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bioRxiv2023

WNK1 enforces macrophage lineage fidelity

The appropriate development of macrophages, the bodys professional phagocyte, is essential for organismal development, especially in mammals. This dependence is exemplified by the observation that loss-of-function mutations in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities owing to an absence of macrophages. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for development of tissue-resident macrophages (TRMs). Myeloid-specific deletion of Wnk1 resulted in a dramatic loss of TRMs, disrupted organ development, systemic neutrophilia, and mortality between 3 and 4 weeks of age. Strikingly, we found that myeloid progenitors or precursors lacking WNK1 not only failed to differentiate into macrophages, but instead differentiated into neutrophils. Mechanistically, the cognate CSF1R cytokine macrophage-colony stimulating factor (M-CSF) stimulates macropinocytosis by both mouse and human myeloid progenitors and precursor cells. Macropinocytosis, in turn, induces chloride flux and WNK1 phosphorylation. Importantly, blocking macropinocytosis, perturbing chloride flux during macropinocytosis, and inhibiting WNK1 chloride-sensing activity each skewed myeloid progenitor differentiation from macrophages into neutrophils. Thus, we have elucidated a role for WNK1 during macropinocytosis and discovered a novel function of macropinocytosis in myeloid progenitors and precursor cells to ensure macrophage lineage fidelity. Highlights- Myeloid-specific WNK1 loss causes failed macrophage development and premature death - M-CSF-stimulated myeloid progenitors and precursors become neutrophils instead of macrophages - M-CSF induces macropinocytosis by myeloid progenitors, which depends on WNK1 - Macropinocytosis enforces macrophage lineage commitment

immunology↗

bioRxiv2025

An N4-like Caulobacter phage requires host smooth lipopolysaccharide biosynthesis for infection

Caulobacter species are Alphaproteobacteria that commonly inhabit plant-associated and aquatic microbial communities. Although Caulobacter is widespread and has long served as a model for the study of bacterial cell biology, our understanding of the diversity of viruses that infect Caulobacter species is limited. Here, we describe the discovery and characterization of Circe, a freshwater N4-like podophage belonging to the Schitoviridae family that infects C. crescentus. We isolated two variants, CirceC and CirceH, that differ by a single nucleotide resulting in an F91I substitution in Gp063, an uncharacterized protein found in diverse bacteriophages and bacteria. While both Circe variants adsorb to C. crescentus with similar efficiency, they produce morphologically distinct plaques and display different infection dynamics. Through forward genetic selection and genome-wide transposon fitness profiling, we identified C. crescentus genes involved in cell envelope assembly, membrane sphingolipid biosynthesis, and envelope polysaccharide biosynthesis that influence susceptibility to Circe infection. Loss-of-function mutations in a predicted nucleoside diphosphate sugar epimerase and multiple genes required for smooth lipopolysaccharide (S-LPS) biosynthesis and export conferred strong resistance to infection. These results support a model in which S-LPS functions as a receptor for phage Circe. Our study expands the known repertoire of Caulobacter phages and adds to a growing understanding of the role of envelope polysaccharides in bacterial infection by N4-family phages. IMPORTANCEViruses that infect bacteria and archaea, known as phages, shape microbial community structure and function. Phages initiate infection by binding to specific molecules on the surface of host cells. Yet for many microbes, the identities of infecting phages and their corresponding host receptors remain poorly defined. Caulobacter spp. are ecologically important bacteria that can produce a variety of protein surface structures, including pili, a flagellum, and a surface layer (S-layer), all of which have been identified as phage receptors in this genus. We discovered Caulobacter phage Circe and provide evidence that it relies on host smooth lipopolysaccharide to infect C. crescentus. This study broadens understanding of phage-host interactions in Caulobacter and establishes Circe as a new system to investigate the molecular mechanisms by which phages engage with bacterial cells.

microbiology↗

bioRxiv2024

fluxTrAM: Integration of tracer-based metabolomics data into atomically resolved genome-scale metabolic networks for metabolic flux analysis

