Cell

Linking cellular phenotype to genotype and gene expression is a central goal in biomedical research. Live-cell imaging captures dynamic cellular phenotypes, whereas single-cell sequencing measures genome-wide molecular states, but pairing these readouts for the same cell remains challenging. Here we present Waldo, a genetically encoded single-cell barcoding strategy that can be read out by both live-cell imaging and single-cell sequencing to connect single cells across both modalities. Waldo barcodes are generated by combinatorial expression of synthetic cell-surface receptors, each detected by a cognate peptide probe coupled either to a fluorescent dye or a DNA barcode. Iterative fluorescent probe staining achieves large barcode diversity, while preserving fluorescence channels for phenotypic reporters. As a proof-of-principle, we use Waldo to link cell division phenotypes after chromosome missegregation to the karyotype and transcriptome of the same cells. Waldo provides a general strategy for connecting imaging phenotypes to genome-wide sequencing readouts in single cells.

Bacteria commit to costly behaviors before they can benefit from them. In Escherichia coli, flagellar synthesis is expensive, yet the genes are expressed in stochastic pulses and only a small minority of pulses result in motility. Despite this noise, we find that lineage-related cells begin swimming at similar times, in groups of two to eight cells. Rare, high-amplitude pulses commit a single cell within one generation, but more commonly, pulses rise slowly and fail to trigger swimming before division. Rather than decaying, their output accumulates as cells inherit hook-basal bodies (HBBs) and continue to build new ones. This structural inheritance acts as a physical integrator, summing ongoing transcriptional activity across generations, so that an incomplete flagellar cascade is carried forward at division rather than lost. HBB accumulation sets when cells swim, and the anti-sigma factor FlgM sets how sharply. By holding late flagellar genes off until several HBBs have accumulated, FlgM makes motility depend steeply on early flagellar gene expression, so that relatives inheriting similar sets of HBBs become motile together. Without FlgM, this sharp dependence is largely lost, and activations of the full cascade do not reliably produce swimming. Together, our results show how the accumulation of a molecular machine integrates fluctuations, and how a checkpoint on its assembly sharpens them into a decision, setting both the speed and heritability of a behavioral trait.

Soil bacteria colonize plants without causing visible disease, yet many suppress host growth. The prevalence and evolutionary reach of this cryptic inhibition are unknown, because phenotyping hundreds of isolates in plants is prohibitively slow. Here we use the unicellular algae Chlamydomonas reinhardtii as a scalable proxy for the green lineage. We screened 148 plant-associated bacteria on Chlamydomonas lawns. Seven of eight Chlamydomonas inhibitors also inhibited the vascular model plant Arabidopsis thaliana. In one of these dual inhibitors, Burkholderia sola MF6, transposon screening and proteomics implicated type VI secretion and tight-adherence pili, and over half the non-inhibitory mutants also failed against Arabidopsis. In the algae, infection triggers rapid deflagellation, then non-lytic regulated cell death. The algal zinc/iron transporter ZIP3 promotes inhibition, whereas the cathepsin X protease CEP12 restrains it, and ZIP3 is epistatic to CEP12. Cryptic inhibition is thus ancient and widespread, and a unicellular algae uncovers it at scale.

T cells persist in the central nervous system (CNS) and can drive both protection and neurological disease. How these cells are organized in humans and what they recognize is largely unknown. Here, we profiled CD8 T cells across anatomically distinct CNS regions, obtained through on-site autopsies and temporal lobe resection surgeries, using single-cell RNA sequencing, paired T cell receptor sequencing, and DNA-barcoded tetramers. Resident memory T cells (TRM) specific for Epstein-Barr virus, cytomegalovirus, influenza A, and SARS-CoV-2 were identified across CNS compartments. Anatomical location was the strongest correlate of TRM cell state, with leptomeningeal cells adopting a cytokine-poised TRM program, whereas brain TRM cells were transcriptionally restrained. Cells of the same clonotype spanned tissues yet adopted local transcriptional states. Viral specificity added another layer of TRM heterogeneity with GZMK/GZMA-expressing EBV-specific populations and interferon-stimulated gene signatures in SARS-CoV-2 and Influenza A-specific cells. The human CNS thus harbors regionally distinct CD8+ TRM shaped by common viral exposures.

