Redhai S, Hirschmüller N, Wang T
… +15 more, Jackson T, Peidli S, Valentini E, Bahuguna S, Leible S, Möller S, Ko C, Holzem M, Kharuk S, Bräckow L, Port F, Ibberson D, Le H, Huber W, Boutros M
Intestinal stem cells (ISCs) continuously renew the gut epithelium by producing specialised cell types, yet the mechanisms that couple ISC renewal with lineage commitment remain poorly characterised. Here, we identify a...Intestinal stem cells (ISCs) continuously renew the gut epithelium by producing specialised cell types, yet the mechanisms that couple ISC renewal with lineage commitment remain poorly characterised. Here, we identify a self-limiting transcriptional program, mediated by the zinc-finger transcription factor Chronophage (Cph), that promotes both ISC maintenance and differentiation into enteroendocrine (EE) cells in the Drosophila midgut. Cph expression is transiently induced by the proneural factor scute at the onset of ISC-to-EE specification. Genetic and single-cell transcriptomic approaches revealed that Cph is required to reprogramme ISCs and sustain normal lifespan. Cph binds to genes involved in proliferation and differentiation, and directly represses its own expression. This autoinhibitory feedback safeguards ISCs from accumulating autophagosomes and undergoing cell death, thus preserving ISC function. Our findings uncover a key regulatory mechanism that balances stem cell maintenance and differentiation, highlighting principles relevant to regenerating tissues.
During macroautophagy, the de novo formation of the autophagosome at a membrane contact site (MCS) with the endoplasmic reticulum requires directional lipid flux for the growth of the initial phagophore before its sealin...During macroautophagy, the de novo formation of the autophagosome at a membrane contact site (MCS) with the endoplasmic reticulum requires directional lipid flux for the growth of the initial phagophore before its sealing into an autophagosome and subsequent fusion with the lysosome/vacuole. It remains unclear, however, how the formation of this specialized MCS and the directionality of the lipid flux are controlled. Here, we present the structure of the key lipid transfer protein Atg2 from yeast solved together with its Atg18 binding partner, a phosphatidylinositol-3-phosphate (PtdIns3P) effector, using cryo-electron microscopy. We reveal a new interface in Atg2 that, together with PtdIns3P, is required for Atg18 recruitment and lipid transfer activity. Furthermore, we visualize lipid densities along the internal hydrophobic cavity of Atg2, providing structural evidence that Atg2 cavity is filled with lipids throughout the entire length, even when Atg2 is cytosolic. Finally, molecular dynamics simulations show that the complex generates membrane curvature, efficiently positioning the lipid channel of Atg2 towards the membrane to promote lipid transfer into the elongating phagophore.
Feng X, Zhang W, Guan H
… +21 more, Yang H, Wu N, Chen Y, Mao X, Xiao H, Li M, Xiong H, Li Y, Zhang Z, Liu M, Jia L, Cha S, She Y, Zhang X, Liao H, Sun G, Liu Y, Wang W, Xu Y, Zhang Y, Lu Y
SUMOylation is critical for plant growth and defense, and the specific substrate recognition mediated by SUMO ligases dictates the biological processes in which SUMOylation functions. However, only four SUMO ligases invo...SUMOylation is critical for plant growth and defense, and the specific substrate recognition mediated by SUMO ligases dictates the biological processes in which SUMOylation functions. However, only four SUMO ligases involved in SUMOylation have been reported in plants. Here, we report MPEL1 as a new SUMO ligase in maize, which paradoxically exhibits SUMO ligase activity despite its sequence homology to SUMO-targeted ubiquitin ligases (STUbL). MPEL1 stabilizes the plant-specific mitogen-activated protein kinase 16 (MAPK16) via SUMOylation at Lys526 within C-terminal domain (CTD). MAPK16 directly binds to JAZ20 via its C-terminal domain and phosphorylates JAZ20 at Thr12/Ser13 residues, thereby triggering its proteasomal degradation and activating broad-spectrum resistance against two Fusarium pathogens. These pathogens cause devastating maize ear rot and stalk rot, leading to severe yield losses and serious food safety concerns worldwide. Notably, MAPK16 overexpression and JAZ20 knockout enhance disease resistance without yield penalty, highlighting their potential for crop improvement. These findings expand the plant SUMO ligase family and provide targets for breeding Fusarium-resistant crops.
