Cell reports最新文献

Applying cryoelectron microscopy (cryo-EM) to small protein complexes is usually challenging due to their lack of features for particle alignment. Here, we characterized antibody responses to 21 kDa human immunodeficiency virus (HIV) membrane-proximal external region germline-targeting (MPER-GT) immunogens through cryo-EM by complexing them with 10E8 or Fabs derived from MPER-GT-immunized animals. Distinct antibody-antigen interactions were analyzed using atomic models generated from cryo-EM maps. Mutagenesis screening revealed that off-target monoclonal antibodies (mAbs), which do not compete with 10E8, bind non-MPER epitopes, and the binding of two most dominant epitopes were verified by cryo-EM. The structures of 10E8-class on-target Fabs showed binding patterns that resemble the YxFW motif in the 10E8 heavy chain complementarity-determining region 3 (HCDR3) loop. Additionally, we demonstrate that high-resolution maps can be generated from heterogeneous samples with pooled competing Fabs. Overall, our findings will facilitate the optimization of MPER-GT antigens and push the size limit for cryo-EM-based epitope mapping with smaller antigens and heterogeneous antibody mixes.

Despite recent advances in cell migration mechanics, the principles governing rapid T cell movement remain unclear. Efficient migration is critical for antitumoral T cells to locate and eliminate cancer cells. To investigate the upper limits of cell speed, we develop a hybrid stochastic-mean field model of bleb-based cell motility. Our model suggests that cell-matrix adhesion-free bleb migration is highly inefficient, challenging the feasibility of adhesion-independent migration as a primary fast mode. Instead, we show that T cells can achieve rapid migration by combining bleb formation with adhesion-based forces. Supporting our predictions, three-dimensional gel experiments confirm that T cells migrate significantly faster under adherent conditions than in adhesion-free environments. These findings highlight the mechanical constraints of T cell motility and suggest that controlled modulation of tissue adhesion could enhance immune cell infiltration into tumors. Our work provides insights into optimizing T cell-based immunotherapies and underscores that indiscriminate antifibrotic treatments may hinder infiltration.

We optogenetically mapped the function and spatial organization of inhibitory circuits formed by interneurons (INs) within the claustrum, a highly interconnected but poorly understood brain region. INs expressing parvalbumin or somatostatin attenuate claustrum output by inhibiting projection neurons (PNs), while INs expressing vasoactive intestinal peptide promote claustrum output by disinhibiting PNs. The spatial organization and degree of convergence differ for each interneuronal circuit. A computational model incorporating measured circuit properties predicts that differential inhibition of PNs by INs toggles claustrum output between cortical and subcortical brain regions and that the spatial organization of IN circuits nonlinearly filters claustrum output according to the strength and spatial distribution of excitatory input. Experimental measurements show that the claustrum spatially filters cortical input as predicted by the model. We conclude that the organization of its inhibitory circuits allows the claustrum to serve as a filter that improves the signal-to-noise ratio of signals transmitted to its downstream targets.

Age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Genome-wide association studies (GWASs) of AMD have identified dozens of risk loci that may house disease targets. However, variants at these loci are largely noncoding, making it difficult to assess their function and whether they are causal. Here, we present a single-cell gene expression and chromatin accessibility atlas of human retinal pigment epithelium (RPE) and choroid to systematically analyze both coding and noncoding variants implicated in AMD. We employ HiChIP and activity-by-contact modeling to map enhancers in these tissues and predict cell and gene targets of risk variants. We further perform allele-specific self-transcribing active regulatory region sequencing (STARR-seq) to functionally test variant activity in RPE cells, including in the context of complement activation. Our work nominates pathogenic variants and mechanisms in AMD and offers a rich and accessible resource for studying diseases of the RPE and choroid.

PDX1 is a key transcription factor regulating insulin expression in response to glucose. Our previous work showed that PDX1 is also stimulated by amino acids (aa). Here, we demonstrate that PDX1 broadly mediates aa-regulated transcriptional programs in β cells, especially those controlling β cell proliferation and function. Mechanistically, mTORC1 phosphorylates PDX1 at serine 61 (S61), enhancing its protein stability and transcriptional activity. A certain monogenic diabetes mutation disrupts this phosphorylation and impairs PDX1 function. To investigate its physiological role, we generated mice carrying S61A and S61E mutations, mimicking unphosphorylated and phosphorylated states. S61 phosphorylation promoted insulin expression and β cell proliferation, leading to Western diet-induced hyperinsulinemia, obesity, and hepatic steatosis. These findings reveal the central role of aa-mTORC1-PDX1 signaling in coordinating β cell proliferation and function under both physiological and pathological conditions.