Human acrocentric short arms harbor K111, an endogenous retrovirus that became an integral component of nucleolar organizer region (NOR)-associated architecture across all five acrocentric chromosome types. Here we combine complete human and primate genomes, haplotype-resolved population assemblies and parent-offspring trios to reconstruct its evolution and transmission. Predominantly full-length ancestral elements underwent human-specific expansion and remodeling, generating recurrent mosaics and individual-specific multicopy configurations. Population structural variation is concentrated in surrounding satellite landscapes rather than within K111-derived sequences themselves. Trio-resolved genomes reveal direct parental transmission, single-parent remodeling and, crucially, maternal-paternal remodeling in distinct configurations: large reciprocal domains with contrasting parental affinities and de novo sequence exchange within an offspring locus. Raw long reads support both maternal-paternal configurations. K111-derived loci associate with nucleolin-positive compartments. Together, these findings establish K111 as an evolutionarily dynamic component and molecular record of acrocentric NOR architecture, revealing unrecognized remodeling and sequence generation across human generations.

Viral development links gene expression and genome replication to particle formation within a cell. Here we construct virtual viruses in a JCVI-syn3A-based spatial minimal cell. For mycoplasma virus P1, molecular events connect expression and replication to coarse-grained packaging, followed by a spatial reconstruction of membrane opening and particle release. Individual finite-size particles coexist, move across the boundary and undergo partial crossings and returns before complete exit. In contrast, Acholeplasma phage MV-L1 (L1) expresses its genes and accumulates genomes as the host continues to grow and develops a constricted shape. The coexistence of viral activity and continued host growth is qualitatively consistent with the non-lytic biology of group 1 acholeplasmaviruses. These reconstructions extend the virtual cell to the viruses that develop within it, linking molecular activity, particle behavior and host growth.

East Asian populations, representing over 20% of the global population, remain critically underrepresented in human genomic studies, limiting our understanding of population-stratified genetic variation and its implications for health and disease. Here we present the first phase of the Asian Pan-Genome project (APG), comprising 320 nearly complete, fully phased haploid genome assemblies from 160 East Asian individuals. These assemblies achieve unprecedented quality, with an average contig N50 of 144.3 megabase pairs and an average quality value of 64.5. Leveraging these superior assemblies, we reveal previously uncharacterized diversity in human repeatome, including population-stratified patterns in centromere satellites and rDNA arrays. Compared to existing global human genome assemblies, the newly generated genomes supplement 152 million base pairs of novel sequences, 355 gene gains, 18,300 structural variation loci and 26 large euchromatic inversions missing from current human pangenomes. We perform population stratification analyses of structural variations, and further resolve the structural haplotypes of complex genomic regions such as Major Histocompatibility Complex and Survival Motor Neuron loci across global pangenomes, exemplifying tandem-duplicate and inversion-rich complex locus architectures in the human genome, respectively. This resource provides a critical foundation for human genetic studies, especially for East Asian populations, promoting more accurate variant discovery, reducing bias, and ultimately advancing the equity and efficacy of genomic medicine.