Elliott L, Yamada M, Kalde M
… +11 more, Hála M, Smertenko A, Rozier F, Evry J, Irani N, Jaillais Y, Hussey PJ, Žárský V, Moore I, Pleskot R, Kirchhelle C
Cytokinesis is a key process in the development of multicellular organisms, through both formative and proliferative divisions. In land plants, successful cytokinesis requires precise targeting of Golgi-derived vesicles...Cytokinesis is a key process in the development of multicellular organisms, through both formative and proliferative divisions. In land plants, successful cytokinesis requires precise targeting of Golgi-derived vesicles to the future cell division plane by the phragmoplast, a cytoskeletal structure that undergoes continuous remodeling. Endomembrane trafficking and phragmoplast remodeling are tightly coupled, indicating the existence of active crosstalk. However, although many molecular regulators of membrane trafficking and cytoskeletal organization are known, it remains poorly understood how these processes are coordinated. Here, we describe a regulatory module consisting of the membrane-associated GTPase RAB-A2a, cytoskeleton-associated Class II Kinesin-12 proteins, and the Fused kinase ortholog TIO. We provide evidence that the interaction between these molecules at the midzone, and differential functions of Class II Kinesin-12 members at the leading and lagging cell plate domains, are essential for cytokinesis initiation and progression in Arabidopsis by simultaneously targeting vesicles to the midzone and coupling phragmoplast remodeling to vesicle delivery.
The cellular and biochemical processes that define the speed at which embryos develop, tissues form, and cells differentiate remain largely unknown. Using the speed of progression of a differentiation front in the develo...The cellular and biochemical processes that define the speed at which embryos develop, tissues form, and cells differentiate remain largely unknown. Using the speed of progression of a differentiation front in the developing Drosophila eye to measure developmental speed, we identified genetic perturbations that slowed the progression of this front. Inhibiting the electron transport chain (ETC), and more generally energy production in mitochondria, resulted in reduced developmental speed. Defective ETC activity led to increased NADH/NAD ratio, whereas ATP levels remained constant due to a compensatory increase in glycolysis. Targeted perturbations showed that the metabolic state of the cells ahead of and/or at the differentiation front determined its speed. Genetic and diet-based perturbations of NAD metabolism indicated that developmental speed was limited by NAD availability. Thus, developmental speed appeared constrained by the cellular redox state and the demand for NAD in the developing Drosophila eye. Our findings therefore show that the NADH/NAD ratio is key to regulating developmental speed and highlight the importance of NAD availability for this regulation in Drosophila.
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 c...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 traditional models insufficient to meet the mounting demands for rapid and precise model systems to evaluate TCR specificities. In this study, we developed a streamlined method that uses adeno-associated virus (AAV) and CRISPR/Cas9-mediated genome editing to precisely integrate pre-rearranged TCRα/β sequences into the mouse TCRβ (Trb) locus, enabling the rapid generation of TCR knock-in mice with physiological TCR expression and functional T-cell differentiation upon antigenic challenge. This approach bypasses the need for screening multiple founders for faithful TCR expression, enhancing the versatility and utility of monoclonal TCR mice in basic immunology and preclinical research, such as in the fields of cancer immunotherapy and vaccine development.
Transposable elements (TEs) are ubiquitous, mobile DNA elements that often exist as multiple copies within host genomes. To persist despite ongoing mutational decay, these genomic parasites must continuously generate new...Transposable elements (TEs) are ubiquitous, mobile DNA elements that often exist as multiple copies within host genomes. To persist despite ongoing mutational decay, these genomic parasites must continuously generate new insertions into the germline genome, a process that risks compromising host reproductive capacity by disrupting coding regions and regulatory sequences and by reshaping chromosomal architecture. Accordingly, hosts have evolved mechanisms to repress TE activity and reduce its fitness costs. In principle, however, such antagonism could lead to lineage-specific TE extinction, which may be suboptimal for hosts, as TEs possess aspects that confer beneficial functions to them. Therefore, hosts not only resist but also tolerate and even exploit the presence of TEs. In parallel, TEs themselves have acquired and deployed unique strategies that promote their own persistence while limiting harm to the host. This review explores how "peaceful" coexistence between hosts and TEs is achieved, focusing on resistance, tolerance, and the associated trade-offs from both host and TE perspectives.
