Biology subjects

Beck, M.

Publications and source records attributed to Beck, M..

53 records · Page 3Linked to original sources

bioRxiv2021

Co-translational assembly counteracts promiscuous interactions

During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examined structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally tested candidate structural motifs and identified several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assembled co-translationally in only one but not all of the relevant assembly pathways. Our results highlight the regulatory complexity of assembly pathways.

molecular biology↗

bioRxiv2021

A Modular Platform for Streamlining Automated Cryo-FIB Workflows

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, current preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. The software enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM guided preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

biophysics↗

bioRxiv2020

3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ~1000 proteins, termed nucleoporins, and has a mass of over 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of 3D super-resolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of over ten thousand individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.

cell biology↗

bioRxiv2020

Operation of a TCA cycle subnetwork in the mammalian nucleus

Nucleic acid and histone modifications critically depend on central metabolism for substrates and co-factors. Although a few enzymes related to the formation of these required metabolites have been reported in the nucleus, the corresponding metabolic pathways are considered to function elsewhere in the cell. Here we show that a substantial part of the mitochondrial tricarboxylic acid (TCA) cycle, the biosynthetic hub of epigenetic modification factors, is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass-spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins, supporting their nuclear location. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus warranting a revision of the canonical view on metabolic compartmentalization and gene expression regulation.

systems biology↗

bioRxiv2020

Nuclear pores constrict upon energy depletion

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins that form an intricate cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter were reported, but the physiological circumstances and the molecular details remain unknown. Here we combined cryo-electron tomography and subtomogram averaging with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. While actively transporting NPCs adopt a dilated conformation, they strongly constrict upon cellular energy depletion. Fluorescence recovery after photo bleaching experiments show that NPC constriction is concomitant with reduced diffusion and active transport across the nuclear envelope. Our data point to a model where the energy status of cells is linked to the conformation of NPC architecture.

cell biology↗

bioRxiv2020

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

microbiology↗

bioRxiv2020

In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 [A]ngstrom, and is publicly available at EMPIAR-10453.

molecular biology↗

bioRxiv2020

Prion protein lowering is a disease-modifying therapy across prion strains, disease stages, and endpoints

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that less than 25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.

neuroscience↗

bioRxiv2020

In cell architecture of the nuclear pore complex and snapshots of its turnover

Nuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.

biophysics↗

bioRxiv2019

Proteome-wide structural probing of low-abundant protein interactions by crosslinking

Proteome-wide crosslinking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In a paradigm shift, we demonstrate both experimentally and by a kinetic model that this bias is caused by the propensity of crosslinks to preferentially form on high abundant proteins and not by the inability to detect crosslinks due to limitations in current technology. We further show, by using both an in-vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that parameters optimized towards a pseudo 1st order kinetics model result in a significant 3 to 10-fold overall increase in the detection of lower-abundant proteins on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide crosslinking studies and demonstrates how to address a larger part of the proteome by crosslinking.

systems biology↗

bioRxiv2019

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution

The evolutionary origin of metazoan cell types such as neurons, muscles, digestive, and immune cells, remains unsolved. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identify 18 distinct cell types comprising four major families. This includes nitric-oxide sensitive contractile cells, digestive cells active in macropinocytosis, and a family of amoeboid-neuroid cells involved in innate immunity. We uncover presynaptic genes in an amoeboid-neuroid cell type, and postsynaptic genes in digestive choanocytes, suggesting asymmetric and targeted communication. Corroborating this, long neurite-like extensions from neuroid cells directly contact and enwrap choanocyte microvillar collars. Our data indicate a link between neuroid and immune functions in sponges, and suggest that a primordial neuro-immune system cleared intruders and controlled ciliary beating for feeding.

developmental biology↗

bioRxiv2019

Benchmarking tomographic acquisition schemes for high-resolution structural biology

Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution, in principle, can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available at EMPIAR-10277 as well as EMD-10207 and might be used to further develop processing routines.

molecular biology↗

bioRxiv2019

An ESCRT-LEM domain inner nuclear membrane protein surveillance system is poised to directly monitor the integrity of the nuclear envelope barrier and nuclear transport system

