Stringent control of T cell activity in the tumor microenvironment is essential for the generation of protective antitumor immunity. However, the identity, differentiation, and functions of the cells that create critical fibroblastic niches promoting tumor-infiltrating T cells remain elusive. Here, we show that CCL19-expressing fibroblastic reticular cells (FRCs) generate interconnected T cell environments (TEs) in human non-small cell lung cancer, including tertiary lymphoid structures and T cell tracks. Analysis of the FRC-T cell interactome in TEs indicated molecular networks regulating niche-specific differentiation of CCL19-expressing fibroblasts and T cell activation pathways. Single-cell transcriptomics and cell fate-mapping analyses in mice confirmed that FRCs in TEs originate from mural and adventitial progenitors. Ablation of intratumoral FRC precursors decreased antitumor T cell activity, resulting in reduced tumor control during coronavirus vector-based immunotherapy. In summary, specialized FRC niches in the tumor microenvironment govern the quality and extent of antitumor T cell immunity.

The sense of taste generally shows diminishing sensitivity to prolonged sweet stimuli, referred to as sweet adaptation. Yet, its mechanistic landscape remains incomplete. Here, we report that glia-like type I cells provide a distinct mode of sweet adaptation via intercellular crosstalk with chemosensory type II cells. Using the microfluidic-based intravital tongue imaging system, we found that sweet adaptation is facilitated along the synaptic transduction from type II cells to gustatory afferent nerves, while type I cells display temporally delayed and prolonged activities. We identified that type I cells receive purinergic input from adjacent type II cells via P2RY2 and provide inhibitory feedback to the synaptic transduction of sweet taste. Aligning with our cellular-level findings, purinergic activation of type I cells attenuated sweet licking behavior, and P2RY2 knockout mice showed decelerated adaptation behavior. Our study highlights a veiled intercellular mode of sweet adaptation, potentially contributing to the efficient encoding of prolonged sweetness.

Cryptochromes (CRYs) are blue-light receptors that regulate diverse aspects of plant growth. However, whether and how non-photoexcited CRYs function in darkness or non-blue-light conditions is unknown. Here, we show that CRY2 affects the Arabidopsis transcriptome even in darkness, revealing a non-canonical function. CRY2 suppresses cell division in the root apical meristem to downregulate root elongation in darkness. Blue-light oligomerizes CRY2 to de-repress root elongation. CRY2 physically interacts with FORKED-LIKE 1 (FL1) and FL3, and these interactions are inhibited by blue light, with only monomeric but not dimeric CRY2 able to interact. FL1 and FL3 associate with the chromatin of cell division genes to facilitate their transcription. This pro-growth activity is inhibited by CRY2's physical interaction with FLs in darkness. Plants have evolved to perceive both blue-light and dark cues to coordinate activation and repression of competing developmental processes in above- and below-ground organs through economical and dichotomous use of ancient light receptors.

We present replication-aware single-molecule accessibility mapping (RASAM), a method to nondestructively measure replication status and protein-DNA interactions on chromatin genome-wide. Using RASAM, we uncover a genome-wide state of single-molecule "hyperaccessibility" post-replication that resolves over several hours. Combining RASAM with cellular models for rapid protein degradation, we demonstrate that histone chaperone CAF-1 reduces nascent chromatin accessibility by filling single-molecular "gaps" and generating closely spaced dinucleosomes on replicated DNA. At cis-regulatory elements, we observe unique modes by which nascent chromatin hyperaccessibility resolves: at CCCTC-binding factor (CTCF)-binding sites, CTCF and nucleosomes compete, reducing CTCF occupancy and motif accessibility post-replication; at active transcription start sites, high chromatin accessibility is maintained, implying rapid re-establishment of nucleosome-free regions. Our study introduces a new paradigm for studying replicated chromatin fiber organization. More broadly, we uncover a unique organization of newly replicated chromatin that must be reset by active processes, providing a substrate for epigenetic reprogramming.

Stress induces aversive memory overgeneralization, a hallmark of many psychiatric disorders. Memories are encoded by a sparse ensemble of neurons active during an event (an engram ensemble). We examined the molecular and circuit processes mediating stress-induced threat memory overgeneralization in mice. Stress, acting via corticosterone, increased the density of engram ensembles supporting a threat memory in lateral amygdala, and this engram ensemble was reactivated by both specific and non-specific retrieval cues (generalized threat memory). Furthermore, we identified a critical role for endocannabinoids, acting retrogradely on parvalbumin-positive (PV+) lateral amygdala interneurons in the formation of a less-sparse engram and memory generalization induced by stress. Glucocorticoid receptor antagonists, endocannabinoid synthesis inhibitors, increasing PV+ neuronal activity, and knocking down cannabinoid receptors in lateral amygdala PV+ neurons restored threat memory specificity and a sparse engram in stressed mice. These findings offer insights into stress-induced memory alterations, providing potential therapeutic avenues for stress-related disorders.