Topoisomerase 1 (TOP1) is essential for relieving DNA supercoils during replication and transcription. However, its transient reaction intermediates (TOP1 cleavage complexes or TOP1-DNA covalent complexes, i.e., TOP1ccs)...Topoisomerase 1 (TOP1) is essential for relieving DNA supercoils during replication and transcription. However, its transient reaction intermediates (TOP1 cleavage complexes or TOP1-DNA covalent complexes, i.e., TOP1ccs) become highly genotoxic when stabilized. While mechanisms that resolve chemotherapy-induced TOP1ccs are well-characterized, how cells prevent their accumulation under physiological conditions for securing genomic stability has remained elusive. Here, we elucidate a novel regulatory pathway in which CHK1-mediated phosphorylation of TOP1 at Serine-320 regulates its religation activity and hence limits steady-state TOP1cc levels during unperturbed cellular metabolism. We further demonstrate a distinct mechanism of TOP1cc stabilization, which escapes recognition by proteasomal and autophagic machineries, while being susceptible to CtIP, SPRTN, and p97-mediated removal. Defective phosphorylation of TOP1 at S320 impairs replication-fork progression, leading to replication- and transcription-associated DSBs, R-loop stabilization, genomic instability, and hypersensitivity to TOP1 poisons. Overall, our study assigns a new function to CHK1 in direct regulation of human TOP1cc dynamics, with critical implications for genomic integrity and combinatorial chemotherapy.
Within cells, across diverse organisms, macromolecular condensation enables spatial and temporal organization of biochemical reactions by organizing proteins and nucleic acids into compositionally distinct membraneless b...Within cells, across diverse organisms, macromolecular condensation enables spatial and temporal organization of biochemical reactions by organizing proteins and nucleic acids into compositionally distinct membraneless biomolecular condensates. In the gut bacterium Bacteroides thetaiotaomicron, condensate formation by the transcription termination factor Rho (BtRho) increases its termination activity and promotes B. thetaiotaomicron fitness in the mammalian gut. Here, we elucidate the molecular mechanism governing carbon starvation-induced BtRho phase separation. We establish that short, specific amino acid sequences within BtRho's intrinsically disordered region (IDR) control BtRho condensation via complex coacervation. The identified sequences participate in RNA and intra-IDR regulatory interactions that drive condensate formation in vitro and in vivo. We also report that the signaling molecule ppGpp is essential for BtRho phase separation in vivo, binds to purified BtRho in an IDR-dependent manner, and promotes RNA-dependent BtRho condensation in vitro. Our findings demonstrate how specific short sequences within an IDR dictate phase separation in response to nutritional cues.
The isolation of single plant cells from complex tissues is prone to selective enrichment and sampling biases, which complicates accurate profiling of the large diversity in cell types. Optimizing methodologies for cell...The isolation of single plant cells from complex tissues is prone to selective enrichment and sampling biases, which complicates accurate profiling of the large diversity in cell types. Optimizing methodologies for cell enrichment and single-cell transcriptomics is therefore critical for single-cell studies addressing plant cell heterogeneity. Here, we systematically compared protoplast enrichment technologies (including conventional and image-based flow cytometry, as well as magnetic cell sorting) and single-cell RNA sequencing (scRNA-seq) platforms (10X Genomics Chromium, BD Rhapsody) using Arabidopsis roots. Image-based flow cytometry offered increased precision due to customizable gating strategies, while magnetic sorting provided faster processing and enhanced representation of cell size heterogeneity. Both scRNA-seq platforms captured root cell heterogeneity and yielded reproducible gene expression profiles, but showed platform-associated differences in cell type composition. Notably, single-nucleotide polymorphism analysis of a mixed ecotype sample revealed that, among cells identified as doublets by computational algorithms, two-thirds were likely to have been misclassified. These insights identify key biases in plant cell purification and scRNA-seq workflows and provide practical guidance for improving data quality across plant species.