The integrity of the nuclear envelope membranes coupled to the diffusion barrier and selective transport properties of nuclear pore complexes (NPCs) are a prerequisite for the robust segregation of nucleoplasm and cytoplasm. Recent work supports that mechanical membrane disruption or perturbation to NPC assembly can trigger an ESCRT-dependent surveillance system that seals nuclear envelope pores: how these pores are sensed and sealed remains to be fully defined. Here, we show that the principal components of the nuclear envelope surveillance system in yeast, which includes the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1, are spatially segregated by the nuclear transport system. Specifically, at steady state Chm7 is actively restricted from the nucleus by Crm1/Xpo1. Consistent with the idea that it is the exposure of the INM that triggers surveillance, the expression of a transmembrane anchor and the winged helix domain of Heh1 is sufficient to recruit and activate Chm7 at a membrane interface. Correlative light electron tomography under conditions of Chm7 hyper-activation further show the formation of an elaborate network of fenestrated sheets at the INM and suggest ER-membrane delivery at sites of nuclear envelope herniation. Our data point to a model in which exposure of Chm7 to Heh1, driven by any perturbation in the nuclear envelope barrier would lead to local nuclear envelope remodeling to promote membrane sealing. Our findings have implications for disease mechanisms associated with defects in NPC assembly and nuclear envelope integrity.

cell biology↗

bioRxiv2018

Dead or Alive? Molecular life-dead distinction in human stool samples reveals significantly different composition of the microbial community

The gut microbiome is strongly interwoven with human health. Conventional gut microbiome analysis generally involves 16S rRNA gene targeting next generation sequencing (NGS) of stool microbial communities, and correlation of results with clinical parameters. However, some microorganisms may not be alive at the time of sampling, and thus their impact on the human health is potentially less significant. As conventional NGS methods do not differentiate between viable and dead microbial components, retrieved results provide only limited information.\n\nPropidium monoazide (PMA) is frequently used in food safety monitoring and other disciplines to discriminate living from dead cells. PMA binds to free DNA and masks it for subsequent procedures. In this article we show the impact of PMA on the results of 16S rRNA gene-targeting NGS from human stool samples and validate the optimal applicable concentration to achieve a reliable detection of the living microbial communities.\n\nFresh stool samples were treated with a concentration series of zero to 300 M PMA, and were subsequently subjected to amplicon-based NGS. The results indicate that a substantial proportion of the human microbial community is not intact at the time of sampling. PMA treatment significantly reduced the diversity and richness of the sample depending on the concentration and impacted the relative abundance of certain important microorganisms (e.g. Akkermansia, Bacteroides). Overall, we found that a concentration of 100 M PMA was sufficient to quench signals from disrupted microbial cells.\n\nThe optimized protocol proposed here can be easily implemented in classical microbiome analyses, and helps to retrieve an improved and less blurry picture of the microbial community composition by excluding signals from background DNA.

microbiology↗

bioRxiv2018

Rapid proteotyping reveals cancer biology and drug response determinants in the NCI-60 cells

We describe the rapid and reproducible acquisition of quantitative proteome maps for the NCI-60 cancer cell lines and their use to reveal cancer biology and drug response determinants. Proteome datasets for the 60 cell lines were acquired in duplicate within 30 working days using pressure cycling technology and SWATH mass spectrometry. We consistently quantified 3,171 proteotypic proteins annotated in the SwissProt database across all cell lines, generating a data matrix with 0.1% missing values, allowing analyses of protein complexes and pathway activities across all the cancer cells. Systematic and integrative analysis of the genetic variation, mRNA expression and proteomic data of the NCI-60 cancer cell lines uncovered complementarity between different types of molecular data in the prediction of the response to 240 drugs. We additionally identified novel proteomic drug response determinants for clinically relevant chemotherapeutic and targeted therapies. We anticipate that this study represents a significant advance toward the translational application of proteotypes, which reveal biological insights that are easily missed in the absence of proteomic data.

systems biology↗

bioRxiv2017

In situ architecture of the algal nuclear pore complex

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate nucleocytoplasmic exchange. They are a hallmark of eukaryotes and are deeply rooted in the evolutionary origin of cellular compartmentalization. NPCs have an elaborate architecture that has been well studied in vertebrates. Whether this architecture is unique or varies significantly in other eukaryotic kingdoms remains unknown, predominantly due to missing in situ structural data. Here, we report the architecture of the algal NPC from the early branching eukaryote Chlamydomonas reinhardtii and compare it to the human NPC. We find that the inner ring of the Chlamydomonas NPC has an unexpectedly large diameter, and the outer rings exhibit an asymmetric oligomeric state that is unprecedented compared to all previously proposed models of NPC architecture. Our study provides evidence that the NPC is subject to substantial structural variation between species. The divergent and conserved features of NPC architecture provide insights into the evolution of the nucleocytoplasmic transport machinery.

cell biology↗

bioRxiv2017

Postmitotic Nuclear Pore Assembly Proceeds By Radial Dilation Of Small ER Membrane Openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

cell biology↗