Phytohormone auxin and its directional transport mediate much of the remarkably plastic development of higher plants. Positive feedback between auxin signaling and transport is a prerequisite for (1) self-organizing processes, including vascular tissue formation, and (2) directional growth responses such as gravitropism. Here, we identify a mechanism by which auxin signaling directly targets PIN auxin transporters. Via the cell-surface AUXIN-BINDING PROTEIN1 (ABP1)-TRANSMEMBRANE KINASE 1 (TMK1) receptor module, auxin rapidly induces phosphorylation and thus stabilization of PIN2. Following gravistimulation, initial auxin asymmetry activates autophosphorylation of the TMK1 kinase. This induces TMK1 interaction with and phosphorylation of PIN2, stabilizing PIN2 at the lower root side, thus reinforcing asymmetric auxin flow for root bending. Upstream of TMK1 in this regulation, ABP1 acts redundantly with the root-expressed ABP1-LIKE 3 (ABL3) auxin receptor. Such positive feedback between cell-surface auxin signaling and PIN-mediated polar auxin transport is fundamental for robust root gravitropism and presumably for other self-organizing developmental phenomena.

Epithelial-mesenchymal transition (EMT) is a fundamental mechanism involved in the morphogenesis of metazoans. Through this evolutionarily conserved multi-stage process, cells acquire quasi-epithelial to multiple intermediate morphologies with epithelial and mesenchymal attributes, rarely reaching a complete mesenchymal phenotype. Complex evolutionary-conserved morphogenetic movements in gastrulation are described extensively, as they exemplify the extent of epithelial cell plasticity in the animal kingdom. Nonetheless, a single-gene knockout can modify the mode of gastrulation while achieving the same body plan. Numerous interconnected mechanisms drive different degrees of EMT, including surface receptor signaling, metabolism, and epigenetics. EMT is reactivated in adult tissues during repair and disease, particularly in cancer initiation, progression to metastasis, and refractoriness to treatment. EMT also contributes to dormancy and drug tolerance, leading to minimal residual disease at the origin of recurrences. Multiple EMT states coexist in tumors, creating a dynamic ecosystem for generating an inflammatory microenvironment, stemness, invasion, and metastasis. This review provides an in-depth description of these aspects along with recent controversies and offers new opportunities to further explore the multiple functions of EMT. Examining the potential attributes of EMT in tissue repair, fibrosis, and cancer progression can provide new opportunities for therapeutic intervention.

Alzheimer's disease (AD) has long resisted effective treatments due to its pathological heterogeneity and cell-type-specific regulatory changes. In this issue of Cell, Li et al. leverage single-cell RNA sequencing and drug repurposing to propose a promising combination therapy, validated through real-world evidence and mouse models, that targets multiple AD-relevant cell types.

After millennia of domestication, dissemination, and improvement, soybean has evolved into a globally significant leguminous crop. Addressing how soybeans adapt to diverse planting environments and breeding objectives will facilitate future breeding advancements. Here, we systematically investigated the genes under selection of 8,105 soybean accessions underlying domestication, dissemination, and improvement. The analyses revealed that black soybeans serve as a critical domestication intermediate, and soybean domestication traits were selected in a stepwise manner. Comparisons across accessions from diverse geographical areas and historical eras identified numerous selected genes that have contributed to trait enhancement and environmental adaptation during the global dissemination and unveiled a temporal shift of breeding priorities in soybean improvement in China. To highlight the allele utilization among soybean varieties, we constructed a variation map and quantitative trait nucleotide (QTN) library. Our findings provide valuable insights and serve as a critical resource for understanding soybean domestication and informing breeding strategies.