Nonsense-mediated RNA decay (NMD) was originally discovered by virtue of its "quality control" function of degrading aberrant mRNAs with premature termination codons (PTCs). NMD was subsequently found to be a highly sele...Nonsense-mediated RNA decay (NMD) was originally discovered by virtue of its "quality control" function of degrading aberrant mRNAs with premature termination codons (PTCs). NMD was subsequently found to be a highly selective and conserved RNA turnover pathway that also degrades subsets of normal mRNAs harboring stop codons in specific contexts. The discovery that many normal mRNAs encoding full-length normal proteins are degraded by NMD has led to a search for biological functions for NMD. In this review, we focus on the evidence for NMD's roles in early embryonic development, nervous system development, spermatogenesis, thymic development, and other developmental processes in mice. NMD also has roles in stem cells, including dictating self-renewal vs. differentiation decisions in embryonic and neural stem cells. We also discuss evidence for NMD's roles in some adult functions, such as circadian rhythm and neuronal activities. Finally, we highlight NMD's causative roles in some human diseases and how therapeutic intervention of this critical pathway can be modeled in mice.
Dimethylsulfoniopropionate (DMSP) is a ubiquitous marine organosulfur compound central to microbial stress responses, chemotaxis, and nutrient cycling. Its catabolism produces dimethylsulfide (DMS), a climate-active gas,...Dimethylsulfoniopropionate (DMSP) is a ubiquitous marine organosulfur compound central to microbial stress responses, chemotaxis, and nutrient cycling. Its catabolism produces dimethylsulfide (DMS), a climate-active gas, and plays a key role in the global sulfur cycle. However, the molecular basis of DMSP import, underpinning its microbial metabolism, remains poorly understood. Here, we identify and characterize the BCCT-family transporter DddT from Psychrobacter sp. D2, a marine gamma-proteobacterium that utilizes DMSP as a carbon source. DddT is essential for DMSP uptake and functions as a Na-coupled symporter driven by the transmembrane sodium gradient. Using cryo-electron microscopy, we determined DddT structures in multiple conformational states, revealing its Na-dependent transport mechanism involving two sodium ions, one coordinated by a previously uncharacterized binding site. Sequence analysis shows that DddT-like proteins with conserved sodium-binding features are widespread in marine bacteria, suggesting this Na-coupled transport mechanism represents a broadly conserved feature of the BCCT family. Our findings provide mechanistic insights into sodium-driven substrate uptake and marine sulfur cycling.
Faithful DNA replication is essential for genome stability, yet replication forks face constant stress. The Bloom syndrome helicase (BLM) safeguards fork integrity, but excessive BLM activity can itself induce replicatio...Faithful DNA replication is essential for genome stability, yet replication forks face constant stress. The Bloom syndrome helicase (BLM) safeguards fork integrity, but excessive BLM activity can itself induce replication stress. We identify SLX4IP as a genome-wide regulator that restrains BLM to maintain replication fork stability. SLX4IP localizes broadly across chromatin with recruitment enhanced under replication stress. Loss of SLX4IP slows replication forks, remodels the replisome, and generates post-replicative single-stranded DNA gaps that are accompanied by elevated nuclear ADP ribose, reflecting compromised replication integrity. These defects are driven by dysregulated BLM activity, establishing SLX4IP as a negative regulator of BLM-dependent replication stress. At ALT telomeres, SLX4IP deficiency triggers ATR signaling, telomere fragility, and accumulation of ALT-associated PML bodies. Here, SLX4IP functions in parallel with FANCM to restrain BLM at ALT telomeres, with co-depletion of SLX4IP and FANCM causing synthetic lethality in ALT-positive cells, a phenotype fully rescued by BLM loss. Together, our results define SLX4IP as a critical genome-wide regulator of replication fork integrity and reveal SLX4IP as a potential vulnerability in ALT-positive cancers.
Cytosolic DNA, derived from cellular damage or microbial infection, functions as a pivotal trigger for the host innate immune responses by activating intracellular DNA-sensing machinery, including the cGAS-STING pathway....Cytosolic DNA, derived from cellular damage or microbial infection, functions as a pivotal trigger for the host innate immune responses by activating intracellular DNA-sensing machinery, including the cGAS-STING pathway. However, whether cytosolic DNA is involved in DNA-sensing pathway-independent biological processes remains largely unknown. Here, we show that cytosolic DNA interacts with UBTF and POLR1A, two essential components of the RNA polymerase I transcription machinery, and sequesters these two proteins in the cytoplasm. This retention decreases nuclear UBTF and POLR1A, inhibits rDNA transcription, suppresses protein synthesis, and curtails cell proliferation. Furthermore, we demonstrate that STING-induced autophagy specifically eliminates cytosolic DNA and restores nuclear UBTF and POLR1A, thereby abolishing the inhibitory effects of cytosolic DNA on rDNA transcription, protein synthesis, and cell proliferation. Thus, our findings uncover a novel role of cytosolic DNA in rDNA transcription, suggesting that cytosolic DNA not only activates immune responses but also interferes with cell metabolism.
p62/SQSTM1 self-assembles with polyubiquitin into liquid-like condensates ("p62 bodies") that function as stress-signaling hubs and selective autophagy cargo. We show that TBK1-dependent phosphorylation at Ser403 acts as...p62/SQSTM1 self-assembles with polyubiquitin into liquid-like condensates ("p62 bodies") that function as stress-signaling hubs and selective autophagy cargo. We show that TBK1-dependent phosphorylation at Ser403 acts as a threshold-dependent modulator of a condensate's physical properties and promotes their rapid autophagic clearance. Phosphorylation within p62 bodies drives a transition from large, fluid droplets to compact, gel-like condensates that efficiently capture LC3-positive isolation membranes and accelerate the autophagic removal of ubiquitinated proteins. PP2A holoenzymes containing PPP2R5A/B/E, recruited via a KEAP1 bridge, counteract TBK1 by dephosphorylating Ser403. Homozygous p62S403E/S403E knock-in embryonic stem cells differentiate into post-mitotic neurons enriched in miniaturized, gel-like p62 bodies. Consistently, phosphorylation-mimetic knock-in mice show similar remodeling of p62 condensates in vivo, demonstrating that this phosphorylation-driven mechanism maintains proteostasis across scales. We propose that Ser403 phosphorylation functions as a molecular switch that couples the material state of p62 condensates to their stability and serves as a central control point for p62-mediated protein degradation.
Methionine restriction has emerged as a promising strategy for extending lifespan and enhancing cancer therapy. LAT4, an amino acid transporter encoded by SLC43A2, is frequently overexpressed in multiple cancers and crit...Methionine restriction has emerged as a promising strategy for extending lifespan and enhancing cancer therapy. LAT4, an amino acid transporter encoded by SLC43A2, is frequently overexpressed in multiple cancers and critically contributes to systemic methionine accumulation. However, the structural basis of LAT4 function remains poorly understood, and no effective inhibitors have been developed to date. In this study, we present high-resolution cryo-electron microscopy structures of LAT4 and the related SLC43A3-encoded purine transporter ENBT1. The phenylalanine-bound structure of LAT4 enables the characterization of the substrate binding pocket. Comparison of the outward-facing ENBT1 and inward-facing LAT4 structures identifies key residues involved in the methionine transport process. Structural analysis of digitonin binding to the central cavity of LAT4 enabled identification of tubeimoside-1 (TBM-1) as a potent inhibitor of LAT4-mediated methionine uptake. We demonstrate that tubeimoside-1 reduces methionine uptake in B16F10 cancer cells. Furthermore, TBM-1 suppresses tumor progression in the MMTV-PyVT mouse model of breast cancer through systemic methionine restriction. Our study provides insights into the LAT4 transport mechanism and identifies tubeimoside-1 as a potent inhibitor of methionine uptake and establishes a foundation for developing LAT4-targeting therapeutics to restrict methionine uptake.