PIWI-interacting RNAs (piRNAs) guide PIWI endoribonucleases to destroy transposon transcripts, ensuring animal fertility. Here, we report the cryo-electron microscopy structure of the MIWI-pachytene piRNA complex isolated from mouse testes. The piRNA is held via non-specific charge-based interactions with the RNA backbone and by specific recognition of the first nucleotide uridine by residues within the MID and PIWI domains. The first six nucleotides of the guide RNA take up the A-form conformation to facilitate pairing with the target. The RNA channel is wider than that observed in insect PIWI proteins, explaining the tolerance for piRNA seed:target mismatches. The PIWI endonuclease domain is in an inactive "un-plugged" state, with the loop containing a catalytic residue (E671) requiring structural re-orientation for activity. Furthermore, the PIWI domain reveals a conserved pre-formed pocket that may serve to accommodate a conserved tryptophan from the interacting factor GTSF1 to promote small RNA-guided endoribonuclease activity.

Quantifying enzymatic activity at the single-cell level remains challenging. Building on previous development of metal-tagged activity-based probes (time-of-flight [TOF]-probes) compatible with mass cytometry, we adapt this technology to quantify individual proteases in immune cells. We optimize and validate TOF-probes targeting catalytically active cysteine and serine proteases in two primary immune populations: neutrophils and monocyte-derived macrophages. Using this platform, we observe striking shifts in cathepsin activity during monocyte-to-macrophage differentiation, along with a functional rewiring from apoptotic to pyroptotic protease programs. In neutrophils, unexpectedly, we detect high levels of active serine proteases even in the resting state, highlighting their potential role as pre-armed effector cells poised for rapid inflammatory responses. These findings lay the foundation for using TOF-probes to quantify active proteases in situ at the single-cell level and provide new insights into protease activation states across immune cells.

While high-fiber diets (HfiDs) promote weight loss, their long-term efficacy is limited by rapid weight regain upon returning to high-fat diets (HFDs). Using C57BL/6J mice in diet-switching paradigms, we characterized tissue-specific responses to HfiD-to-HFD transitions through single-nucleus and spatial transcriptomics. HfiD pre-feeding enhanced mesenteric white adipose tissue progenitor/adipocyte sensitivity to subsequent HFD exposure. In the intestine, HfiD prevented HFD-induced immune-enterocyte expansion in the duodenum and reversed the enterocyte-to-goblet cell shift in the colon while maintaining persistent epigenetic reprogramming. Although HfiD-induced microbiome changes were largely reversed by HFD, we identified sexually dimorphic remodeling of adipose cell populations during diet transitions. Our findings demonstrate that prior HfiD feeding fundamentally reprograms adipose and intestinal responses to subsequent HFD challenge, providing mechanistic insights into dietary intervention outcomes. This work establishes a spatiotemporal resource for understanding tissue plasticity during dietary changes, offering new perspectives for obesity management strategies.

NLRP3 inflammasome activation requires both transcriptional priming and complex assembly, but how RNA m⁶A methylation coordinates these steps remains unclear. Here, we show that m⁶A levels increase during macrophage NLRP3 inflammasome activation and that METTL3 loss suppresses this activation. Myeloid-specific Mettl3 knockout mice display reduced inflammation and improved metabolic outcomes in lipopolysaccharide (LPS)-induced sepsis, monosodium urate (MSU)-induced arthritis, and diet-induced obesity. Integrated chromatin-associated RNA sequencing (chrRNA-seq), kethoxal-assisted single-stranded DNA sequencing (KAS-seq), and chrRNA-methylated RNA immunoprecipitation (MeRIP)-seq analyses show that METTL3 installs m⁶A co-transcriptionally on nascent Jak1, Nlrp3, and Il1β RNAs and that METTL3 regulates dynamic transcription and chromatin accessibility while selectively maintaining Nlrp3/Il1β transcription. YTHDF1-driven translation of Jak1 activates the JAK1-STAT3-C/EBPβ axis to initiate Nlrp3/Il1β transcription, and m⁶A-YTHDF1 translation of Nlrp3/Il1β amplifies protein output, forming a coupled transcriptional-translational circuit. Pharmacologic STAT3 inhibition and METTL3 catalytic rescue validate this pathway and identify METTL3-mediated m⁶A as a therapeutic target for inflammasome-driven diseases.

Ruminants thrive in diverse ecosystems by leveraging their rumen microbiome to ferment fibrous plants. However, the spatial biogeography of rumen microbiome and the genetic diversity of the ventral rumen epithelium remain unknown. Here, we present a multi-omics study in roe deer, sika deer, and sheep, integrating region-resolved microbiome and metabolome across 11 ruminal sacs, as well as single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and bulk RNA sequencing (RNA-seq) of ventral epithelium. We reveal species-specific microbial compositions and metabolic capacities contributing to differences in short-chain fatty acid and vitamin B production. We uncover functional divergence, genomic specialization, and metabolic changes across the microbiome of distinct ruminal sacs. Single-cell profiling reveals changes of immune responses and structural remodeling of the ruminal ventral epithelium. We demonstrate that vitamin B₁₂ promotes epithelial growth and we identify genes enhancing stem cell differentiation. Our results highlight variation in microbial ecology and epithelial architecture among three ruminant species, offering insights to improve livestock productivity.