Biology subjects

Spang, A.

Publications and source records attributed to Spang, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Biased agonism of carvedilol in the beta1-adrenergic receptor is governed by conformational exclusion

Only two non-visual arrestins recognize many hundreds of different, intracellularly phosphorylated G protein-coupled receptors (GPCRs). Due to the highly dynamic nature of GPCR*arrestin complexes, the critical determinants of GPCR-arrestin recognition have remained largely unclear. We show here that arrestin2 recruitment to the {beta}1-adrenergic receptor ({beta}1AR) can be induced by an arrestin-activating phosphopeptide that is not covalently linked to the receptor and that the recruitment is independent of the presence and type of the orthosteric receptor ligand. Apparently, the arrestin-receptor interaction is driven by the conformational switch within arrestin induced by the phosphopeptide, whereas the electrostatic attraction towards the receptor phosphosites may only play an auxiliary role. Extensive NMR observations show that in contrast to previous static GPCR*arrestin complex structures, the {beta}1AR complex with the beta-blocker carvedilol and arrestin2 is in a G protein-inactive conformation. The insensitivity to the specific receptor conformation provides a rationale for arrestins promiscuous recognition of GPCRs and explains the arrestin-biased agonism of carvedilol, which largely blocks G protein binding, while still enabling arrestin engagement. Significance statementG protein-coupled receptors regulate cellular signaling through G proteins and arrestins. While G protein interactions are well understood, the molecular basis of arrestin recognition remains unclear due to the dynamic nature of GPCR*arrestin complexes. We show that arrestin recognition of the {beta}1-adrenergic receptor occurs independently of the receptors conformational state or ligand binding. Using NMR, cryo-EM, and biochemical assays, we find that arrestin engagement is driven by a conformational change within arrestin itself, triggered by a non-receptor-attached phosphopeptide, whereas electrostatic attraction towards receptor phosphosites may only play an auxiliary role. These findings provide new insights into arrestin activation, explain its ability to recognize diverse GPCRs, with significant implications for understanding biased signaling mechanisms and designing of selective therapeutic strategies.

biochemistry↗

bioRxiv2024

Chimeric Origins and Dynamic Evolution of Central Carbon Metabolism in Eukaryotes

The origin of eukaryotes was a key event in the history of life. Current leading hypotheses propose that a symbiosis between an asgardarchaeal host cell and an alphaproteobacterial endosymbiont represented a crucial step in eukaryotic origins and invoke a central role for syntrophic interactions - that is, metabolic cross-feeding between the partners as the basis for their subsequent evolutionary integration. A major unanswered question is whether the metabolism of modern eukaryotes bears any vestige of this ancestral interaction. To investigate this question in detail, we systematically analyze the evolutionary origins of the eukaryotic gene repertoires mediating central carbon metabolism. Our phylogenetic and sequence analyses reveal that this gene repertoire is chimeric, with ancestral contributions from Asgardarchaeota and Alphaproteobacteria operating predominantly in glycolysis and TCA, respectively. Furthermore, our analyses reveal diverse additional contributions from other prokaryotic sources as well as the extent to which this ancestral metabolic interplay has been remodeled via gene loss, transfer, and subcellular retargeting in the >2Ga since the origin of eukaryotic cells. Together, our work demonstrates that in contrast to previous assumptions, the eukaryotic metabolism preserves information about the nature of the original asgardarchaeal-alphaproteobacterial interactions, and supports syntrophy scenarios on the origin of the eukaryotic cell.

evolutionary biology↗

bioRxiv2024

Coordination between ESCRT function and Rab conversion during endosome maturation

The endosomal pathway is essential for regulating cell signaling and cellular homeostasis. Rab5-positive early endosomes receive proteins from the plasma membrane. Dependent on a ubiquitin mark on the protein, they will be either recycled or sorted into intraluminal vesicles (ILVs) by ESCRT. During endosome maturation Rab5 will be replaced by Rab7 on endosomes that are able to fuse with lysosomes to form endolysosomes. Whether ESCRT-driven ILV formation and Rab5-to-Rab7 conversion are coordinated remains unknown. Here we show that loss of early ESCRTs leads to enlarged Rab5-positive endosomes and prohibit Rab conversion. Reduction of ubiquitinated cargo alleviated this phenotype. Moreover, ubiquitinated proteins on the endosomal limiting membrane prevented the displacement of the Rab5GEF RABX-5 by the Rab7GEF SAND-1/CCZ-1. Overexpression of Rab7 could partially overcome this block, even in the absence of SAND-1, whereby the HOPS complex presumably acts as Rab7GEF. Our data reveal a hierarchy of events in which cargo corralling by ESCRTs is upstream of Rab conversion and thereby ESCRT-0 and ubiquitinated cargo could act as timers determining the onset of Rab conversion.