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution, hindering unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here, we develop reverse-padlock amplicon-encoding fluorescence in situ hybridization (RAEFISH), an image-based spatial transcriptomics method with whole-genome coverage and single-molecule resolution in intact tissues. We demonstrate the spatial profiling of transcripts from 23,000 human or 22,000 mouse genes in single cells and tissue sections. Our analyses reveal transcript-specific subcellular localization, cell-type-specific and cell-type-invariant zonation-dependent transcriptomes, and gene programs underlying preferential cell-cell interactions. Finally, we further develop our technology for the direct spatial readout of guide RNAs (gRNAs) in an image-based, high-content CRISPR screen. Overall, these developments offer a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNAs in many biomedical contexts.

Mycobacterium tuberculosis (Mtb) has co-evolved with humans for thousands of years and is characterized by variation in virulence, transmissibility, and disease phenotypes. To identify bacterial contributors to phenotypic diversity, we developed new RNA sequencing (RNA-seq) and phylogenomic tools to capture hundreds of Mtb isolate transcriptomes, link transcriptional and genetic variation, and find associations between variants and epidemiologic traits. Across 274 Mtb clinical isolates, we uncovered unexpected diversity in virulence gene expression, which we linked to known and unknown regulators. Surprisingly, we found that many isolates harbor variants associated with decreased expression of EsxA (Esat6) and EsxB (Cfp10), which are virulence effectors, dominant T cell antigens, and immunodiagnostic targets. Across >55,000 isolates, these variants associate with increased transmissibility, especially in drug-resistant Mtb strains. Our data suggest expression of Mtb virulence genes is evolving in response to drug-linked pressure, raising concerns about use of these targets in immunodiagnostics and next-generation vaccines.

Transposon-encoded TnpB nucleases gave rise to type V CRISPR-Cas12 effectors through multiple independent domestication events. These systems use different RNA molecules as guides for DNA targeting: transposon-derived right-end RNAs (reRNAs or omega RNAs) for TnpB and CRISPR RNAs for type V CRISPR-Cas systems. However, the molecular mechanisms bridging transposon activity and CRISPR immunity remain unclear. We identify TranCs (transposon-CRISPR intermediates) derived from distinct IS605- or IS607-TnpB lineages. TranCs utilize both CRISPR RNAs and reRNAs to direct DNA cleavage. The cryoelectron microscopy (cryo-EM) structure of LaTranC from Lawsonibacter sp. closely resembles that of the ISDra2 TnpB complex; however, unlike a single-molecule reRNA, the LaTranC guide RNA is functionally split into a tracrRNA and crRNA. An engineered RNA split of ISDra2 TnpB enabled activity with a CRISPR array. These findings indicate that functional RNA splitting was the primary molecular event driving the emergence of diverse type V CRISPR-Cas systems from transposons.

Understanding cell diversification from a common genome in metazoans requires single-cell transcriptional analysis. We introduce single-cell full-length EU-labeled nascent RNA sequencing (scFLUENT-seq), a single-cell nascent RNA sequencing method using brief 10-min metabolic labeling to capture genome-wide transcription. Surprisingly, individual cells-from splenic lymphocytes to pluripotent stem cells-transcribe only ∼0.02%-3.1% of the genome, versus >80% in bulk, revealing limited genome engagement and profound cell-type and cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is pervasive and stochastic. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription involves both gene readthrough and independent initiation, while distal intergenic transcription is largely independent of neighboring genes and correlates with increased transcriptional diversity, a hallmark of cellular plasticity. Although global RNA synthesis and turnover are coupled in bulk, individual mRNA transcription and decay are poorly coordinated in single cells, suggesting noise-buffering mechanisms. Overall, scFLUENT-seq uncovers complex coding and noncoding transcriptional dynamics that underlie single-cell heterogeneity and state transitions.

The molecular mechanisms underlying the pathogenesis of Alzheimer's disease (AD), the most common form of dementia, remain poorly understood. Proteomics offers a crucial approach to elucidating AD pathogenesis, as alterations in protein expression are more directly linked to phenotypic outcomes than changes at the genetic or transcriptomic level. In this study, we develop multiscale proteomic network models for AD by integrating large-scale matched proteomic and genetic data from brain regions vulnerable to the disease. These models reveal detailed protein interaction structures and identify putative key driver proteins (KDPs) involved in AD progression. Notably, the network analysis uncovers an AD-associated subnetwork that captures glia-neuron interactions. AHNAK, a top KDP in this glia-neuron network, is experimentally validated in human induced pluripotent stem cell (iPSC)-based models of AD. This systematic identification of dysregulated protein regulatory networks and KDPs lays down a foundation for developing innovative therapeutic strategies for AD.

