Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA.

Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.

Supramolecular assembly of pyrene-DNA conjugates: influence of pyrene substitution pattern and implications for artificial LHCs.

The supramolecular self-assembly of pyrene-DNA conjugates into nanostructures is presented. DNA functionalized with different types of pyrene isomers at the 3'-end self-assemble into nano-objects. The shape of the nanostructures is influenced by the type of pyrene isomer appended to the DNA. Multilamellar vesicles are observed with the 1,6- and 1,8-isomers, whereas conjugates of the 2,7-isomer exclusively assemble into spherical nanoparticles. Self-assembled nano-spheres obtained with the 2,7-dialkynyl pyrene isomer were used for the construction of an artificial light-harvesting complex (LHC) in combination with Cy3 as the energy acceptor.

Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.

Early molecular layer interneuron hyperactivity triggers Purkinje neuron degeneration in SCA1.

Toxic proteinaceous deposits and alterations in excitability and activity levels characterize vulnerable neuronal populations in neurodegenerative diseases. Using in vivo two-photon imaging in behaving spinocerebellar ataxia type 1 (Sca1) mice, wherein Purkinje neurons (PNs) degenerate, we identify an inhibitory circuit element (molecular layer interneurons [MLINs]) that becomes prematurely hyperexcitable, compromising sensorimotor signals in the cerebellum at early stages. Mutant MLINs express abnormally elevated parvalbumin, harbor high excitatory-to-inhibitory synaptic density, and display more numerous synaptic connections on PNs, indicating an excitation/inhibition imbalance. Chemogenetic inhibition of hyperexcitable MLINs normalizes parvalbumin expression and restores calcium signaling in Sca1 PNs. Chronic inhibition of mutant MLINs delayed PN degeneration, reduced pathology, and ameliorated motor deficits in Sca1 mice. Conserved proteomic signature of Sca1 MLINs, shared with human SCA1 interneurons, involved the higher expression of FRRS1L, implicated in AMPA receptor trafficking. We thus propose that circuit-level deficits upstream of PNs are one of the main disease triggers in SCA1.

Morphofunctional changes at the active zone during synaptic vesicle exocytosis.

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria.

The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores; Actinobacteria, forming exospores; Cyanobacteria, forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.

Complex DNA Architectonics─Self-Assembly of Amphiphilic Oligonucleotides into Ribbons, Vesicles, and Asterosomes.

The precise arrangement of structural subunits is a key factor for the proper shape and function of natural and artificial supramolecular assemblies. In DNA nanotechnology, the geometrically well-defined double-stranded DNA scaffold serves as an element of spatial control for the precise arrangement of functional groups. Here, we describe the supramolecular assembly of chemically modified DNA hybrids into diverse types of architectures. An amphiphilic DNA duplex serves as the sole structural building element of the nanosized supramolecular structures. The morphology of the assemblies is governed by a single subunit of the building block. The chemical nature of this subunit, i.e., polyethylene glycols of different chain length or a carbohydrate moiety, exerts a dramatic influence on the architecture of the assemblies. Cryo-electron microscopy revealed the arrangement of the individual DNA duplexes within the different constructs. Thus, the morphology changes from vesicles to ribbons with increasing length of a linear polyethylene glycol. Astoundingly, attachment of a N-acetylgalactosamine carbohydrate to the DNA duplex moiety produces an unprecedented type of star-shaped architecture. The novel DNA architectures presented herein imply an extension of the current concept of DNA materials and shed new light on the fast-growing field of DNA nanotechnology.

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes.

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.

Cryo-EM structure of the octameric pore of Clostridium perfringens ß-toxin.

Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens ß-toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo-electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo-octameric pores like the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded ß-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin-like family. In addition, we show that the ß-barrel protrusion domain can be modified without affecting the pore-forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD.

ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset.

Neurons as a model system for cryo-electron tomography.

Cryo-electron tomography (Cryo-ET) provides unique opportunities to image cellular components at high resolution in their native state and environment. While many different cell types were investigated by cryo-ET, here we review application to neurons. We show that neurons are a versatile system that can be used to investigate general cellular components such as the cytoskeleton and membrane-bound organelles, in addition to neuron-specific processes such as synaptic transmission. Furthermore, the synapse provides a rich environment for the development of cryo-ET image processing tools suitable to elucidate the functional and spatial organization of compositionally and morphologically heterogeneous macromolecular complexes involved in biochemical signaling cascades, within their native, crowded cellular environments.

Wt1 transcription factor impairs cardiomyocyte specification and drives a phenotypic switch from myocardium to epicardium.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.

Tetraphenylethylene-DNA conjugates: influence of sticky ends and DNA sequence length on the supramolecular assembly of AIE-active vesicles.