cell biology↗

bioRxiv2024

Bacterial RNA promotes proteostasis through inter-tissuecommunication in C. elegans

Life expectancy has been increasing over the last decades, which is not matched by an increase in healthspan. Besides genetic composition, environmental and nutritional factors influence both health- and lifespan. Diet is thought to be a major factor for healthy ageing. Here, we show that dietary RNA species extend healthspan in C. elegans. Inherent bacterial-derived double stranded RNA reduces protein aggregation in a C. elegans muscle proteostasis model. This beneficial effect depends on low levels of systemic selective autophagy, the RNAi machinery in the germline, even when the RNA is delivered through ingestion in the intestine and the integrity of muscle cells. Our data suggest a requirement of inter-organ communication between the intestine, the germline and muscles. Our results demonstrate that bacterial-derived RNAs elicit a systemic response in C. elegans, which protects the animal from protein aggregation during ageing. We provide evidence that low stress levels are beneficial for healthspan. One-Sentence SummaryBacteria-derived dietary cues and inter-tissue communication promote proteostasis and fitness in C. elegans

cell biology↗

bioRxiv2024

Endosomal LRBA regulates the endo-lysosomal pathway

Deleterious mutations in the LRBA (Lipopolysaccharide Responsive Beige-like Anchor protein) gene cause severe childhood immune dysregulation. The clinical manifestations of LRBA deficiency syndrome are highly variable. Thus, the complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, does not colocalize with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway yielding the accumulation of enlarged endolysosomes. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency. SummaryLRBA-deficient patients exhibit enlarged functional endolysosomes due to defects in recycling to the plasma membrane. LRBA localization on Rab4+ endosomes depends on Arf1 and Arf3, and is essential for a functional endosomal-lysosomal pathway. Our results could inform new treatment options.

immunology↗

bioRxiv2023

Selective lipid recruitment by an archaeal DPANN symbiont from its host

The symbiont Ca. Nanohaloarchaeum antarcticus is obligately dependent on its host Halorubrum lacusprofundi for lipids and other metabolites due to its lack of certain biosynthetic genes. However, it remains unclear which specific lipids or metabolites are acquired from its host, and how the host responds to infection. Here, we explored the lipidome dynamics of the Ca. Nha. antarcticus - Hrr. lacusprofundi symbiotic relationship during co-cultivation. By using a comprehensive untargeted lipidomic methodology, our study reveals that Ca. Nha. antarcticus selectively recruits 110 lipid species from its host, i.e. nearly two-thirds of the total number of host lipids. Lipid profiles of co-cultures displayed shifts in abundances of bacterioruberins and menaquinones and changes in degree of bilayer-forming glycerolipid unsaturation. This likely results in increased membrane fluidity and improved resistance to membrane disruptions, consistent with compensation for higher metabolic load and mechanical stress on host membranes when in contact with Ca. Nha. antarcticus cells. Notably, our findings differ from previous observations of other DPANN symbiont-host systems, where no differences in lipidome composition were reported. Altogether, our work emphasizes the strength of employing untargeted lipidomics approaches to provide details into the dynamics underlying a DPANN symbiont-host system.

microbiology↗

bioRxiv2023

An evolutionary timescale for Bacteria calibrated using the Great Oxidation Event