Bispecific antibodies and chimeric antigen receptor T cells are some of the most potent cancer immunotherapeutics in clinical use, yet most cancers remain poorly targetable. High-affinity antibodies required to maximize killing detect low antigen expression in normal tissue, risking "on-target, off-cancer" toxicity. This compels identification of cancer-restricted cell-surface protein antigens, which are rare. Tumor-associated carbohydrate antigens (TACAs) are the most abundant and widespread cancer antigens known but are poorly targetable by antibodies. Here, we describe glycan-dependent T cell recruiter (GlyTR) pan-cancer immunotherapeutics that utilize high-avidity "velcro-like" lectin binding to kill cells with high but not low TACA expression. GlyTR1 and GlyTR2 bind immunosuppressive β1,6GlcNAc-branched N-glycans or multiple TACAs (Tn, sialyl-Tn, LacDiNAc, and GD2), respectively, overcome immunosuppressive mechanisms in the tumor microenvironment and trigger target-density-dependent T cell-mediated pan-cancer killing, yet they lack toxicity in mice with human-like TACA expression. Density-dependent lectin binding to TACAs provides highly potent and safe pan-cancer immunotherapeutics.

Primary cilia are critical organelles found on most human cells. Their dysfunction is linked to hereditary ciliopathies with a wide phenotypic spectrum. Despite their significance, the specific roles of cilia in different cell types remain poorly understood due to limitations in analyzing ciliary protein composition. We employed antibody-based spatial proteomics to expand the Human Protein Atlas to primary cilia. Our analysis identified the subciliary locations of 715 proteins across three cell lines, examining 128,156 individual cilia. We found that 69% of the ciliary proteome is cell-type specific, and 78% exhibited single-cilia heterogeneity. Our findings portray cilia as sensors tuning their proteome to effectively sense the environment and compute cellular responses. We reveal 91 cilia proteins and found a genetic candidate variant in CREB3 in one clinical case with features overlapping ciliopathy phenotypes. This open, spatial cilia atlas advances research on cilia and ciliopathies.

Astrocyte Ca2+ dynamics control synaptic circuits and behavior, yet the underlying biology remains poorly understood. By combining volumetric high-resolution electron microscopy and two-photon Ca2+ imaging, we characterize astrocyte leaflets that interface with synapses. These convoluted structures with ≤250 nm diameter originate from astrocytic shafts or cell bodies, contain minuscule endoplasmic reticulum saccules expressing IP3 receptors but not mitochondria, and are often interconnected via gap junctions forming domains with cytosolic continuity. Leaflets enwrap 90% of synapses in clusters and only 10% individually. By fast imaging of astrocyte peripheral microvolumes, we identify leaflet-specific Ca2+ events that were synaptically induced, IP3R1-mediated, and often displayed separate originations merging into large, long-lasting Ca2+ elevations. Using combined axon-leaflet Ca2+ imaging, we show that these complex events reflect integration of incoming inputs from different neurons. The astrocyte leaflet organization may thus coordinate, via Ca2+ signals, multiple synapses and circuits active at different spatiotemporal scales, executing computations distinct from neurons.

