Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1

High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding.

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called "MultiCLEM," allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.This work was financially supported by grants from the Deutsche Forschungsgemeinschaft (SFB1129 Z2 to J.A.G. Briggs), EMBL (to J.A.G. Briggs), the Medical Research Council (MC_UP_1201/16 to J.A.G. Briggs), and the German Ministry of Education and Research (031A605 to K.R. Patil). The Schuldiner laboratory is supported by the European Research Council CoG 646604 Peroxisystem, the Deutsche Forschungsgemeinschaft (grant SFB1190 and a Deutsch-Israelische Projektkooperation [DIP] collaborative grant). N. Gabrielli was supported by the EMBL interdisciplinary postdoctoral program. M. Schuldiner is an incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics

Phenological shifts of abiotic events, producers and consumers across a continent

Ongoing climate change can shift organism phenology in ways that vary depending on species, habitats and climate factors studied. To probe for large-scale patterns in associated phenological change, we use 70,709 observations from six decades of systematic monitoring across the former Union of Soviet Socialist Republics. Among 110 phenological events related to plants, birds, insects, amphibians and fungi, we find a mosaic of change, defying simple predictions of earlier springs, later autumns and stronger changes at higher latitudes and elevations. Site mean temperature emerged as a strong predictor of local phenology, but the magnitude and direction of change varied with trophic level and the relative timing of an event. Beyond temperature-associated variation, we uncover high variation among both sites and years, with some sites being characterized by disproportionately long seasons and others by short ones. Our findings emphasize concerns regarding ecosystem integrity and highlight the difficulty of predicting climate change outcomes. The authors use systematic monitoring across the former USSR to investigate phenological changes across taxa. The long-term mean temperature of a site emerged as a strong predictor of phenological change, with further imprints of trophic level, event timing, site, year and biotic interactions.Peer reviewe

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.Wellcome Trus

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.Wellcome Trus

The ER protein Ema19 facilitates the degradation of non-imported mitochondrial precursor proteins

For the biogenesis of mitochondria, hundreds of proteins need to be targeted from the cytosol into the various compartments of this organelle. The intramitochondrial targeting routes these proteins take to reach their respective location in the organelle are well understood. However, the early targeting processes, from cytosolic ribosomes to the membrane of the organelle, are still largely unknown. In this study, we present evidence that an integral membrane protein of the endoplasmic reticulum (ER), Ema19, plays a role in this process. Mutants lacking Ema19 show an increased stability of mitochondrial precursor proteins, indicating that Ema19 promotes the proteolytic degradation of non-productive precursors. The deletion of Ema19 improves the growth of respiration-deficient cells, suggesting that Ema19-mediated degradation can compete with productive protein import into mitochondria. Ema19 is the yeast representative of a conserved protein family. The human Ema19 homolog is known as sigma 2 receptor or TMEM97. Though its molecular function is not known, previous studies suggested a role of the sigma 2 receptor as a quality control factor in the ER, compatible with our observations about Ema19. More globally, our data provide an additional demonstration of the important role of the ER in mitochondrial protein targeting

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens

Prevalence and genetic diversity of Wolbachia endosymbiont and mtDNA in Palearctic populations of Drosophila melanogaster

Abstract Background Maternally inherited Wolbachia symbionts infect D. melanogaster populations worldwide. Infection rates vary greatly. Genetic diversity of Wolbachia in D. melanogaster can be subdivided into several closely related genotypes coinherited with certain mtDNA lineages. mtDNA haplotypes have the following global distribution pattern: mtDNA clade I is mostly found in North America, II and IV in Africa, III in Europe and Africa, V in Eurasia, VI is global but very rare, and VIII is found in Asia. The wMel Wolbachia genotype is predominant in D. melanogaster populations. However, according to the hypothesis of global Wolbachia replacement, the wMelCS genotype was predominant before the XX century when it was replaced by the wMel genotype. Here we analyse over 1500 fly isolates from the Palearctic region to evaluate the prevalence, genetic diversity and distribution pattrern of the Wolbachia symbiont, occurrence of mtDNA variants, and finally to discuss the Wolbachia genotype global replacement hypothesis. Results All studied Palearctic populations of D. melanogaster were infected with Wolbachia at a rate of 33–100%. We did not observe any significant correlation between infection rate and longitude or latitude. Five previously reported Wolbachia genotypes were found in Palearctic populations with a predominance of the wMel variant. The mtDNA haplotypes of the I_II_III clade and V clade were prevalent in Palearctic populations. To test the recent Wolbachia genotype replacement hypothesis, we examined three genomic regions of CS-like genotypes. Low genetic diversity was observed, only two haplotypes of the CS genotypes with a ‘CCG’ variant predominance were found. Conclusion The results of our survey of Wolbachia infection prevalence and genotype diversity in Palearctic D. melanogaster populations confirm previous studies. Wolbachia is ubiquitous in the Palearctic region. The wMel genotype is dominant with local occurrence of rare genotypes. Together with variants of the V mtDNA clade, the variants of the ‘III+’ clade are dominant in both infected and uninfected flies of Palearctic populations. Based on our data on Wolbachia and mtDNA in different years in some Palearctic localities, we can conclude that flies that survive the winter make the predominant symbiont contribution to the subsequent generation. A comprehensive overview of mtDNA and Wolbachia infection of D. melanogaster populations worldwide does not support the recent global Wolbachia genotype replacement hypothesis. However, we cannot exclude wMelCS genotype rate fluctuations in the past