Most of lifes diversity and history is microbial but it has left a meagre fossil record, greatly hindering understanding of evolution in deep time. However, the co-evolution of life and the Earth system has left signatures of bacterial metabolism in the geochemical record, most conspicuously the Great Oxidation Event (GOE) [~]2.33 billion years ago (Ga, (Poulton et al. 2021)), in which oxygenic photosynthesis and tectonism (Eguchi, Seales, and Dasgupta 2019) transformed Earths biosphere from dominantly anaerobic to aerobic. Here, we combine machine learning and phylogenetic reconciliation to infer ancestral transitions to aerobic lifestyles during bacterial evolution. Linking these transitions to the GOE provides new constraints to infer the timetree of Bacteria. We find that extant bacterial phyla are truly ancient, having radiated in the Archaean and the Proterozoic: the oldest include Bacillota (Firmicutes), which radiated 3.1-3.7 Ga, Cyanobacteria (3.3-3.5 Ga) and Patescibacteria (3-3.5 Ga). We show that most bacterial phyla were ancestrally anaerobic and that most transitions to an aerobic lifestyle post-dated the GOE. Our analyses trace oxygen production and consumption back to Cyanobacteria. From that starting point, horizontal transfer seeded aerobic lifestyles across bacterial diversity over hundreds of millions of years. Our analyses demonstrate that the diversification of aerobes proceeded in two waves corresponding to the GOE and to a second sustained rise in atmospheric O2 at the dawn of the Palezoic (Krause et al. 2022).

evolutionary biology↗

bioRxiv2023

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER encounter structure (ERMES) and Tom70 are part of two parallel and partially redundant ER-to-mitochondria transfer routes. When ER-to-mitochondria transfer is prevented, many mitochondrial precursor proteins associate with ER membranes, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

biochemistry↗

bioRxiv2023

ATP synthase evolution on a cross-braced dated tree of life

The timing of early cellular evolution from the divergence of Archaea and Bacteria to the origin of eukaryotes remains poorly constrained. The ATP synthase complex is thought to have originated prior to the Last Universal Common Ancestor (LUCA) and analyses of ATP synthase genes, together with ribosomes, have played a key role in inferring and rooting the tree of life. Here we reconstruct the evolutionary history of ATP synthases using an expanded sampling of Archaea, Bacteria, and eukaryotes. We developed a phylogenetic cross-bracing approach making use of endosymbioses and ancient gene duplications of the major ATP synthase subunits to infer a highly resolved, dated species tree and establish an absolute timeline for ATP synthase evolution. Our analyses show that the divergence of the ATP synthase into F- and A/V-type lineages, was a very early event in cellular evolution dating back to more than 4Ga potentially predating the diversification of Archaea and Bacteria. Our cross-braced, dated tree of life also provides insight into more recent evolutionary transitions including eukaryogenesis, showing that the eukaryotic nuclear and mitochondrial lineages diverged from their closest archaeal (2.67-2.19Ga) and bacterial (2.58-2.12Ga) relatives at roughly the same time, with the nuclear stem being moderately longer.

evolutionary biology↗

bioRxiv2023

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding ofproteins with alpha helical domains

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we have quantified the expression of RPL39L in human pluripotent cells, cancer cell lines and tissue samples, and in mouse germ cells. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread proteasomal activity. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

molecular biology↗

bioRxiv2023

The power and limitations of species tree-aware phylogenetics

Species tree-aware phylogenetic methods model how gene trees are generated along the species tree by a series of evolutionary events, including the duplication, transfer and loss of genes. Over the past ten years these methods have emerged as a powerful tool for inferring and rooting gene and species trees, inferring ancestral gene repertoires, and studying the processes of gene and genome evolution. However, these methods are complex and can be more difficult to use than traditional phylogenetic approaches. Method development is rapid, and it can be difficult to decide between approaches and interpret results. Here, we review ALE and GeneRax, two popular packages for reconciling gene and species trees, explaining how they work, how results can be interpreted, and providing a tutorial for practical analysis. It was recently suggested that reconciliation-based estimates of duplication and transfer frequencies are unreliable. We evaluate this criticism and find that, provided parameters are estimated from the data rather than being fixed based on prior assumptions, reconciliation-based inferences are in good agreement with the literature, recovering variation in gene duplication and transfer frequencies across lineages consistent with the known biology of studied clades. For example, published datasets support the view that transfers greatly outnumber duplications in most prokaryotic lineages. We conclude by discussing some limitations of current models and prospects for future progress. Significance statementEvolutionary trees provide a framework for understanding the history of life and organising biodiversity. In this review, we discuss some recent progress on statistical methods that allow us to combine information from many different genes within the framework of an overarching phylogenetic species tree. We review the advantages and uses of these methods and discuss case studies where they have been used to resolve deep branches within the tree of life. We conclude with the limitations of current methods and suggest how they might be overcome in the future.

evolutionary biology↗

bioRxiv2023

Genetic analysis of rab7 mutants in zebrafish

Vascular network formation requires the fusion of newly formed blood vessels and the emergence of a patent lumen between the newly established connections so that blood flow can start. Lumen formation has been shown to depend on the late endosomal/lysosomal pathway in various organs of animal tubular systems. Here, we identified a late endosomal/lysosomal vesicular fraction (Rab7/Lamp2) in early zebrafish angiogenic sprouts, which appears to contribute to apical membrane growth during lumen formation. To study the effect of the late endocytic pathway on vascular development, we generated mutant alleles for all three rab7 genes in zebrafish (rab7a, rab7ba, rab7bb). All rab7 genes are expressed in wild-type zebrafish and we did not detect any compensatory effects by the other rab7 isoforms in single knockout mutants, which were all viable. Only the triple mutant was lethal suggesting some functional redundancy. However, the different rab7 isoforms fulfil also at least partially independent functions because eggs laid from mothers lacking two rab7 (rab7a and/or rab7bb). showed reduced survival and contained enlarged yolk granules, suggesting maternal contribution of these two rab7. Finally, we observed minor effects on lumen formation in embryos which still express one copy of rab7. Our results support the notion that the late endocytic/lysosomal compartment contributes to lumen expansion.

genetics↗

bioRxiv2023

The intracellular lifestyle of an archaeal symbiont

DPANN Archaea are a diverse group of organisms typically characterised by small cells and reduced genomes. To date, all cultivated DPANN Archaea are ectosymbionts that require direct cell contact with an archaeal host species for proliferation. However, the dynamics of DPANN - host interactions and the impacts of these interactions on the host species are poorly understood. Here, we show that one DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. Our data also suggest that these interactions involve invasion of the host cell by the nanohaloarchaeon. This is the first reported instance of such a predatory-like lifestyle amongst Archaea and indicates that some DPANN Archaea may interact with host populations in a manner similar to viruses.

microbiology↗

bioRxiv2022

Distinguishing between canonical and non-canonical tRNA genes reveals that Thermococcaceae adhere to the standard archaeal tRNA gene set

Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.

genomics↗

bioRxiv2022

The small GTPase Arf1 regulates ATP synthesis and mitochondria homeostasis by modulating fatty acid metabolism

Lipid mobilization through fatty acid {beta}-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where {beta}-oxidation products enter mitochondria and fuel the TCA cycle. Little is known about the physical and metabolic cooperation between these organelles. We found that expression of fatty acid transporters and of the rate-limiting enzyme involved in {beta}-oxidation are decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. As a consequence, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although {beta}-oxidation occurs mainly in mitochondria in mammals, Arf1s role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact-sites.

cell biology↗

bioRxiv2021

A novel live cell imaging assay reveals regulation of endosome maturation

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1- and Rab11-dependent endosomal recycling occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

cell biology↗

bioRxiv2021

Unraveling the critical growth factors for stable cultivation of (nano-sized) Micrarchaeota

Micrarchaeota are members of the archaeal DPANN superphylum. These so far poorly characterized archaea have been found to have reduced genomes and likely depend on interactions with host organisms for growth and survival. Here we report on the enrichment of the first stable co-culture of a member of the Micrarchaeota together with its host, as well as the isolation of the latter. Electron microscopic analysis suggest that growth is dependent on the physical interaction of the two organisms within a biofilm. The interaction seems to be ensured by the necessity to grow in form of a biofilm. Furthermore, transcriptomic analyses indicate a shift towards biofilm formation of the host as a result of co-cultivation. Finally, genomic, metabolomic, extracellular polymeric substance (EPSs) and lipid content analyses reveal that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host and thereby provide first insights into the basis of symbiont-host interactions.

microbiology↗

bioRxiv2021

The C. elegans PTCHD homolog PTR-4 is required for proper organization of the pre-cuticular apical extracellular matrix

The Patched-related (PTR) superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other PTR or Patched domain (PTCHD) family members. C. elegans has a large number of PTR/PTCHD proteins, despite lacking a canonical hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the pre-cuticle apical extracellular matrix (aECM), a transient and molecularly distinct aECM that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with pre-cuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when pre-cuticle matrix is present. Finally, PTR-4 acts in a cell non-autonomous manner to properly localize the secreted ZP domain protein LET-653 to the pre-cuticle aECM. We propose that PTR-4 exports lipids or other hydrophobic components of the pre-cuticle aECM.

cell biology↗