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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2021

Cell geometry distinguishes migration-associated heterogeneity in two-dimensional systems

BackgroundIn vitro migration assays are a cornerstone of cell biology and have found extensive utility in research. Over the past decade, several variations of the two-dimensional (2D) migration assay have improved our understanding of this fundamental process. However, the ability of these approaches to capture the functional heterogeneity during migration and their accessibility to inexperienced users has been limited. MethodsWe downloaded published time-lapse 2D cell migration datasets and subjected them to feature extraction with the Fiji software. We used the Analyze Particles tool to extract ten cell geometry features (CGFs), which were grouped into shape, size and position descriptors. Next, we defined the migratory status of cells using the MTrack2 plugin. All data obtained from Fiji were further subjected to rigorous statistical analysis with R version 4.0.2. ResultsWe observed consistent associative trends between size and shape descriptors and validated the robustness of our observations across four independent datasets. We used these descriptors to resolve the functional heterogeneity during migration by identifying and characterizing non-migrators (NM) and migrators (M). Statistical analysis allowed us to identify considerable heterogeneity in the NM subset, that has not been previously reported. Interestingly, differences in 2D-packing appeared to affect CGF trends and heterogeneity of the migratory subsets for the datasets under investigation. ConclusionWe developed an analytical pipeline using open source tools, to identify and morphologically characterize functional migratory subsets from label-free, time-lapse migration data. Our quantitative approach identified a previously unappreciated heterogeneity of non-migratory cells and predicted the influence of 2D-packing on migration.

cancer biology↗

bioRxiv2021

The terminal heme synthetic enzyme, Coproheme Decarboxylase, coordinates heme synthesis and uptake in response to iron in Mycobacteria

Heme is both an essential cofactor and an abundant source of nutritional iron for the human pathogen Mycobacterium tuberculosis (Mtb). While heme is required for Mtb survival and virulence, it is also potentially cytotoxic. Since Mtb has the ability to both make and uptake heme, the de novo synthesis of heme and its acquisition from the host must be balanced in order to mitigate heme toxicity. However, the mechanisms employed by Mtb to regulate heme uptake, synthesis, and bioavailability are poorly understood. By integrating ratiometric heme sensors with mycobacterial genetics, cell biology, and biochemistry, we determined that the terminal heme biosynthetic enzyme, coproheme decarboxylase (ChdC), plays a role in regulating both heme bioavailability and uptake in Mtb. Moreover, we found that Mtb has a preference for scavenging reduced ferrous heme and exhibits a cell surface heme reductase activity that is regulated by ChdC. In Mtb, ChdC expression is down-regulated when iron is limiting, which in-turn increases both heme import and bioavailability. Such a mechanism may serve to protect cells from heme toxicity while trying to meet the nutritional demand for iron. Our results demonstrate that heme synthesis and uptake are tightly integrated in mycobacteria and represent the first example of a heme synthetic enzyme playing a role in controlling heme uptake. Significance StatementHeme is an essential but potentially cytotoxic cofactor and iron source for the pathogen, Mycobacterium tuberculosis (Mtb). To understand how Mtb coordinates heme uptake and synthesis to mitigate heme toxicity, we integrated heme sensors with mycobacterial genetics and biochemical approaches to probe the interplay between heme synthesis and scavenging. We discovered that the terminal heme synthetic enzyme, coproheme decarboxylase (ChdC), negatively regulates heme uptake and utilization in response to iron availability through a mechanism involving control of a ferric heme reductase. During iron limitation, ChdC is downregulated, thereby enhancing exogenous heme reduction, uptake and utilization while simultaneously suppressing heme synthesis, which allows Mtb to avoid heme toxicity. Our results highlight the close coordination between heme synthesis and uptake in mycobacteria. ClassificationBiological sciences : Biochemistry

microbiology↗

bioRxiv2022

intra-single cell sequencing (iSCseq) spotlights transcriptomic and epigenetic heterogeneity inside multinucleated osteoclast

Single-cell RNA-seq (scRNA-seq) has clarified cellular heterogeneity within cell populations. However, scRNA-seq and spatial transcriptomics cannot capture the dynamic transcriptomic changes inside living cells. To decode subcellular gene expression, we developed intra-single cell sequencing (iSCseq), a novel approach that combines confocal imaging, repeatedly picking up cellular components inside living cells, and next-generation sequencing (intra single-cell RNA-seq; iSCseq). iSCseq illustrated the subcellular heterogeneity of gene expression. iSCseq revealed not only multiple differentiation stages embedded in the same cell, but also physical cytoskeletal connections, physiological activity of mitochondria, and intracellular calcium, as confirmed by transcriptomic evidence. Inclusive iSCseq with in vivo scRNA-seq datasets identified new osteoclast subsets in physiological and pathological bones. Network analysis with centrality provided insights into the connection between subcellular components, and clearly divided differentiation and fusion processes in multinucleation. The iSCseq approach has the potential to enhance cell biology at subcellular resolution and identify new therapeutic targets. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/506360v4_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@115c1d8org.highwire.dtl.DTLVardef@1695ad2org.highwire.dtl.DTLVardef@1c9d257org.highwire.dtl.DTLVardef@1107ddb_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefintra-single cell sequencing (iSCseq) enhances single-cell technology by combining live cell imaging, subcellular sampling from living cells and sequencing, offering deeper insights into cell functions and pathology at subcellular resolution through inclusive analysis with scRNA-seq and advanced centrality-focused network analysis. HighlightsO_LIintra-single cell sequencing (iSCseq) clarifies subcellular heterogeneity C_LIO_LIiSCseq connects morphological and physiological features with transcriptome C_LIO_LIInclusive iSCseq unveils osteoclast subsets in physiological and pathological bones C_LIO_LILinkage at subcellular resolution reveals key players in characteristic fusion C_LI

genomics↗

bioRxiv2026

3D Human Neuromuscular Architecture Encoded by Compliant Patterned Hydrogel Scaffolds

Engineered human neuromuscular models are limited by a trade-off between biological fidelity and architectural control: self-assembled organoids organize stochastically, while engineered 3D tissues impose anisotropy through passive tension at the edges without internal guidance. Here we encode architectural guidance into human neuromuscular tissues by coupling a three-dimensional, cell-laden biomatrix to a xolographically printed hydrogel substrate featuring sub-millimeter surface topography. We demonstrate that grooved substrates increase human primary myotube alignment and maturation in the overlying 3D tissue, while guiding directional axon extension from human iPSC-derived motor neuron spheroids. We show that combining both cell types along a shared alignment axis establishes functional neuromuscular connectivity, evidenced by acetylcholine receptor clustering, glutamate-evoked contraction and its blockade by d-tubocurarine (curare), and yields innervation coverage of centimeter scale tissues. Overall, this platform combines compliant substrates, mesoscale tissue architectural guidance, and human-relevant cell biology in a single untethered construct for studies of neuromuscular tissue development and function.

bioengineering↗

bioRxiv2023

Amphiphiles formed from synthetic DNA-nanomotifs mimic the dispersal of transcriptional clusters in the cell nucleus

Stem cells exhibit prominent clusters controlling the transcription of genes into RNA. These clusters form by a phase-separation mechanism, and their size and shape are controlled via an amphiphilic effect of transcribed genes. Here, we construct amphiphile-nanomotifs purely from DNA, and achieve similar size and shape control for phase-separated droplets formed from fully synthetic, self-interacting DNA-nanomotifs. Low amphiphile concentrations induce rounding of droplets, followed by splitting and, ultimately, full dispersal at higher concentrations. Super-resolution microscopy data obtained from zebrafish embryo stem cells reveal a comparable transition for transcriptional clusters with increasing transcription levels. Brownian dynamics and lattice simulations further confirm that addition of amphiphilic particles is sufficient to explain the observed changes in shape and size. Our work reproduces key aspects of the complex organization of transcription in biological cells using relatively simple, DNA sequence-programmable nanostructures, opening novel ways to control mesoscopic organization of synthetic nanomaterials. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/525851v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a8cc4borg.highwire.dtl.DTLVardef@fc6651org.highwire.dtl.DTLVardef@a17c74org.highwire.dtl.DTLVardef@1f1cf14_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Coupling metastasis to pH sensing G Protein-coupled receptor-68 through first in class inhibitor identified in an in vivo chemical genetic screen

An acidic milieu is a hallmark of the glycolytic metabolism that occurs in cancerous cells. The acidic environment is known to promote cancer progression, but the underlying signaling and cell biological underpinnings of these phenomena are not well understood. Here, we describe ogremorphin, a first-in-class small-molecule inhibitor of GPR68, an extracellular proton-sensing and mechanosensing G protein-coupled receptor. Ogremorphin was discovered in a chemical genetic zebrafish screen for its ability to perturb neural crest development, which shares basic cell behaviors of migration and invasion with cancer metastasis. Ogremorphin also inhibited migration and invasive behavior of neural crest-derived human melanoma cells in vitro. Furthermore, in phenome-wide association studies (PheWAS), we identified an aberrantly activated variant of GPR68, which is associated with cancer metastasis in vivo and promotes invasive phenotypes of cancer cells in vitro. Thus, extracellular proton-sensing GPR68 signaling promotes cell migration and invasion during embryonic development and may do likewise in cancer progression.

cancer biology↗

bioRxiv2026

A broad-host-range Rhizobium rhizogenes strain enables transient expression across diverse crops and establishes functional assays in faba bean

Agrobacterium-mediated transient expression has revolutionized plant research, enabling numerous landmark discoveries across diverse areas of plant biology. Yet this powerful approach remains largely confined to solanaceous species, leaving most economically important crop families without a comparable rapid assay platform. Here, we show that an engineered Rhizobium rhizogenes strain, AS109, mediates efficient transient expression across diverse dicot species spanning multiple taxonomic families, consistently outperforming commonly used laboratory agrobacterial strains. Leveraging the broad host range of AS109, we establish a suite of functional assays in faba bean (Vicia faba), including protein localisation, RNA interference-mediated gene silencing, cell-surface elicitor recognition screens, nucleotide-binding leucine-rich repeat receptor (NLR) activation, and infection cell biology at the host-pathogen interface. We further demonstrate that both singleton NLRs and sensor-helper NLR pairs from Solanaceae retain effector recognition and cell death activity when transferred into faba bean, establishing a rapid platform for evaluating cross-family transferability of disease-resistance genes. AS109 thus provides an accessible and versatile chassis for functional genomics in non-model crops, bridging the widening gap between hypothesis generation and experimental validation across diverse plant species.

plant biology↗

bioRxiv2020

Cytokinin inhibits fungal development and virulence by targeting cellular trafficking

Cytokinin (CK) is an important plant developmental regulator, having activities in many aspects of plant life and its response to the environment. CKs are involved in diverse processes in the plant, including stem-cell maintenance, vascular differentiation, growth and branching of roots and shoots, leaf senescence, nutrient balance and stress tolerance. In some cases, phytopathogens secrete CKs. It has been suggested that to achieve pathogenesis in the host, CK-secreting biotrophs manipulate CK signaling to regulate the host cell cycle and nutrient allocation. CK is known to induce host plant resistance to several classes of phytopathogens from a handful of works, with induced host immunity via salicylic acid signaling suggested to be the prevalent mechanism for this host resistance. Here, we show that CK directly inhibits the growth, development, and virulence of fungal phytopathogens. Focusing on Botrytis cinerea (Bc), we demonstrate that various aspects of fungal development can be reversibly inhibited by CK. We also found that CK affects both budding and fission yeast in a similar manner. Investigating the mechanism by which CK influences fungal development, we conducted RNA-NGS on mock and CK treated B. cinerea samples, finding that CK inhibits the cell cycle, cytoskeleton, and endocytosis. Cell biology experiments demonstrated that CK affects cytoskeleton structure and cellular trafficking in Bc, lowering endocytic rates and endomembrane compartment sizes, likely leading to reduced growth rates and arrested developmental programs. Mutant analyses in yeast confirmed that the endocytic pathway is altered by CK. Our work uncovers a remarkably conserved role for a plant growth hormone in fungal biology, suggesting that pathogen-host interactions resulted in fascinating molecular adaptations on fundamental processes in eukaryotic biology. ImportanceCytokinins (CKs), important plant growth/ developmental hormones, have previously been associated with host disease resistance. Here, we demonstrate that CK directly inhibits the growth, development, and virulence of B. cinerea (Bc) and many additional phytopathogenic fungi. Molecular and cellular analyses revealed that CK is not toxic to Bc, but rather, Bc likely recognizes CK and responds to it, resulting in cell cycle and individual cell growth retardation, via downregulation of cytoskeletal components and endocytic trafficking. Mutant analyses in yeast confirmed that the endocytic pathway is a CK target. Our work demonstrates a conserved role for CK in yeast and fungal biology, suggesting that suggesting that pathogen-host interactions may cause molecular adaptations on fundamental processes in eukaryotic biology.

microbiology↗

bioRxiv2019

Dynamics of chromosome replication and its relationship to predatory attack lifestyles in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a small Gram-negative, an obligate predatory bacterium that is largely found in wet, aerobic environments (i.e. soil). This bacterium attacks and invades other Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle of B. bacteriovorus consists of two phases: a free-living non-replicative attack phase wherein the predatory bacterium searches for its prey, and a reproductive phase, in which B. bacteriovorus degrades a hosts macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of chromosome replication in B. bacteriovorus. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of B. bacteriovorus. Our results confirm that in B. bacteriovorus chromosome replication fires only during the reproductive phase, and show for the first time that this predatory bacterium exhibits a novel spatiotemporal arrangement of chromosome replication. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. This chromosome replication is not coincident with the predator-cell division, and it terminates shortly before synchronous predator-filament septation occurs. In addition, we demonstrate that if this lifecycle fails in some cells of B. bacteriovorus, they can instead use two prey cells sequentially to complete their life cycle.\n\nImportanceNew strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium, Bdellovibrio bacteriovorus, which kills other bacteria including pathogens, is considered promising for bacterial infections. The B. bacteriovorus life cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the hosts macromolecules and reuses them for its own growth. To understand the use of B. bacteriovorus as a living antibiotic, it is first necessary to dissect its life cycle including chromosome replication. Here, we present for the first time a real-time investigation into subcellular localization of chromosome replication in a single cells of B. bacteriovorus. This process initiates at the invasion pole of B. bacteriovorus and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of B. bacteriovorus require two prey cells sequentially to complete their life cycle.

microbiology↗

bioRxiv2019

In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here we present a comprehensive phosphoproteomic dataset for budding yeast, comprised of over 30,000 high confidence phosphorylation sites identified by mass spectrometry. This single dataset nearly doubles the size of the known phosphoproteome in budding yeast and defines a set of cell cycle-regulated phosphorylation events. With the goal of enhancing the identification of functional phosphorylation events, we performed computational positioning of phosphorylation sites on available 3D protein structures and systematically identified events predicted to regulate protein complex architecture. Results reveal a large number of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic \"clashes\" predicted to disrupt the interaction. Phosphorylation site mutants experimentally validate our predictions and support a role for phosphorylation in negatively regulating protein-protein interactions. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

biochemistry↗

bioRxiv2019

Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine

Selenoproteins contain the amino acid selenocysteine and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a non-canonical translation mechanism requiring suppression of the UGA stop codon, and a selenocysteine insertion sequence (SECIS) element in the 3 untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element, and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged selenocysteine (DMNB-Sec) at the UAG amber stop codon. Recombinantly expressed selenoproteins can be photoactivated in living cells with spatial and temporal control. Using this approach, the native selenoprotein methionine-R-sulfoxide reductase 1 is generated and activated in mammalian cells. The ability to site-specifically introduce selenocysteine directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

biochemistry↗

bioRxiv2026

MinJ is a conserved nine-pass transmembrane protein that contains a putative transmembrane β-sheet

The Min system disassembles FtsZ-rings after septation in Bacillus subtilis and is localized to the nascent division plane and cell poles by the protein MinJ. The N-terminal region of MinJ contains transmembrane segments while the C-terminal region of MinJ contains a PDZ domain but its topology and functional domains are poorly understood. Here we empirically test MinJ topology based on a variety of transmembrane prediction models and find that the data is most consistent with Alphafold3, which predicts a 9-pass transmembrane protein with an external N-terminus and internal C-terminus. Deletion analysis indicates that all regions of the protein tested are required for function but deletion of the PDZ domain alone preserves polar localization and interaction with both MinD and DivIVA. Moreover, Alphafold predicts that transmembrane segments 6 and 7 comprise staves of an unusual transmembrane {beta}-sheet and deletion of the putative {beta}-sheet in the absence of MinD results in a minicell frequency that exceeds mutation of MinD alone. Bioinformatic analysis indicates that MinJ is highly conserved within Firmicutes and is co-conserved with MinD and DivIVA with which it interacts. Our data clarify the structure of MinJ and support models in which MinJ has functions in addition to restricting the activity of the Min system. IMPORTANCEFaithful positioning of the bacterial division site is important for cell growth and is coordinated by the conserved Min system. Although the Min system of Bacillus subtilis has been extensively studied, MinJ, the membrane protein that links the division inhibitor MinCD to the polar determinant DivIVA, remains the least well-understood. Here we experimentally define the membrane topology of MinJ and show that our data are most consistent with a nine-pass transmembrane architecture predicted by AlphaFold3. We further provide genetic, cell biological, and evolutionary evidence supporting that two of the staves form a highly conserved putative transmembrane {beta}-sheet, a structure normally excluded from the plasma membrane. Our findings refine MinJ structural organization and provide a framework for understanding its conserved functions in bacterial cell division.

microbiology↗

bioRxiv2023

Development and utilization of new O2-independent bioreporters

Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells and organisms, as well as gene regulation. GFP and all its derivatives are now standard tools in cell biology, immunology, molecular biology and microbiology laboratories around the world. A common feature of these proteins is their O2-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and in cellulo characterization of genetic circuits encoding the O2-independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7 based constructions can be used to produce fluorescing cells of V. diazotrophicus, a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world. ImportanceFluorescent proteins are used since decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. GFP and all its derivatives share a common feature: they rely on the presence of O2 for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O2-independant Flavin-Binding Fluorescent Protein. We demonstrated that Escherichia coli cells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated that Vibrio diazotrophicus NS1, a facultative anaerobe, is fluorescent in the absence of O2 when KOFP-7 is produced. Altogether, the development of new genetic circuits allowing O2-independent fluorescence will open new perspective to study anaerobic processes.

molecular biology↗

bioRxiv2021

Comprehensive Benchmarking of CITE-seq versus DOGMA-seq Single Cell Multimodal Omics

The recently developed transcription, epitopes, and chromatin accessibility by sequencing (TEA-seq) and similar DOGMA-seq single-cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but independent optimization, benchmarking and evaluation are lacking. We explored the utility, pros and cons of DOGMA-seq compared to the bimodal cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) assay in activated and stimulated human peripheral blood T cells. We identified an optimal incubation time and concentration of digitonin (DIG) for cell permeabilization and found that single-cell trimodal omics measurements after DIG permeabilization were generally better than after an alternative "low-loss lysis" (LLL) permeabilization condition. Next, we found that DOGMA-seq with optimized DIG permeabilization and its ATAC library provides more information, even though its mRNA and cell surface protein antibody-derived tag (ADT) libraries have slightly inferior quality, compared to CITE-seq. Finally, we recognized the additional value of DOGMA-seq for studying lineage-specific T helper cells.

genomics↗

bioRxiv2023

Automatic Detection of Cell-cycle Stages using Recurrent Neural Networks

Mitosis is the process by which eukaryotic cells divide to produce two similar daughter cells with identical genetic material. Research into the process of mitosis is therefore of critical importance both for the basic understanding of cell biology and for the clinical approach to manifold pathologies resulting from its malfunctioning, including cancer. In this paper, we propose an approach to study mitotic progression automatically using deep learning. We used neural networks to predict different mitosis stages. We extracted video sequences of cells undergoing division and trained a Recurrent Neural Network (RNN) to extract image features. The use of RNN enabled better extraction of features. The RNN-based approach gave better performance compared to classifier based feature extraction methods which do not use time information. Evaluation of precision, recall, and F-score indicates the superiority of the proposed model compared to the baseline. To study the loss in performance due to confusion between adjacent classes, we plotted the confusion matrix as well, to quantify the amount of misclassification. In addition, we visualized the feature space to understand why RNNs are better at classifying the mitosis stages than other classifier models, which indicated the formation of strong clusters for the different classes, clearly confirming the advantage of the proposed RNN-based approach.

bioinformatics↗

bioRxiv2023

Trifunctional sphinganine: a new tool to dissect sphingolipid function

Functions of the sphingolipids sphingosine and sphinganine in cells are not well established. While some signaling roles for sphingosine have been elucidated, the closely related sphinganine has been described only insofar as it does not elicit many of the same signaling responses. The underlying mechanisms behind the cell biological differences between these lipids are not well understood. Here, we prepared multifunctionalized derivatives of the two lipid species that only differ in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distribution within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid-protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies on the functions of sphingosine and sphinganine.

biochemistry↗

bioRxiv2025

Generating cognate epitope sequences of T-cell receptors with a generative transformer

Single-cell TCR sequencing enables high-resolution analysis of T Cell Receptor (TCR) diversity and clonality, offering valuable insights into immune responses and disease mechanisms. However, identifying cognate epitopes for individual TCRs requires complex and costly functional assays. We address this challenge with EpitopeGen, a largescale transformer model based on the GPT-2 architecture that generates potential cognate epitope sequences directly from TCR sequences. To overcome the scarcity of TCR-epitope binding pairs ({approx} 100, 000), EpitopeGen uses a semi-supervised learning method, termed BINDSEARCH, which searches over 70 billion potential pairs and incorporates high binding affinity predictions as pseudo-labels. To incorporate CD8+ T cell biology into the model as an inductive bias, EpitopeGen employs a novel data balancing method, termed Antigen Category Filter, that carefully controls antigen category ratios in its training dataset. EpitopeGen significantly outperforms baseline approaches, generating epitopes with high binding affinity, diversity, naturalness, and biophysical stability. Notably, the epitopes generated by EpitopeGen follow biologically plausible antigen category distributions, a crucial feature not achieved by other models. Using EpitopeGen, we directly identify subsets of clonally expanded tumorinfiltrating lymphocytes that recognize tumor-associated antigens, exhibiting elevated cytotoxicity and reduced exhaustion markers. From COVID-19 patients, EpitopeGen detects T cells that recognize COVID-19 spike proteins and non-structural proteins with distinct transcriptomic characteristics. In conclusion, EpitopeGen represents the first computational method that enables direct inference of antigen recognition profiles of CD8+ T cells from plain TCR repertoires.

immunology↗

bioRxiv2025

Zinc deficiency induces spatially distinct responses in roots and impacts ZIP12-dependent zinc homeostasis in Arabidopsis

How zinc (Zn) deficiency shapes root development remains unclear, with conflicting reports on its effect on primary root growth in Arabidopsis thaliana (Arabidopsis). The impact of Zn shortage on the root apical meristem (RAM) in particular has not been systematically explored. Using an integrative approach combining cell biology, transcriptomics, and ionomics, we dissected how Zn deficiency alters root zonation and function. We showed that Zn deficiency triggers a striking reorganization of the root tip (RT): the RAM size is reduced, yet meristematic activity and local Zn levels are preserved. This is accompanied by promoted cell elongation and differentiation. Transcriptome profiling revealed a distinct Zn deficiency response in the RAM-enriched RT compared to mature root tissues, with ZIP12 emerging as the most strongly induced gene in the RT. Functional analysis of zip12 mutants uncovered major defects in root growth, RAM structure, expression of Zn-responsive genes, and metal partitioning. Our work unveiled a new layer of root developmental plasticity under Zn deficiency and identified ZIP12 as a central player in maintaining Zn homeostasis and root meristem function in Arabidopsis. These findings provide a framework to better understand how plants adapt root growth to fluctuating micronutrient availability.

plant biology↗