The discovery of antibiotics and their subsequent therapeutic use revolutionized our ability to treat once deadly infectious diseases, and antibiotics have become one of the most commonly prescribed drug classes. Unfortu...The discovery of antibiotics and their subsequent therapeutic use revolutionized our ability to treat once deadly infectious diseases, and antibiotics have become one of the most commonly prescribed drug classes. Unfortunately, these compounds not only target pathogenic strains, but also non-pathogenic bacteria that fulfill important functions for the human host. As such, antibiotic treatment can cause severe collateral damage, resulting in dysbiosis, for example, in the human gut microbiome. Given the immense importance of the gut microbiome for human health, antibiotic-induced dysbiosis can cause a variety of detrimental health outcomes. In addition, antibiotic (over-)use causes selection of antibiotic-resistant strains, and the human gut microbiome has become a major reservoir for resistance determinants that can transfer to pathogenic isolates and cause hard-to-treat infections. In this review, we describe various adverse effects that antibiotic use has on the human gut microbiome, how we can approach this problem experimentally, and discuss pathways to mitigate antibiotic-induced collateral damage.
Metabolic heterogeneity across scales is a key driver of biological and physiological processes from tissues down to single cells. Recent advances in mass spectrometry imaging (MSI) allow researchers to map metabolic gra...Metabolic heterogeneity across scales is a key driver of biological and physiological processes from tissues down to single cells. Recent advances in mass spectrometry imaging (MSI) allow researchers to map metabolic gradients and centres of activity in intact tissues otherwise masked in bulk analyses. This Comment provides a practical guide to spatial metabolomics, discussing technical challenges and their resolution from experimental design to sample preparation and data analysis.
Lysosomes and peroxisomes are essential for cellular homeostasis, yet how their activities are coordinated remains poorly understood. Here, we identify peroxisome-derived ether lipids as key regulators of lysosomal funct...Lysosomes and peroxisomes are essential for cellular homeostasis, yet how their activities are coordinated remains poorly understood. Here, we identify peroxisome-derived ether lipids as key regulators of lysosomal function. A genome-wide CRISPR/Cas9 screen in LYSET-deficient mucolipidosis V cells revealed that disruption of ether lipid synthesis genes or peroxins markedly reduces lysosome accumulation and restores degradative capacity. Genetic or pharmacological inhibition of ether lipid synthesis enhanced lysosomal exocytosis and promoted the clearance of undigested material independently of mannose-6-phosphate trafficking. Conversely, supplementation with the ether lipid precursor hexadecylglycerol increased lysosome abundance, while reducing their degradative capacity. These findings uncover a peroxisome-lysosome metabolic axis, in which ether lipids act as bidirectional regulators of lysosomal number and function independently of the lysosomal master regulator TFEB. Our findings reveal how peroxisome-localized lipid metabolism modulates lysosomal homeostasis, and suggest potential new strategies to combat lysosomal and peroxisomal disorders.
Complex tissue architecture is achieved through multiple rounds of morphological transitions. Here, we analyzed cellular flows and tissue mechanics during avian skin development by employing chicken and transgenic quail...Complex tissue architecture is achieved through multiple rounds of morphological transitions. Here, we analyzed cellular flows and tissue mechanics during avian skin development by employing chicken and transgenic quail skin explant models. We demonstrate how novel cellular flows initiate chemo-mechanical circuits that guide epithelial protrusion, folding, invagination, and spatial cell fate specification. During initial feather bud formation, stiff dermal condensates protrude vertically from the locally softened epithelial sheet. As the bud elongates, it stretches the epithelial cells at the base, thus mechanically activating YAP, which causes the epithelial sheet to fold downward and form a stiff cylindrical wall that invaginates into the skin. This stiff epithelial tongue is essential for the compaction and formation of the tightly packed dermal papillae. These topological transformational events are mechanically interconnected, and the completion of one circuit initiates the next. In contrast, during scale development, the rigid epithelial sheet restricts dermal cell flows, preventing further topological transformation. Based on these findings, we developed a topological transformation model describing how this process enabled the evolution of feather follicles from scales.