This unsupervised detection paves the way for systematic unbiased recognition of macromolecular complexes in situ.

Computational Methods Toward Unbiased Pattern Mining and Structure Determination in Cryo-Electron Tomography Data.

Cryo-electron tomography can uniquely probe the native cellular environment for macromolecular structures. Tomograms feature complex data with densities of diverse, densely crowded macromolecular complexes, low signal-to-noise, and artifacts such as the missing wedge effect. Post-processing of this data generally involves isolating regions or particles of interest from tomograms, organizing them into related groups, and rendering final structures through subtomogram averaging. Template-matching and reference-based structure determination are popular analysis methods but are vulnerable to biases and can often require significant user input. Most importantly, these approaches cannot identify novel complexes that reside within the imaged cellular environment. To reliably extract and resolve structures of interest, efficient and unbiased approaches are therefore of great value. This review highlights notable computational software and discusses how they contribute to making automated structural pattern discovery a possibility. Perspectives emphasizing the importance of features for user-friendliness and accessibility are also presented.

Structural basis of a transcription pre-initiation complex on a divergent promoter.

Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.

In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography.

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.

An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective ß-Barrel Porin Requirements for Polymer Secretion.

Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In the social predatory bacterium Myxococcus xanthus, the exopolysaccharide (EPS) pathway WzaX, major spore coat (MASC) pathway WzaS, and biosurfactant polysaccharide (BPS) pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning α-helices. Comparative genomics across all bacteria (>91,000 OPX proteins identified and analyzed), complemented with cryo-electron tomography cell-envelope analyses, revealed such "truncated" WzaX/S/B architecture to be the most common among three defined OPX-protein structural classes independent of periplasm thickness. Fold recognition and deep learning revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded beside wzaX/S/B, respectively) to be integral OM ß-barrels, with structural homology to the poly-N-acetyl-d-glucosamine synthase-dependent pathway porin PgaA. Such bacterial porins were identified near numerous genes for all three OPX protein classes. Interior MXAN_7418/3226/1916 ß-barrel electrostatics were found to match properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 herein shown to mediate EPS translocation, we have designated this new secretion machinery component "Wzp" (i.e., Wz porin), with the final step of M. xanthus EPS/MASC/BPS secretion across the OM now proposed to be mediated by WzpX/S/B (i.e., MXAN_7418/3226/1916). Importantly, these data support a novel and widespread secretion paradigm for polysaccharide biosynthesis pathways in which those containing OPX components that cannot span the OM instead utilize ß-barrel porins to mediate polysaccharide transport across the OM. IMPORTANCE Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. In the prototypic Escherichia coli Group-1-capsule biosynthesis system, eight copies of this canonical OPX protein cross the OM with an α-helix, forming a polysaccharide-export pore. Herein, we instead reveal that most OPX proteins across all bacteria lack this α-helix, raising questions as to the manner by which most secreted polysaccharides actually exit cells. In the model developmental bacterium Myxococcus xanthus, we show this process to depend on OPX-coupled OM-spanning ß-barrel porins, with similar porins encoded near numerous OPX genes in diverse bacteria. Knowledge of the terminal polysaccharide secretion step will enable development of antimicrobial compounds targeted to blocking polymer export from outside the cell, thus bypassing any requirements for antimicrobial compound uptake by the cell.

Rhoptry secretion system structure and priming in Plasmodium falciparum revealed using in situ cryo-electron tomography.

Apicomplexan parasites secrete contents of the rhoptries, club-shaped organelles in the apical region, into host cells to permit their invasion and establishment of infection. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is unknown whether a similar molecular machinery exists in the malaria parasite Plasmodium. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct apical vesicle docking the tips of the two rhoptries to the RSA. We also discover two additional rhoptry organizations that lack the apical vesicle. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to different rhoptry organizations. Our results highlight previously unknown steps in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

Archaic chaperone-usher pili self-secrete into superelastic zigzag springs.

Adhesive pili assembled through the chaperone-usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria1-3. Archaic chaperone-usher pathway pili, the most diverse and widespread chaperone-usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii-a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections.