Biology subjects

Wagner, A.

Publications and source records attributed to Wagner, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

A rugged yet easily navigable fitness landscape of antibiotic resistance

A fitness landscape is a biological analogue of a physical landscape, in which each genotype occupies a location whose elevation corresponds to fitness. Theoretical models predict that rugged fitness landscapes with multiple peaks should impair Darwinian evolution, because natural selection prevents evolving populations from traversing the valleys that lie between peaks. Experimental tests of this prediction are very limited. Here we combine CRISPR-Cas9 genome editing and deep sequencing to map the fitness landscape of more than 260000 genotypes of the E. coli folA gene in an environment harboring the antibiotic trimethoprim. The folA gene encodes the key metabolic enzyme dihydrofolate reductase (DHFR), which is also a target of this antibiotic. With 514 mostly low fitness peaks, the DHFR fitness landscape is rugged. Despite this ruggedness, its highest fitness peaks are easily accessible to evolving populations. Fitness-increasing paths to high fitness peaks are abundant, and individual peaks have large basins of attractions. The basins of different peaks overlap, which renders the outcome of adaptive evolution highly contingent on chance events. In sum, ruggedness need not be an obstacle to Darwinian evolution but can reduce its predictability. If true in general, evolutionary biology and other fields of sciences in which landscapes play an important role may have to re-appraise the complexity of optimization problems on realistic landscapes.

evolutionary biology↗

bioRxiv2022

Not quite lost in translation: Mistranslation alters adaptive landscape topography and the dynamics of evolution

Mistranslation - the erroneous incorporation of amino acids into nascent proteins - is a source of protein variation that is orders of magnitude more frequent than DNA mutation. Like other sources of nongenetic variation, it can affect adaptive evolution. We study the evolutionary consequences of mistranslation with experimental data on mistranslation rates applied to three empirical adaptive landscapes. We find that mistranslation generally flattens adaptive landscapes by reducing the fitness of high fitness genotypes and increasing that of low fitness genotypes, but it does not affect all genotypes equally. Most importantly, it increases genetic variation available to selection by rendering many neutral DNA mutations non-neutral. Mistranslation also renders some beneficial mutations deleterious and vice versa. It increases the probability of fixation of 3 to 8 percent of beneficial mutations. Even though mistranslation increases the incidence of epistasis, it also allows populations evolving on a rugged landscape to evolve modestly higher fitness. Our observations show that mistranslation is an important source of non-genetic variation that can affect adaptive evolution on fitness landscapes in multiple ways.

evolutionary biology↗

bioRxiv2022

The Formation of Biogenic Guanine Crystals Closely Resembles Melanosome Morphogenesis

Animals precisely control the morphology and assembly of highly reflective guanine crystals to produce diverse optical phenomena used in coloration and vision. However, little is known about how organisms regulate crystallization to produce optically useful crystal morphologies which express highly reflective crystal faces. Guanine crystals form inside iridosome vesicles within specialized chromatophore cells called iridophores. By following the formation of iridosomes in developing scallop eyes we show that, despite their different physical properties, the morphogenesis of iridosomes bears a striking resemblance to melanosome morphogenesis in vertebrates. A key finding is that pre-assembled intravesicular fibrillar sheets template crystal nucleation, growth, and orientation, dictating the formation of optically functional, but thermodynamically disfavored plate-like habits. The common control mechanisms for melanin and guanine formation inspire new approaches for manipulating the morphologies and properties of molecular materials.

cell biology↗

bioRxiv2022

Integrative Single Cell Multiomic Profiling Analysis Reveals HOX-PBX Gene Regulatory Network Contributing to the Survival of mTOR Hyperactive Cells

Lymphangioleiomyomatosis (LAM) is a rare, debilitating lung disease that predominantly affects women of reproductive age. LAM is characterized by the infiltration of the lungs by abnormally proliferating smooth muscle-like cells of unknown origin via an estrogen-dependent metastatic mechanism. LAM cells carry deleterious mutations of tuberous sclerosis complex (TSC1/TSC2) genes, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) and ultimately dysregulated cell growth. Sirolimus, an FDA approved mTORC1 inhibitor and current best-choice medication for LAM stabilizes lung function in most LAM patients. However, it requires sustained application and remains inefficacious in some patients. The greatest barriers to finding a cure for LAM include its undetermined origin and unclear underlying pathogenesis. Our study aims to advance knowledge on the origin of LAM, and ultimately serve as a premise for the development of novel therapeutic targets for LAM. Single cell RNA sequencing (scRNA-seq) is a powerful tool in biomedical research that informs gene expression differences at the cellular level and may provide insights into the most fundamental origin of LAM cells. Our scRNA-seq analysis of LAM cells revealed a unique population of cells (LAMCORE), expressing uterine-similar homeobox transcription factors (HOX) and Pre-B-cell leukemia homeobox 1 (PBX1), which are absent in normal lung, suggesting that the uterus is the primary origin of LAM. PBX1 is a transcription factor critical for female reproductive tract development and maintenance, and its overexpression is implicated in some female reproductive cancers. In this study we hypothesize that PBX1 promotes survival and lung colonization of LAM (TSC2-null) cells. Using LAM patient-derived cells, we validated the transcriptional profile, gene expression and protein levels of PBX1. We have the first functional evidence that PBX1 and its downstream targets are upregulated in LAM cells. In a mouse model of LAM, we monitored the effect of suppression of PBX1 by short hairpin RNA-mediated gene silencing on lung colonization and tumor growth. We also found that pharmacological suppression of PBX1 attenuates LAM lung colonization and promotes death of LAM cells in vivo and vitro. Our data collectively suggests that PBX1 is a critical regulator of LAM progression.

cancer biology↗

bioRxiv2022

Cellular senescence modulates progenitor cell expansion during axolotl limb regeneration

Axolotl limb regeneration is accompanied by the transient induction of cellular senescence within the blastema, the structure which nucleates regeneration. The precise role of this blastemal senescent cell (bSC) population, however, remains unknown. Here, through a combination of gain- and loss-of-function assays, we elucidate the functions and molecular features of cellular senescence in vivo. We demonstrate that cellular senescence plays a positive role during axolotl regeneration, by creating a pro-proliferative niche that supports progenitor cell expansion and blastema outgrowth. Senescent cells impact on their microenvironment via Wnt pathway modulation. Further, we uncover a link between Wnt signalling and senescence induction, and propose that bSC-derived Wnt signals facilitate the proliferation of neighbouring cells in part by preventing their induction into senescence. This work defines the roles of cellular senescence in regeneration of complex structures.

developmental biology↗

bioRxiv2022

Adipose tissue mitochondrial respiration in Atlantic salmon: implications for sex-dependent life-history variation

Adipose tissue is essential for energy homeostasis, with mitochondria having a central role in its function. Mitochondria-mediated white adipose tissue dysfunction has been linked to several metabolic disorders in humans but surprisingly little is known about natural variation in mitochondrial function in wild animal populations, and its evolutionary significance. Early sexual maturation (low age-at-maturity) in Atlantic salmon (Salmo salar) is promoted by higher adiposity and has a strong genetic association with the vgll3 locus. This makes Atlantic salmon a convenient wild model to study the potential role of mitochondria-mediated adipose tissue processes in relation to the timing of maturation. Yet, mitochondrial respiration has not been measured in the adipose tissue in fish, and the lack of data is restricting the development of informed hypotheses. Here, using 13 Atlantic salmon individuals reared in common-garden conditions, we first verified the feasibility of measuring mitochondrial respiration in the adipose tissue. As expected, the respiration level was generally low, but nonetheless we successfully quantified its biological variation in the adipose tissue. Next, we analysed the potential association of mitochondrial respiration with mitochondrial DNA (mtDNA) content, adipocyte size, sex, and the vgll3 genotype. Despite low samples sizes, mitochondrial respiration, leak respiration, and coupling capacity (P/E ratio) were marginally significantly decreased in immature females carrying with the vgll3 early maturation compared to the alternative genotype. Based on these results, we suggested two new hypotheses on how the coupling capacity of oxidative phosphorylation could be linked with the timing of maturation via adiposity and pave the way to study the mechanistic relationships between life-history variation and mitochondrial bioenergetics in wild populations.

physiology↗

bioRxiv2022

Guided construction of single cell reference for human and mouse lung

Accurate cell type identification is a key and rate-limiting step in single cell data analysis. Single cell references with comprehensive cell types, reproducible and functional validated cell identities, and common nomenclatures are much needed by the research community to optimize automated cell type annotation and facilitate data integration, sharing, and collaboration. In the present study, we developed a novel computational pipeline to utilize the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples and constructed "LungMAP CellRef" and "LungMAP CellRef Seed" for both normal human and mouse lungs. "CellRef Seed" has an equivalent prediction power and produces consistent cell annotation as does "CellRef" but improves computational efficiency and simplifies its utilization for fast automated cell type annotation and online visualization. This atlas set incorporates 48 human and 40 mouse well-defined lung cell types catalogued from diverse anatomic locations and developmental time points. Using independent datasets, we demonstrated the utility of our CellRefs for automated cell type annotation analysis of both normal and disease lungs. User-friendly web interfaces were developed to support easy access and maximal utilization of the LungMAP CellRefs. LungMAP CellRefs are freely available to the pulmonary research community through fast interactive web interfaces to facilitate hypothesis generation, research discovery, and identification of cell type alterations in disease conditions.

bioinformatics↗

bioRxiv2022

Rapid evolutionary change in trait correlations of a single protein

Many organismal traits are genetically determined and covary in evolving populations. The resulting trait correlations can either help or hinder evolvability - the ability to bring forth new and adaptive phenotypes. The evolution of evolvability requires that trait correlations themselves must be able to evolve, but we know little about this ability. To learn more about it, we here study one of the simplest evolvable systems, a gene encoding a single protein, and two traits of this protein, namely the ability to emit yellow and green light. We show that correlations between these two traits can evolve rapidly through both mutation and selection on short evolutionary time scales. In addition, we show that these correlations are driven by a proteins ability to fold, because single mutations that alter foldability can dramatically change trait correlations. Since foldability is important for most proteins and their traits, mutations affecting protein folding may alter trait correlations mediated by many other proteins. Thus, mutations that affect protein foldability may also help shape the correlations of complex traits that are affected by hundreds of proteins.

evolutionary biology↗

bioRxiv2022

Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for {beta}-glucuronidase activity. We identified SN243, a genuine {beta}-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family had no recorded evidence of {beta}-glucuronidases at the outset of this study, showing that a functional metagenomic approach can shed light on assignments that are currently unpredictable by bioinformatics. Kinetic analyses of SN243 characterised it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous GH3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.

biochemistry↗

bioRxiv2022

Bartonella taylorii: A model organism for studying Bartonella infection in vitro and in vivo

Bartonella spp. are Gram-negative facultative intracellular pathogens that infect diverse mammals and cause a long-lasting intra-erythrocytic bacteremia in their natural host. These bacteria translocate Bartonella effector proteins (Beps) into host cells via their VirB/VirD4 type 4 secretion system (T4SS) in order to subvert host cellular functions, thereby leading to the downregulation of innate immune responses. Most studies on the functional analysis of the VirB/VirD4 T4SS and the Beps were performed with the major zoonotic pathogen Bartonella henselae for which efficient in vitro infection protocols have been established. However, its natural host, the cat, is unsuitable as an experimental infection model. In vivo studies were mostly confined to rodent models using rodent-specific Bartonella species, while the in vitro infection protocols devised for B. henselae are not transferable for those pathogens. The disparities of in vitro and in vivo studies in different species has hampered progress in our understanding of Bartonella pathogenesis. Here we describe the murine-specific strain B. taylorii IBS296 as a new model organism facilitating the study of bacterial pathogenesis both in vitro in cell cultures and in vivo in laboratory mice. We implemented the split NanoLuc luciferase-based translocation assay to study BepD translocation through the VirB/VirD4 T4SS. We found increased effector-translocation into host cells if the bacteria were grown on tryptic soy agar (TSA) plates and experienced a temperature shift immediately before infection. The improved infectivity in vitro could be correlated to an upregulation of the VirB/VirD4 T4SS. Using our adapted infection protocols, we showed BepD-dependent immunomodulatory phenotypes in vitro. In mice, the implemented growth conditions enabled infection by a massively reduced inoculum without having an impact on the course of the intra-erythrocytic bacteremia. The established model opens new avenues to study the role of the VirB/VirD4 T4SS and the translocated Bep effectors in vitro and in vivo.

microbiology↗

bioRxiv2022

Light activation of Orange Carotenoid Protein reveals initial C8'-C7' double bond trans/cis photoisomerization

Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9-C8 and C7-C6 single bonds in the bicycle-pedal (BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. The isomerization and its transient nature are confirmed in OCP crystals and solution by FTIR and UV/Vis spectroscopy. This study reveals the single bond isomerization of the carotenoid in the BP manner and subsequent thermal structural reactions as the basis of OCP photoreception. Understanding and potentially controlling the OCP dynamics offers the prospect of novel applications in biomass engineering as well as in optogenetics and bioimaging.

biophysics↗

bioRxiv2022

Ontogenetic Color Switching in Lizards as a by-Product of Guanine Cell Development

Many animals undergo dramatic changes in colour during development1,2. Changes in predation risk during ontogeny are associated with spectacular switches in defensive colours, typically involving the replacement of skin or the production of new pigment cells3. Ontogenetic colour systems are ideal models for understanding the evolution and formation mechanisms of animal colour which remain largely enigmatic2. We show that defensive colour switching in lizards arises by reorganization of a single photonic system, as an incidental by-product of chromatophore maturation. The defensive blue tail colour of hatchling A. beershebensis lizards is produced by light scattering from premature guanine crystals in underdeveloped iridophore cells. Camouflaged adult tail colours emerge upon reorganization of the guanine crystals into a photonic reflector during chromatophore maturation. The substituent guanine crystals form by the attachment of individual nanoscopic plates, which coalesce during growth to form single crystals. Our results show that the blue colour of hatchlings is a fortuitous, but necessary, precursor to the development of adult colour. Striking functional colours in animals can thus arise not as distinct evolutionary innovations but via exploitation of the timing of naturally occurring changes in chromatophore cell development.

developmental biology↗

bioRxiv2021

Single-cell Atlas Unveils Cellular Heterogeneity and Novel Markers in Human Neonatal and Adult Intervertebral Discs

The origin, composition, distribution, and function of cells in the human intervertebral disc (IVD) has not been fully understood. Here, cell atlases of both human neonatal and adult IVDs have been generated and further assessed by gene ontology pathway enrichment, pseudo-time trajectory, histology, and immunofluorescence. Comparison of cell atlases revealed the presence of two sub-populations of notochordal cells (NCs) and their associated markers in both the neonatal and adult IVDs. Developmental trajectories predicted 7 different cell states that describe the developmental process from neonatal to adult cells in IVD and analyzed the NCs role in the IVD development. A high heterogeneity and gradual transition of annulus fibrosus cells (AFCs) in the neonatal IVD was detected and their potential relevance in IVD development assessed. Collectively, comparing single-cell atlases between neonatal and adult IVDs delineates the landscape of IVD cell biology and may help discovering novel therapeutic targets for IVD degeneration.

developmental biology↗

bioRxiv2021

Translation stalling proline motifs are enriched in slow-growing, thermophilic, and multicellular bacteria

Rapid bacterial growth depends on the speed at which ribosomes can translate mRNA into proteins. mRNAs that encode successive stretches of proline can cause ribosomes to stall, substantially reducing translation speed. Such stalling is especially detrimental for species that must grow and divide rapidly. Here we focus on di-prolyl motifs (XXPPX) and ask whether their incidence varies with growth rate. To find out we conducted a broad survey of such motifs in >3000 bacterial genomes across 36 phyla. Indeed, fast-growing species encode fewer motifs than slow-growing species, especially in highly expressed proteins. We also found many di-prolyl motifs within thermophiles, where prolines can help maintain proteome stability. Moreover, bacteria with complex, multicellular lifecycles also encode many di-prolyl motifs. This is especially evident in the slow-growing phylum Myxococcota. Bacteria in this phylum encode many serine-threonine kinases, and many di-prolyl motifs at potential phosphorylation sites within these kinases. Serine-threonine kinases are involved in cell signaling and help regulate developmental processes linked to multicellularity in the Myxococcota. Altogether, our observations suggest that weakened selection on translational rate, whether due to slow or thermophilic growth, may allow di-prolyl motifs to take on new roles in biological processes that are unrelated to translational rate.