Quantitative inference of intracellular reaction rates is essential for characterising metabolic phenotypes. The classical experimental method for measuring metabolic fluxes makes use of stable-isotope tracing of metabolites through the metabolic network, followed by mass spectrometry analysis. The most common 13C-based metabolic flux analysis requires multidisciplinary knowledge in analytical chemistry, cell biology, and mathematical modelling, as well as the use of multiple independent tools for handling mass spectrometry data. Besides, flux analysis is usually carried out within a small network to validate a specific biological hypothesis. To overcome interdisciplinary barriers and extend flux interpretation towards a genome-scale level, we developed fluxTrAM, a semi-automated pipeline for processing tracer- based metabolomics data and integrating it with atomically resolved genome-scale metabolic networks to enable flux predictions at genome-scale. fluxTrAM integrates different software packages inside and outside of the COBRA Toolbox v3.4 for the generation of metabolite structure and reaction databases for a genome-scale model, labelled mass spectrometry data processing into standardised mass isotopologue distribution data (MID), and metabolic flux analysis. To demonstrate the utility of this pipeline, we generated 13C-labeled metabolomics data on an in vitro human induced pluripotent stem cell (iPSC)-derived dopaminergic neuronal culture and processed 13C-labeled MID datasets. In parallel, we generated a cheminformatic database of standardised and context-specific metabolite structures, and atom-mapped reactions for a genome-scale dopaminergic neuronal metabolic model. MID data could be exported into established flux inference software for conventional flux inference on a core model scale. It could also be integrated into the atomically resolved metabolic model for flux inference at genome-scale using moiety fluxomics method. The core model flux solution and moiety flux solution were then compared to two additional flux solutions predicted via flux balance analysis and entropic flux balance analysis. The extensive computational flux analysis and comparison helped to better evaluate the obtained flux feasibility of the neuron-specific genome-scale model and suggested new tracer-based metabolomics experiments with novel labeling configurations, such as labelling a moiety within the thymidine metabolite. Overall, fluxTrAM enables the automation of labelled liquid chromatography (LC)-mass spectrometry (MS) data processing into MID datasets and atom mapping for any given genome-scale metabolic model. It contributes to the standardisation and high throughput of metabolic flux analysis at genome- scale.

systems biology↗

bioRxiv2021

Revisiting the role of beta-tubulin in Drosophila development: beta-tubulin60D is not an essential gene, and its novel Pin1 allele has a tissue-specific dominant-negative impact

Diversity in cytoskeleton organization and function may be achieved through alternative tubulin isotypes and by a variety of post-translational modifications. The Drosophila genome contains five different {beta}-tubulin paralogs, which may play an isotype tissue-specific function in vivo. One of these genes, the beta-tubulin60D gene, which is expressed in a tissue-specific manner, was found to be essential for fly viability and fertility. To further understand the role of the beta-tubulin60D gene, we generated new beta-tubulin60D null alleles (beta-tubulin60DM) using the CRISPR/Cas9 system and found that the homozygous flies were viable and fertile. Moreover, using a combination of genetic complementation tests, rescue experiments, and cell biology analyses, we identified Pin1, an unknown dominant mutant with bristle developmental defects, as a dominant-negative allele of beta-tubulin60D. We also found a missense mutation in the Pin1 mutant that results in an amino acid replacement from the highly conserved glutamate at position 75 to lysine (E75K). Analyzing the {beta}-tubulin structure suggests that this E75K alteration destabilizes the alpha-helix structure and may also alter the GTP-Mg2+ complex binding capabilities. Our results revisited the credence that beta-tubulin60D is required for fly viability and revealed for the first time in Drosophila, a novel dominant-negative function of missense beta-tubulin60D mutation in bristle morphogenesis. Author summaryDiversity in cell microtubule cytoskeleton organization and function may be achieved through alternative tubulin isotypes and by a variety of post-translational modifications. The expression pattern of different tubulin isotypes (both and {beta} subunits) can vary according to cell type and stage of development, which contribute significantly to cell-specific MT organization and function. In this study, we revisited the role of one of the beta-tubulin isotopes in Drosophila, namely, beta-tubulin60D. This is the first study where a well molecularly defined protein null allele of {beta}Tub60D was generated and characterized. This well-characterized {beta}Tub60D allele demonstrated unambiguity that {beta}Tub60D is not an essential gene, as was described before. Moreover, we identified Pin1, an unknown dominant mutant with bristle developmental defects, as a dominant-negative allele of beta-tubulin60D. We also found a missense mutation in the Pin1 mutant that results in an amino acid (E75K). Analyzing the {beta}-tubulin structure suggests that this E75K alteration destabilizes the alpha-helix structure and may also alter GTP-Mg2+ complex binding capabilities. Thus, our results also revealed for the first time in Drosophila, a novel dominant-negative function of a missense beta-tubulin60D mutation, which has a tissue-specific function.