The supramolecular assembly of DNA conjugates, functionalized with tetraphenylethylene (TPE) sticky ends, into vesicular structures is described. The aggregation-induced emission (AIE) active TPE units allow monitoring the assembly process by fluorescence spectroscopy. The number of TPE modifications in the overhangs of the conjugates influences the supramolecular assembly behavior. A minimum of two TPE residues on each end are required to ensure a well-defined assembly process. The design of the presented DNA-based nanostructures offers tailored functionalization with applications in DNA nanotechnology.

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm.

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

A small ribosome-associated ncRNA globally inhibits translation by restricting ribosome dynamics.

Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in Saccharomyces cerevisiae that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision.

Hijacking of the host cell Golgi by Plasmodium berghei liver stage parasites.

The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we here show an interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasitophorous vacuolar membrane of the parasite. Expression of specific dominant-negative Arf1 and Rab GTPases, which interfere with the host cell Golgi-linked vesicular machinery, results in developmental delay and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the ability of the parasites to induce Golgi fragmentation. Altogether, we demonstrate that the structural integrity of the host cell Golgi and Golgi-associated vesicular traffic is important for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the Golgi structure of the hepatocyte to optimize its own intracellular development. This article has an associated First Person interview with the first author of the paper.

Loss of Claudin-3 Impairs Hepatic Metabolism, Biliary Barrier Function, and Cell Proliferation in the Murine Liver.

BACKGROUND & AIMS: Tight junctions in the liver are essential to maintain the blood-biliary barrier, however, the functional contribution of individual tight junction proteins to barrier and metabolic homeostasis remains largely unexplored. Here, we describe the cell type-specific expression of tight junction genes in the murine liver, and explore the regulation and functional importance of the transmembrane protein claudin-3 in liver metabolism, barrier function, and cell proliferation. METHODS: The cell type-specific expression of hepatic tight junction genes is described using our mouse liver single-cell sequencing data set. Differential gene expression in Cldn3-/- and Cldn3+/+ livers was assessed in young and aged mice by RNA sequencing (RNA-seq), and hepatic tissue was analyzed for lipid content and bile acid composition. A surgical model of partial hepatectomy was used to induce liver cell proliferation. RESULTS: Claudin-3 is a highly expressed tight junction protein found in the liver and is expressed predominantly in hepatocytes and cholangiocytes. The histology of Cldn3-/- livers showed no overt phenotype, and the canalicular tight junctions appeared intact. Nevertheless, by RNA-seq we detected a down-regulation of metabolic pathways in the livers of Cldn3-/- young and aged mice, as well as a decrease in lipid content and a weakened biliary barrier for primary bile acids, such as taurocholic acid, taurochenodeoxycholic acid, and taurine-conjugated muricholic acid. Coinciding with defects in the biliary barrier and lower lipid metabolism, there was a diminished hepatocyte proliferative response in Cldn3-/- mice after partial hepatectomy. CONCLUSIONS: Our data show that, in the liver, claudin-3 is necessary to maintain metabolic homeostasis, retention of bile acids, and optimal hepatocyte proliferation during liver regeneration. The RNA-seq data set can be accessed at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159914.

Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood-brain barrier.

The migration of activated T cells across the blood-brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper.

Multi-scale alignment of respiratory cilia and its relation to mucociliary function.

The tracheobronchial tree is lined by a mucociliary epithelium containing millions of multiciliated cells. Their integrated oscillatory activity continuously propels an overlying pollution-protecting mucus layer in cranial direction, leading to mucociliary clearance - the primary defence mechanism of the airways. Mucociliary transport is commonly thought to co-emerge with the collective ciliary motion pattern under appropriate geometrical and rheological conditions. Proper ciliary alignment is therefore considered essential to establish mucociliary clearance in the respiratory system. Here, we used volume electron microscopy in combination with high-speed reflection contrast microscopy in order to examine ciliary orientation and its spatial organization, as well as to measure the propagation direction of metachronal waves and the direction of mucociliary transport on bovine tracheal epithelia with reference to the tracheal long axis (TLA). Ciliary orientation is measured in terms of the basal body orientation (BBO) and the axonemal orientation (AO), which are commonly considered to coincide, both equivalently indicating the effective stroke as well as the mucociliary transport direction. Our results, however, reveal that only the AO is in line with the mucociliary transport, which was found to run along a left-handed helical trajectory, whereas the BBO was found to be aligned with the TLA. Furthermore, we show that even if ciliary orientation remains consistent between adjacent cells, ciliary orientation exhibits a gradual shift within individual cells. Together with the symplectic beating geometry, this intracellular orientational pattern could provide for the propulsion of highly viscous mucus and likely constitutes a compromise between efficiency and robustness.