Historical crop remains preserved in everyday objects are an underused archive of agrobiodiversity change, yet their degraded DNA resists conventional analysis. We recovered cereal grains and straws used as upholstery padding in a Central European settee dated to about 1870 to 1900. We identified and characterized the specimen by genome skimming. Damage-aware sequencing confirmed authentic historical DNA of modest, late nineteenth-century age. The assembled plastome placed the specimen securely within rye, and a genome-wide SNP panel of rye landraces resolved it inside cultivated Secale cereale subsp. cereale, closest to a Hungarian bred rye and a Slovak upland landrace and remote from wild or eastern lineages. Screening 192 stress-associated loci recovered the structural machinery of resistance, abiotic stress and aluminum tolerance pathways, while heading-date genotyping showed the Weining earliness allele at Hd2R already present. Divergence across 36 domestication sweep regions concentrated at a few improvement loci. Integrating these genomic signals with genebank passport data and an 1891 newspaper record, we interpret the specimen as a tall Central European rye at the threshold between landrace and improved variety. Historical crop material, read this way, preserves both genomes and the traces of human agricultural practice.

Extracellular vesicles (EVs) can deliver RNA and proteins into host cells to manipulate immunity; but how EVs transverse the cell wall is unknown. Using the fungal pathogen Botrytis cinerea that induces cross-kingdom RNA interference in plants, we uncovered that EV-mediated RNA delivery was dependent on cell wall degrading enzymes. Through fluorescence and transmission electron microscopy, and molecular genetic techniques, we demonstrate that EV-associated proteins compromise the plant cell wall, thereby facilitating RNA delivery. Notably, cell wall degrading enzymes are commonly associated with EVs across plant-colonizing bacterial, fungal and oomycete species, indicating a conserved role in EV transport across cell walls. These findings uncover a formerly unknown mechanism by which cell wall degrading enzymes facilitate EVs to transverse the cell wall for cargo delivery in cross-kingdom communication.

Bacteria of the genus Achromatium harbor hundreds of chromosomes that were previously suggested to be genetically diverse. By sequencing multiple regions from individual cells, we demonstrate that chromosomes within a single cell differ in nucleotide and amino acid sequence, reaching levels of divergence well below accepted bacterial species boundaries and below the range associated with homologous recombination. Sequencing of dividing cells further revealed that daughter cells inherit distinct chromosome populations, a mode of inheritance previously associated with sexual reproduction in eukaryotes. Combining subcellular sequencing with high-resolution microscopy, we reconstruct the three-dimensional cellular architecture of Achromatium and show that extensive intracellular heterogeneity arises from the spatial segregation of chromosome populations by the cells internal structure, which limits genome-wide recombination. Our findings establish cellular architecture as a determinant of genome evolution in giant polyploid bacteria and identify spatial genome segregation as a mechanism enabling the maintenance and inheritance of divergent chromosome populations in bacteria.

Many epigenetic regulators play essential roles in animal development, but precisely how they function across different cell types without clear sequence preferences remains a foundational challenge. Here, we employed single-cell RNA sequencing to survey the mutant phenotypes of 30 essential regulatory pathways across over 450 gastrulation stage mouse embryo replicates, including essential factors associated with repressive chromatin states, RNA surveillance/processing, and other genomic roles. We then use this integrated manifold to partition mutant responses into a developmentally-resolved interaction network, revealing a hierarchy of heterochromatin-associated interactions that act orthogonally to Polycomb Repressive Complex-mediated control of embryo patterning. Compared to the embryo proper, we find that the developing placenta and yolk sac can accommodate broad swings in repetitive element expression, a resilience in keeping with the relative evolutionary recency and phenotypic plasticity of these tissues. Dense temporal sampling of Human Silencing Hub (HUSH) mutant embryogenesis further supports the notion that the gastrulating embryo is particularly constrained and depends on multiple layers of insulation, including the need to suppress evolutionarily young genetic elements to stably license highly conserved, phylotypic programs. In combination, our work highlights the power of single cell-resolved developmental genetics to contextualize the roles of epigenetic regulators, which restrict genomic action to support robust cellular identities and embryological forms.

During intracellular infection, host cells adopt different metabolic states that traditional bulk analyses cannot distinguish. Using single-cell spatial metabolomics of human macrophages infected with Legionella pneumophila, we show that bacterial uptake activates host metabolism, whereas the bacterial effectors secreted through the type IV secretion system counteract this response and promote a glycolytic shift. The activity of effectors also generates distinct metabolic states within the infected macrophage population.