Underlying variation in height are regulatory changes to chondrocytes, cartilage cells comprising long-bone growth plates. Currently, we lack knowledge on epigenetic regulation and gene expression of chondrocytes sampled across the human skeleton, and therefore we cannot understand basic regulatory mechanisms controlling height biology. We first rectify this issue by generating extensive epigenetic and transcriptomic maps from chondrocytes sampled from different growth plates across developing human skeletons, discovering novel regulatory networks shaping human bone/joint development. Next, using these maps in tandem with height genome-wide association study (GWAS) signals, we disentangle the regulatory impacts that skeletal element-specific versus global-acting variants have on skeletal growth, revealing the prime importance of regulatory pleiotropy in controlling height variation. Finally, as height is highly heritable, and thus often the test case for complex-trait genetics, we leverage these datasets within a testable omnigenic model framework to discover novel chondrocyte developmental modules and peripheral-acting factors shaping height biology and skeletal growth.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

In transcription-coupled nucleotide excision repair (TC-NER), stalled RNA polymerase II (RNA Pol II) binds CSB and CRL4CSA, which cooperate with UVSSA and ELOF1 to recruit TFIIH. To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also requires STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9-Å cryo-electron microscopy structure shows that STK19 binds the TC-NER complex through CSA and the RPB1 subunit of RNA Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of RNA Pol II for lesion verification. Our analysis of cell-free TC-NER suggests that STK19 couples RNA Pol II stalling to downstream repair events.

Targeted protein degradation strategies leverage endogenous cellular degradation machinery to selectively eliminate a protein of interest. Emerging technologies are opening avenues in drug discovery and functional characterization of intracellular, membrane, and extracellular proteins. To view this SnapShot, open or download the PDF.

In a recently published article in Nature, Bayly-Jones et al. report the cryo-EM structures of a lysosomal cholesterol sensor, LYCHOS, also known as GPR155, which reveals a unique fusion of a plant auxin-transporter-like domain with a seven-transmembrane GPCR-like domain and elucidates mechanistic insights into cellular regulation of mTORC1 activity.

The identification of individual protein-protein interactions (PPIs) began more than 40 years ago, using protein affinity chromatography and antibody co-immunoprecipitation. As new technologies emerged, analysis of PPIs increased to a genome-wide scale with the introduction of intracellular tagging methods, affinity purification (AP) followed by mass spectrometry (MS), and co-fractionation MS (CF-MS). Now, combining the resulting catalogs of interactions with complementary methods, including crosslinking MS (XL-MS) and cryogenic electron microscopy (cryo-EM), helps distinguish direct interactions from indirect ones within the same or between different protein complexes. These powerful approaches and the promise of artificial intelligence applications like AlphaFold herald a future where PPIs and protein complexes, including energy-driven protein machines, will be understood in exquisite detail, unlocking new insights in the contexts of both basic biology and disease.

The ribosome, together with its tRNA substrates, links genotype to phenotype by translating the genetic information carried by mRNA into protein. During the past half-century, the structure and mechanisms of action of the ribosome have emerged from mystery and confusion. It is now evident that the ribosome is an ancient RNA-based molecular machine of staggering structural complexity and that it is fundamentally similar in all living organisms. The three central functions of protein synthesis-decoding, catalysis of peptide bond formation, and translocation of mRNA and tRNA-are based on elegant mechanisms that evolved from the properties of RNA, the founding macromolecule of life. Moreover, all three of these functions (and even life itself) seem to proceed in defiance of entropy. Protein synthesis thus appears to exploit both the energy of GTP hydrolysis and peptide bond formation to constrain the directionality and accuracy of events taking place on the ribosome.

Since the introduction of the central dogma of molecular biology in 1958, various RNA species have been discovered. Messenger RNAs transmit genetic instructions from DNA to make proteins, a process facilitated by housekeeping non-coding RNAs (ncRNAs) such as small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and transfer RNAs (tRNAs). Over the past four decades, a wide array of regulatory ncRNAs have emerged as crucial players in gene regulation. In celebration of Cell's 50th anniversary, this Review explores our current understanding of the most extensively studied regulatory ncRNAs-small RNAs and long non-coding RNAs (lncRNAs)-which have profoundly shaped the field of RNA biology and beyond. While small RNA pathways have been well documented with clearly defined mechanisms, lncRNAs exhibit a greater diversity of mechanisms, many of which remain unknown. This Review covers pivotal events in their discovery, biogenesis pathways, evolutionary traits, action mechanisms, functions, and crosstalks among ncRNAs. We also highlight their roles in pathophysiological contexts and propose future research directions to decipher the unknowns of lncRNAs by leveraging lessons from small RNAs.