genomics↗

bioRxiv2021

Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, Brachydactyly B and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

biochemistry↗

bioRxiv2021

Allergy-induced systemic inflammation impairs tendon quality

Treatment of tendinopathies still present a major challenge, since the aetiology of the disease remains poorly understood. To determine whether the systemic inflammation accompanying predisposing factors including rheumatoid arthritis, diabetes or smoking contributes to the onset of tendinopathy, we studied the effect of a systemic inflammation induced by an allergic episode on tendon properties. To this end, we elicited an allergic response in mice by exposing them to a plant allergen and subsequently analysed both their flexor and Achilles tendons. Biomechanical testing and histological analysis revealed that tendons from allergic mice not only showed a significant reduction of both elastic modulus and tensile stress, but also alterations of the tendon matrix. Moreover, 3D tendon-like constructs treated with sera from allergic mice displayed a matrix-remodelling expression profile and the expression of macrophage-associated markers and matrix metalloproteinase 2 (MMP2) was increased in allergic Achilles tendons. Analysing data from an epidemiologic study comprising data from more than 10.000 persons, we found that persons suffering from an allergic condition appeared to have an increased propensity to develop a tendinopathy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/450910v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1263836org.highwire.dtl.DTLVardef@349621org.highwire.dtl.DTLVardef@6c9142org.highwire.dtl.DTLVardef@1b49bdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Multi-resolution deconvolution of spatial transcriptomics data reveals continuous patterns of inflammation

The function of mammalian cells is largely influenced by their tissue microenvironment. Advances in spatial transcriptomics open the way for studying these important determinants of cellular function by enabling a transcriptome-wide evaluation of gene expression in situ. A critical limitation of the current technologies, however, is that their resolution is limited to niches (spots) of sizes well beyond that of a single cell, thus providing measurements for cell aggregates which may mask critical interactions between neighboring cells of different types. While joint analysis with single-cell RNA-sequencing (scRNA-seq) can be leveraged to alleviate this problem, current analyses are limited to a discrete view of cell type proportion inside every spot. This limitation becomes critical in the common case where, even within a cell type, there is a continuum of cell states that cannot be clearly demarcated but reflects important differences in the way cells function and interact with their surroundings. To address this, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI), a probabilistic method for multi-resolution analysis for spatial transcriptomics that explicitly models continuous variation within cell types. Using simulations, we demonstrate that DestVI is capable of providing higher resolution compared to the existing methods and that it can estimate gene expression by every cell type inside every spot. We then introduce an automated pipeline that uses DestVI for analysis of single tissue slices and comparison between tissues. We apply this pipeline to study the immune crosstalk within lymph nodes to infection and explore the spatial organization of a mouse tumor model. In both cases, we demonstrate that DestVI can provide a high resolution and accurate spatial characterization of the cellular organization of these tissues, and that it is capable of identifying important cell-type-specific changes in gene expression - between different tissue regions or between conditions. DestVI is available as an open-source software package in the scvi-tools codebase (https://scvi-tools.org).

bioinformatics↗

bioRxiv2021

The length scale of multivalent interactions is evolutionarily conserved in fungal and vertebrate phase-separating proteins

One key feature of proteins that form liquid droplets by phase separation inside a cell is the presence of multiple sites - multivalency - that mediate interactions with other proteins. We know little about the variation of multivalency on evolutionary time scales. Here, we investigated the long-term evolution ([~]600 million years) of multivalency in fungal mRNA decapping subunit 2 protein (Dcp2), and in the FET protein family. We found that multivalency varies substantially among the orthologs of these proteins. However, evolution has maintained the length scale at which sequence motifs that enable protein-protein interactions occur. That is, the total number of such motifs per hundred amino acids is higher and less variable than expected by neutral evolution. To help explain this evolutionary conservation, we developed a conformation classifier using machine-learning algorithms. This classifier demonstrates that disordered segments in Dcp2 and FET proteins tend to adopt compact conformations, which is necessary for phase separation. Thus, the evolutionary conservation we detected may help proteins preserve the ability to undergo phase separation. Altogether, our study reveals that the length scale of multivalent interactions is an evolutionarily conserved feature of two classes of phase-separating proteins in fungi and vertebrates.

evolutionary biology↗