Viral-induced DNA damage triggers both eukaryotic and prokaryotic innate immunity. Here, we report that self-DNA ends generated routinely during normal termination of bacterial replication suffice to trigger the P2-encoded OLD antiphage system. Normally, these physiological triggers are cleared by the dual helicase-nuclease activities of the conserved RecBCD enzyme, which acts as an immune safeguard analogous to mammalian TREX1 exonuclease and plant DBR1 debranching enzyme. However, when this clearance fails, the transient self-DNA ends persist and are recognized by ATP-bound OLD dimers via a positively charged channel across their ATPase subunits. ATP hydrolysis then activates OLD to cleave tRNAs, causing cytotoxicity. Single-cell microscopy reveals that dozens of OLD molecules associate with kilobases of adjacent DNA, amplifying the sparse physiological DNA end signal. Our findings reveal how an endogenous byproduct poses a constant autoimmune threat in bacteria, highlighting a universal molecular conflict across kingdoms and the convergent solutions evolved to resolve it.

Mycobacterium tuberculosis (Mtb) secretes antigenic proteins that drive protective T cell responses, yet how Mtb intracellular localization affects the trafficking and spatiotemporal distribution of mycobacterial antigens in macrophages remains elusive. Here, we developed a split fluorescent protein reporter to monitor the dynamics of the TB vaccine lead antigen Ag85B in infected human macrophages. We discovered that Mtb triggers the formation and remodeling of two distinct types of Mtb Antigen Storage Compartment (MASC) that retains Ag85 within macrophage membranes. Using subcellular spatial proteomics, we profiled the composition of the MASC, identifying both the Mtb and macrophage proteins that constitute these compartments. Unexpectedly, MASCs are defined by metabolic compartmentalization and rewiring to lipid metabolism in both the macrophage and Mtb. Thus, Mtb builds via the ESX-1 system a metabolically distinct antigen storage compartment in immune cells, with potential implications for antigen selection and delivery in TB vaccine development.

Pathogenic variants in SCN8A, encoding the voltage-gated sodium channel NaV1.6, cause disease through opposing gain-of-function (GoF) and loss-of-function (LoF) mechanisms, yet the downstream cellular programs that distinguish these directions have not been resolved within a single genetic system. We used an isogenic Scn8a allelic series in which a cis-acting modifier produces a stepwise reduction of NaV1.6 on the N1768D background, isolating GoF, rescued, and LoF states on a shared background, and profiled the hippocampal transcriptome with pathway-level inference. The gain- and loss-of-function extremes engaged mechanistically opposite programs: an active fibro-inflammatory injury cascade in the seizing brain versus suppression of neuronal-signaling and growth programs, including cAMP Response Element-Binding signaling (CREB) in neurons, in the LoF state. The injury core was conserved across genetic backgrounds, appeared only after seizures rather than tracking channel dose, and was durably suppressed in the rescued genotype without erosion after seizure onset. Genetic rescue and the repurposed angiotensin-receptor blocker candesartan converged on the same injury program through distinct routes, and the same program was engaged in an -synuclein model of Parkinson's disease and in human epileptic tissue. These findings show that the two clinical directions of SCN8A disease have distinct tissue-level correlates and call for opposite therapeutic logic, channel or injury-cascade suppression for GoF and restoration of channel output for LoF, and identify a conserved, seizure-driven injury core as a tractable cross-disease target.