Fluorescence microscopy has been widely applied in the life sciences. While intensity as a steady-state signal is widely used, the time-resolved (tr) signal using fluorescence lifetime remains underexplored. Herein, we present a family of time-resolved fluorescent proteins (tr-FPs) with rationally controlled lifetimes. Using a strategy that regulates lifetime without affecting the spectra of FPs, we have developed a series of tr-FPs that cover the visible spectrum and a wide range of lifetimes. The tr-FPs are employed in temporal-spectral resolved microscopy, allowing for the simultaneous imaging of 9 different proteins in live cells and the correlation of multiple activities to cell cycles. Furthermore, tr-FPs enable multiplexing super-resolution microscopy that concurrently visualizes 4 proteins using the lifetime signal and are demonstrated to quantify the stoichiometry of cellular proteins. Our work introduces the concept and development of tr-FPs as a transformative toolset, presenting opportunities to integrate system complexity and quantitative accuracy into biological research.

Characterizing genetic variation in natural populations is vital to evolutionary biology; however, many non-model species lack genomic resources. Here, we demonstrate that reference bias significantly affects population genomic analyses by mapping whole-genome sequence data from gray foxes (Urocyon cinereoargenteus) to a conspecific reference and two heterospecific canid genomes (dog and Arctic fox). Mapping to the conspecific genome improved read pairing by ∼5% and detected 26%-32% more SNPs and 33%-35% more singletons. Nucleotide diversity estimates increased by over 30%, FST increased from 0.189 to 0.197, and effective population size estimates were 30%-60% higher with the conspecific reference. Recombination rates varied by up to 3-fold at chromosome ends with heterospecific references. Importantly, FST outlier detection differed markedly, with heterospecific genomes identifying twice as many unique outlier windows. These findings highlight the impact of reference genome choice and the importance of conspecific genomic resources for accurate evolutionary inference.

In this issue of Cell, Huang and colleagues reveal how ancient hybridization between ancestors of tomato and a related wild species, Solanum etuberosum, enabled the origin of tuber formation and the diversification of potato species. Their genomic analyses highlight how interactions between genes derived from distinct progenitors can drive development of novel traits in hybrids.

Personalized cancer vaccines aim to broaden the anti-tumor T cell repertoire by targeting neoantigens unique to each patient's tumor, but immunogenicity has been inconsistent. In this issue of Cell, Blass, Keskin, Tu et al. evaluate NeoVaxMI, a multi-adjuvant personalized synthetic long-peptide vaccine administered with nivolumab in patients with melanoma. NeoVaxMI elicited stronger CD4+ and CD8+ responses than earlier iterations, and vaccine-induced T cells trafficked to regressing metastatic lesions.

Evaluating the innate value of objects is critical for expressing adaptive behaviors. However, where and how this computation takes place in the brain remain elusive. By recording from virtually every neuron in Drosophila higher olfactory areas, we show that the lateral horn is a site of innate odor value computation, where distinct neurons represent opposing innate values. A connectome-based spiking network model recapitulating the neural activity indicates that representations of aversive odors emerge through specific convergence of feedforward excitation, whereas those of attractive odors emerge through additional local inhibition. This inhibition is broad yet balanced with excitation and implements gain control and thresholding to shape attractive odor tuning. Manipulation of local inhibition biased neuronal and behavioral odor responses according to the prediction of the model. Thus, odors at the opposite ends of the hedonic spectrum are processed in sub-circuits that are not only segregated but also distinct in connectivity motifs.

A significant complication of Crohn's disease (CD) is intestinal fibrosis, which narrows the bowel lumen to form a stricture. Creeping fat (CF) is the wrapping of mesenteric adipose tissue around diseased bowel, of which the role in CD stricture progression is unclear. By constructing a human single-cell CD fibroblast atlas, we identified CF-derived, CTHRC1+ fibroblasts enriched for Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) signatures and localized to a fibrotic CF-bowel wall interface within the stricture. We further showed that analogous Cthrc1+ mouse fibroblasts derive from mesenteric adipose tissue stromal cells, infiltrate fibrotic bowel, and deposit extracellular matrix in a YAP/TAZ-dependent manner in a mouse model of intestinal fibrosis. Our findings identify CF as a key source of pro-fibrotic fibroblasts and raise the possibility of improving future clinical management of stricture progression by targeting not only the bowel but also CF.