Every cell must solve the problem of how to fold its genome. We describe how the folded state of chromosomes is the result of the combined activity of multiple conserved mechanisms. Homotypic affinity-driven interactions lead to spatial partitioning of active and inactive loci. Molecular motors fold chromosomes through loop extrusion. Topological features such as supercoiling and entanglements contribute to chromosome folding and its dynamics, and tethering loci to sub-nuclear structures adds additional constraints. Dramatically diverse chromosome conformations observed throughout the cell cycle and across the tree of life can be explained through differential regulation and implementation of these basic mechanisms. We propose that the first functions of chromosome folding are to mediate genome replication, compaction, and segregation and that mechanisms of folding have subsequently been co-opted for other roles, including long-range gene regulation, in different conditions, cell types, and species.

In this essay, I will put forth what I see as a major conceptual challenge for biology in the next decade, one that is inspired by Crick's Central Dogma: understanding information flow in the cell in the most general sense.

Recombinant DNA technology has profoundly advanced virtually every aspect of biological and medical sciences, from basic research to biotechnology. Here, I discuss conceptual connections linking fundamental discoveries that were enabled by the technology, advances in the understanding of gene regulation in both prokaryotes and eukaryotes, and the recent FDA-approved CRISPR-based gene therapy for sickle cell anemia and β-thalassemia based on transcriptional derepression.

Molecular biology aims to understand the details of life by focusing closely on biopolymers-DNAs, RNAs, and proteins-and how they interact with one another. Advances in this field have enabled dazzling achievements in virtually all areas of biological, biomedical, and clinical sciences. As we draw near to the conclusion of Cell's 50th anniversary, we celebrate the wonders of molecular biology and look ahead to the exciting path forward for a branch of science that is driven by curiosity and has always been an integral part of the journal.

The microbiota in individual habitats differ in both relative composition and absolute abundance. While sequencing approaches determine the relative abundances of taxa and genes, they do not provide information on their absolute abundances. Here, we developed a machine-learning approach to predict fecal microbial loads (microbial cells per gram) solely from relative abundance data. Applying our prediction model to a large-scale metagenomic dataset (n = 34,539), we demonstrated that microbial load is the major determinant of gut microbiome variation and is associated with numerous host factors, including age, diet, and medication. We further found that for several diseases, changes in microbial load, rather than the disease condition itself, more strongly explained alterations in patients' gut microbiome. Adjusting for this effect substantially reduced the statistical significance of the majority of disease-associated species. Our analysis reveals that the fecal microbial load is a major confounder in microbiome studies, highlighting its importance for understanding microbiome variation in health and disease.

Kernel dehydration rate (KDR) is a crucial production trait that affects mechanized harvesting and kernel quality in maize; however, the underlying mechanisms remain unclear. Here, we identified a quantitative trait locus (QTL), qKDR1, as a non-coding sequence that regulates the expression of qKDR1 REGULATED PEPTIDE GENE (RPG). RPG encodes a 31 amino acid micropeptide, microRPG1, which controls KDR by precisely modulating the expression of two genes, ZmETHYLENE-INSENSITIVE3-like 1 and 3, in the ethylene signaling pathway in the kernels after filling. microRPG1 is a Zea genus-specific micropeptide and originated de novo from a non-coding sequence. Knockouts of microRPG1 result in faster KDR in maize. By contrast, overexpression or exogenous application of the micropeptide shows the opposite effect both in maize and Arabidopsis. Our findings reveal the molecular mechanism of microRPG1 in kernel dehydration and provide an important tool for future crop breeding.

β-Hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve the interconversion of BHB and primary energy intermediates. Here, we identify a previously undescribed BHB secondary metabolic pathway via CNDP2-dependent enzymatic conjugation of BHB and free amino acids. This BHB shunt pathway generates a family of anti-obesity ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. The most abundant BHB-amino acid, BHB-Phe, is a ketosis-inducible congener of Lac-Phe that activates hypothalamic and brainstem neurons and suppresses feeding. Conversely, CNDP2-KO mice exhibit increased food intake and body weight following exogenous ketone ester supplementation or a ketogenic diet. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, enzymatic amino acid BHB-ylation defines a ketone shunt pathway and bioactive ketone metabolites linked to energy balance.