Horizontal gene transfer in Acinetobacter baumannii has been attributed to conjugative pili, transduction, and natural transformation. Here, we describe a previously unrecognized type of chromosomal DNA exchange mediated by cell-envelope conduits that establish direct cytoplasmic continuity between neighboring A. baumannii cells. Cryo-electron tomography reveals that these conduits, which were found in multiple clinical isolates, comprise an outer membrane, a peptidoglycan layer, and an inner membrane. The ~65 nm-thick conduits contain two ~2 nm-thick filaments, which super-resolution microscopy identifies as DNA. Interstrain horizontal gene transfer assays coupled with whole-genome sequencing demonstrate the transfer and homologous recombination of chromosomal segments up to 1.1 Mbp, corresponding to as much as 27% of the A. baumannii genome. Together, these findings provide direct structural and functional evidence for a conduit-mediated large-scale chromosomal DNA exchange, thus expanding the known repertoire of horizontal gene transfer strategies that may contribute to the remarkable genomic plasticity of A. baumannii.

Cytokine receptor pairing rules, set by evolution, confine JAK-STAT signaling to a narrow region of a far larger combinatorial space. Of more than 1,200 pairings theoretically possible among the [~]36 JAK-associated human cytokine receptors, only [~]30-40 exist in nature. Using a double-orthogonal platform, we enforced pairings across the full all-by-all receptor matrix and resolved a fine-grained STAT atlas richer than the natural repertoire. Selected non-natural pairings generated emergent T cell states unpredictable from either parental receptor, with pairing orientation encoding signaling specificity. A synthetic IL-21R>IL-2R{beta} pairing, but not its reciprocal, drove a cytotoxic Tc17-like state, whereas natural IL-9R/{gamma}c drove a Tc1 fate despite similar STAT activation, showing that rebalancing quantitative STAT combinatorics can modulate T cell fate. Recombining IL-31R, not expressed in T cells, with STAT-biased receptor partners generated diverse states, several with superior antitumor efficacy. These findings define a non-natural pairing code for engineering synthetic T cell fates.

Many prokaryotes are diderms. They divide using an FtsZ division ring to simultaneously constrict physically coupled inner and outer membranes to form daughter cells with two membranes. Currently, only one archaeon, Ignicoccus hospitalis, is known to have an outer and inner membrane. Here, in exploring Ignicoccus cell division, we show by expansion microscopy that I. hospitalis uses a contractile CdvA-ESCRT-III ring to repeatedly constrict and cut its inner membrane in a way that yields clusters containing multiple cytoplasms within a shared periplasm. The outer bounding membrane then bursts to release the progeny cells, which regain their parental cell architecture via a membrane duplication process. Furthermore, this life cycle appears conserved across Ignicoccus strains, even though the precise timing of events changes. Taken together, these data show that Ignicoccus cells propagate by undergoing dynamic changes in their architecture as they transit between single and multi-cellular states.

Airborne environmental DNA can capture biodiversity across the tree of life, but low sample biomass makes rapid, untargeted detection technically challenging. We combined 45-min air collection, nanopore sequencing and real-time taxonomic analysis in a shotgun metagenomic workflow capable of producing results within 3 hours. Across 77 samples from 13 London sites, including a year of weekly sampling at the Natural History Museum Wildlife Garden, we detected 1,916 species spanning bacteria, fungi, plants and animals. Communities varied spatially and seasonally, shifting from plant dominance in spring to ascomycete dominance in summer and basidiomycete dominance in late autumn and winter. Plant read abundance increased with upwind vegetation, linking airborne signals to surrounding habitat. Detection of catalogued garden plants depended on reference availability, dispersal biology, plant size and proximity to the collector. Together, these findings establish airborne shotgun metagenomics as a platform for rapid, repeated and scalable biodiversity assessment across space and time.

Tracking your position in the environment during navigation requires integrating multiple signals into a spatial representation. This process is thought to occur primarily in the hippocampal formation, but evidence for it has also been observed in other regions. To investigate it across the brain, we recorded from over 20,000 neurons in mice navigating a virtual corridor that decoupled position from correlated signals. Spatial position was encoded in every region, and was more common in neurons tuned to visual landmarks. Most neurons brainwide were also modulated by running speed, likely reflecting arousal, and many by rewards. The hippocampal formation represented spatial position slightly more uniformly than other regions, but less precisely than visual cortex. Spatial position and other navigational signals are thus represented widely across the brain.