developmental biology↗

bioRxiv2026

One-step generation of TCR knock-in mice targeted to the TCRβ locus results in functional mature T lymphocytes

Transgenic mouse models expressing predefined T cell receptors (TCRs) have been instrumental in advancing our understanding of T cell biology. However, these traditional models rely on random genomic insertion of large constructs, require labor-intensive embryo manipulation, and frequently result in aberrant TCR expression and phenotypes. These limitations render TCR transgenic models insufficient to meet the mounting demands for rapid and precise model systems to evaluate TCR specificities. To address these challenges, we developed a streamlined method that combines Adeno-Associated Virus (AAV), coupled with CRISPR/Cas9 genome editing to precisely integrate pre-rearranged TCR/{beta} sequences into the mouse Trb locus, enabling the rapid generation of first-of-its-kind TCR knock-in mice with physiological TCR expression and functional T cell differentiation. This approach bypasses the need for technically advanced embryo manipulation and enables rapid generation of models through a universally optimized AAV vector system, significantly enhancing the versatility and utility of monoclonal TCR mice in basic immunology and preclinical research such as cancer immunotherapy and vaccine development, providing a transformative resource to accelerate discovery and translation across disciplines.

immunology↗

bioRxiv2024

Outside-in engineering of cadherin endocytosis using a conformation strengthening antibody.

P-cadherin, a crucial cell-cell adhesion protein which is overexpressed in numerous malignant cancers, is a popular target for drug delivery antibodies. However, molecular guidelines for engineering antibodies that can be internalized upon binding to P-cadherin are unknown. Here, we use a combination of biophysical, biochemical, and cell biological methods to demonstrate that trapping the cadherin extracellular region in an X-dimer adhesive conformation, triggers cadherin endocytosis via a novel outside-in signaling mechanism. We show that the monoclonal antibody CQY684 traps P-cadherin in an X-dimer conformation and strengthens this adhesive structure. Formation of stable X-dimers results in the dissociation of p120-catenin, a suppressor of cadherin endocytosis, from the X-dimer cytoplasmic region. This increases the turnover of P-cadherin and targets the cadherin-antibody complex to the lysosome. Our results establish a previously unknown outside-in signaling mechanism that provides fundamental insights into how cells regulate adhesion and that can be exploited by anti-cadherin antibodies for intracellular drug delivery.

biophysics↗

bioRxiv2018

OptoGranules reveal the evolution of stress granules to ALS-FTD pathology

Stress granules are non-membranous assemblies of mRNA and protein that form in response to a variety of stressors. Genetic, pathologic, biophysical and cell biological studies have implicated disturbances in the dynamics of membrane-less organelles, such as stress granules, as a pathobiological component of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1-12. This confluence of evidence has inspired the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of stress granules; however, this concept has remained largely untestable in available models of stress granule assembly, which require the confounding variable of exogenous stressors. Here we demonstrate the development and use of a light-inducible stress granule system, termed OptoGranules, which permits discrete, experimental control of the dynamics and material properties of stress granules in living cells in the absence of exogenous stressors. The nucleator in this system is Opto-G3BP1, a light-sensitive chimeric protein assembled from the intrinsically disordered region (IDR) and RNA-binding domain of G3BP1 combined with the light-sensitive oligomerization domain of Arabidopsis thaliana cryptochrome 2 (CRY2) photolyase homology region (PHR). Upon stimulation with blue light, Opto-G3BP1 initiates the rapid assembly of dynamic, cytoplasmic, liquid granules that are composed of canonical stress granule components, including G3BP1, PABP, TIA1, TIAR, eIF4G, eIF3{eta}, ataxin 2, GLE1, TDP-43 and polyadenylated RNA. With this system, we demonstrate that persistent or repetitive assembly of stress granules is cytotoxic and is accompanied by the evolution of stress granules to neuronal cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.

neuroscience↗

bioRxiv2020

Fabrication and electromechanical characterization of free-standing asymmetric membranes

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner where specific properties and processes can be isolated and examined. Here, we describe a new approach to fabricate and characterize planar, free-standing, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic DOPC and anionic DOPG content as compared to previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC/DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison to symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future. O_TEXTBOXSIGNIFICANCE A defining characteristic of the cell membrane is asymmetry in phospholipid composition between the interior and exterior bilayer leaflet. Although several methods have been used to artificially create membranes with asymmetry, there has not been extensive characterization of the impact of asymmetry on membrane material properties. Here, a technique to fabricate free-standing asymmetric membranes is developed which facilitates the visualization and electromechanical characterization of the bilayer. Asymmetry in anionic phospholipid concentration is quantified by measurements of membrane capacitance at varying voltages, which also allows for determination of the membrane compressibility. This method represents an advance in the development of artificial biomembranes by reliably creating phospholipid bilayers with asymmetry and facilitates the interrogation of more complex biological processes in the future. C_TEXTBOX

biophysics↗

bioRxiv2026

Importins recognize the winged-helix fold of ETS transcription factors to mediate nuclear import

Protein trafficking between the cytoplasm and the nucleus is a fundamental process in eukaryotic cell biology. While linear nuclear localization signals (NLSs) are well-characterized, many nuclear proteins lack a predictable NLS. Here, we identify the ETS domain, a DNA-binding winged-helix fold, from ETS family transcription factors as a structure-encoded NLS. We show that ETS domains mediate nuclear import through direct recognition by multiple nuclear transport receptors, including IPO9. Cryo-electron microscopy analysis of the EHF:IPO9 complex reveals that the IPO9 wraps around the ETS domain and engages structural features throughout the winged-helix fold. Biochemical studies demonstrate that the ETS domain DNA-binding helix is critical for importin recognition and for NLS activity in mammalian cells. Comparison of IPO9 bound to EHF and the histone H2A:H2B dimer reveals distinct interaction hotspots, illustrating how IPO9 employs unique combinatorial binding surfaces to accommodate structurally diverse cargos. These findings define a new class of globular NLSs and highlight the adaptability of importins in recognizing distinct protein folds. Significance StatementNuclear import is essential for transcription factor function. However, many nuclear proteins lack recognizable nuclear localization signals (NLSs), leaving their trafficking mechanisms unresolved. Here, we identify the winged-helix DNA-binding domain of ETS transcription factors as a structure-encoded NLS shared across the ETS family of proteins. We show that multiple importins directly interact with this globular domain and define the molecular basis for cargo recognition by determining the cryo-EM structure of an importin bound to an ETS family protein. These studies establish a new class of globular NLSs and shed light on how individual importins can recognize diverse protein folds. We also provide mechanistic insight into nuclear trafficking defects that are caused by disease-linked ETS transcription factor mutations.

biochemistry↗

bioRxiv2020

NetBoxR: Automated Discovery of Biological Process Modules by Network Analysis in R

SummaryLarge-scale sequencing projects, such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), have accumulated a variety of high throughput sequencing and molecular profiling data, but it is still challenging to identify potentially causal genetic mutations in cancer as well as in other diseases in an automated fashion. We developed the NetBoxR package written in the R programming language, that makes use of the NetBox algorithm to identify candidate cancer-related processes. The algorithm makes use of a networkbased approach that combines prior knowledge with a network clustering algorithm, obviating the need for and the limitation of functionally curated gene sets. A key aspect of this approach is its ability to combine multiple data types, such as mutations and copy number alterations, leading to more reliable identification of functional modules. We make the tool available in the Bioconductor R ecosystem for applications in cancer research and cell biology. Availability and implementationThe NetBoxR package is free and open-sourced under the GNU GPL-3 license R package available at https://www.bioconductor.org/packages/release/bioc/html/netboxr.html Contactlium2@mskcc.org; aluna@jimmy.harvard.edu; sander.research@gmail.com Supplementary informationNone

bioinformatics↗

bioRxiv2023

Phosphorylation controls spatial and temporal activities of motor-PRC1 complexes to complete mitosis

During mitosis, spindle architecture alters as chromosomes segregate to daughter cells. The microtubule crosslinker Protein Required for Cytokinesis 1 (PRC1) is essential for spindle stability, chromosome segregation and completion of cytokinesis, but how it recruits motors to the central spindle to coordinate the segregation of chromosomes is unknown. Here, we combine structural and cell biology approaches to show that the human CENP-E motor, which is essential for chromosome capture and alignment by microtubules, binds to PRC1 through a conserved hydrophobic motif. This binding mechanism is also used by Kinesin-4 Kif4A:PRC1. Using in vitro reconstitution, we demonstrate that CENP-E slides antiparallel PRC1-crosslinked microtubules. We find that the regulation of CENP-E -PRC1 interaction is spatially and temporally coupled with relocalization to overlapping microtubules in anaphase. Finally, we demonstrate that the PRC1:microtubule motor interaction is essential in anaphase to control chromosome partitioning, retain central spindle integrity and ensure cytokinesis. Taken together our findings reveal the molecular basis for the cell cycle regulation of motor-PRC1 complexes to couple chromosome segregation and cytokinesis.

biochemistry↗

bioRxiv2018

Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

genetics↗

bioRxiv2015

Learning from heterogeneous data sources: an application in spatial proteomics

Sub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis.\n\nAbbreviations

Bioinformatics↗

bioRxiv2026

Identifying endogenous substrates of the 26S proteasome through site-specific photocrosslinking

The 26S proteasome is the hub for regulated protein turnover in eukaryotic cells. Degradation of proteins by the Ubiquitin-Proteasome System plays critical roles in every aspect of cell biology, such as the regulation of gene transcription, the quality control of translation and protein folding, and protein transport across membranes. While mRNA levels and protein abundances can be readily measured with a robust set of established tools, only a few methodologies exist to identify proteins that are degraded by the proteasome rather than the lysosome as the second major pathway for turnover. Here, we sought to address this by using genetic code expansion to introduce a photo-crosslinkable unnatural amino acid into the yeast 26S proteasome and capture cellular protein substrates as they translocate through the proteasomal ATPase motor. In vitro biochemical experiments confirmed that these modified proteasomes are functional, which allowed us to introduce them into live yeast cells for in vivo crosslinking and the identification of enriched ATP-dependent substrates by mass spectrometry. These experiments revealed a very diverse pool of proteasomal substrates that markedly changed upon cell exposure to endoplasmic reticulum stress. Together, our results represent an exciting avenue for probing the landscape of proteasomal substrates and its changes in response to various cellular conditions and stresses.

biochemistry↗

bioRxiv2022

SARS-CoV-2 Helicase might interfere with cellular nonsense-mediated RNA decay, insights from a bioinformatics study

Unraveling molecular interactions between viral proteins and host cells is key to understanding the pathogenesis of viral diseases. We hypothesized that potential sequence and structural similarities between SARS-CoV2 proteins and proteins of infected cells might influence host cell biology and antiviral defense. Comparing the proteins of SARS-CoV-2 with human and mammalian proteins revealed sequence and structural similarities between viral helicase with human UPF1. The latter is a protein that is involved in nonsense mediated RNA decay (NMD), an mRNA surveillance pathway which also acts as a cellular defense mechanism against viruses. Protein sequence similarities were also observed between viral nsp3 and human Poly ADP-ribose polymerase (PARP) family of proteins. Gene set enrichment analysis on transcriptomic data derived from SARS-CoV-2 positive samples illustrated the enrichment of genes belonging to the NMD pathway compared with control samples. Moreover, comparing transcriptomic data from SARS-CoV2-infected samples with transcriptomic data derived from UPF1 knockout cells demonstrated a significant overlap between datasets. These findings suggest that helicase/UPF1 sequence and structural similarity might have the ability to interfere with the NMD pathway with pathogenic and immunological implications.

bioinformatics↗

bioRxiv2021

A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last [~]15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis. Author SummaryGenomes are in flux, as genes are constantly added and lost throughout evolution. New genes were once thought to arise almost exclusively via the modification or duplication of existing genes. Recently, however, interest has grown in alternative modes of new gene origination, such as de novo evolution from genetic material that previously did not encode proteins. Many de novo genes are expressed in male reproductive tissues, but their significance for fertility is not well understood. We screened likely de novo genes expressed in the Drosophila testis for reproductive roles and found one gene, atlas, essential for male fertility. We leveraged genetic and cell biological experiments to investigate roles for Atlas protein in reproduction and found that it is required during sperm development for proper packaging of DNA in the sperm nucleus. Evolutionary analyses of this gene revealed a complicated history, including loss in some lineages, movement between chromosomes, and fusion with a non-protein-coding gene. Studying both the functions and evolutionary histories of new proteins illustrates how they might evolve critical roles in biological processes despite their relative novelty. Furthermore, the study of atlas identifies an essential genetic player in the fly testis, an important model system for understanding how gametes are produced.

genetics↗

bioRxiv2021

Pre-vaccination Frequency of Circulatory Tfh is associated with Robust Immune Response to TV003 Dengue Vaccine

It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents. Author summaryDengue virus (DENV) is a worldwide threat that causes significant health and economic burden. Currently, there are several challenges in the development of a DENV vaccine including the existence of four different serotypes all; capable of causing disease and antibody dependent enhancement (ADE). For complete protection, a vaccine must be able to generate neutralizing antibodies against all 4 serotypes to avoid ADE. Currently, there is one licensed DENV vaccine, CYD-TDV (DENGVAXIATM). However, this vaccine is only efficacious in protecting against severe disease in DENV seropositive individuals therefore serostatus effect must be further studied for optimal vaccine design. A subset of CD4+ T cells called T-follicular helper (Tfh) cells have been well known to play a major role in aiding high affinity antibody production. Therefore, we chose to look at subsets of Tfh and the cytokines they produce in human blood that can serve as biomarkers for effective vaccine design. We found that DENV sero-positive participants had increased pre-vaccination frequencies of Tfh cells and higher levels of the Tfh related chemokine CXCL13/BLC that plays a role in directing antigen-specific responses. This pre-vaccination Tfh profile and CXCL13/BLC are then correlated positively with the vaccinees ability to produce neutralizing antibody against all four sero-types (breadth of the Response) of DENV, an important goal for all DENV vaccine trials.

immunology↗

bioRxiv2026

Catch-bond engineering surpasses high-affinity maturation for T cell receptor therapies against solid tumors

The sensitivity of T cell receptors (TCRs) has traditionally been attributed to their affinity, a principle that has long underpinned both T cell biology and TCR engineering. Currently, high-affinity maturation remains the predominant strategy employed to enhance TCR sensitivity; however, this approach has been associated with severe off-target toxicities in clinical settings. In this study, using the only FDA-approved TCR-T therapy as a clinical reference, we demonstrate that force-induced catch bonds, rather than static affinity, primarily determine TCR sensitivity and facilitate the development of more effective TCR engineering strategies. Among polar and charged amino acids, histidine was determined to be the most effective residue for pinpointing engineering hotspots. TCRs engineered to form catch bonds exhibited superior performance compared to the FDA-approved TCR subjected to high-affinity maturation, improving the efficacy of TCR-T cell therapies against solid tumors without eliciting off-target toxicity or alloreactivity. Mechanistically, a correlation was observed between TCR sensitivity and the strength of catch bonds, whereas no such relationship was found with affinity. The duration of T cell-tumor cell interactions, immunological synapse formation, and the intensity of subsequent signaling are governed by catch bonds rather than by affinity. Structural and computational investigations have demonstrated that force-induced reconfiguration of the ligand-receptor interface, along with the formation of a novel hydrogen bond network, augments the specificity of interactions between the TCR and antigenic peptides. Furthermore, bispecific T cell engagers utilizing the TCR scaffold also formed catch bonds with tumor antigens, indicating intrinsic mechanosensory properties of the extracellular domains of TCRs and suggesting a novel therapeutic modality based on catch-bond-engineered TCRs.

immunology↗