EMBO Journal最新文献

Bacteria often experience nutrient limitation. While the exponential and stationary growth phases have been characterized in the model bacterium Escherichia coli, little is known about what happens inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the positions of nucleoids and cell division sites become increasingly asymmetric during the transition phase. These asymmetries were accompanied by an asymmetric reorganization of protein, ribosome, and RNA probes in the cytoplasm. Results from live-cell imaging experiments, complemented with genetic and ¹³C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. Live-cell atomic force microscopy analysis, combined with in vitro biochemical experiments, suggests that these phenotypes are due to the propensity of glycogen to phase-separate into soft condensates in the crowded cytoplasm. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase-separation allows cells to store large glucose reserves that are not perceived by the cell as cytoplasmic space.

Delivery of antibacterial effector proteins into competitor cells using the Type VI secretion system (T6SS) is a widespread strategy for inter-bacterial competition. While many enzymatic T6SS effectors have been described, relatively few which form pores in target cell membranes have been reported. Here, we describe a widely-occurring family of T6SS-dependent pore-forming effectors, exemplified by Ssp4 of Serratia marcescens Db10. We show in vitro that Ssp4 forms regulated pores with high selectivity for cations, and use structural models and molecular dynamics simulations to predict how these pores conduct ions. Ssp4 has a broader phylogenetic distribution and is active against a wider range of bacterial species than Ssp6, the other pore-forming effector delivered by the same T6SS, with the two effectors displaying distinct ion selectivities and impacts on intoxicated cells. Finally, identification of Ssp4-resistant mutants revealed that a mucA mutant of Pseudomonas fluorescens is protected against T6SS attacks. We propose that deployment of two distinct T6SS-dependent pore-forming toxins is a common strategy to ensure effective de-energisation of closely- and distantly-related competitors.

Plasma C-reactive protein (CRP) is widely used as a biomarker for bacterial infections due to its massive induction during infections. However, the biological function of CRP remains largely undefined. Here we show that CRP enables liver resident macrophages (Kupffer cells) to capture and eliminate a wide range of invasive bacteria from the bloodstream of mice, and thereby provides rapid and sterilizing immunity. Mechanistically, CRP binds to at least 20 capsule types of Gram-positive and -negative pathogens, and shuffles the encapsulated bacteria to Kupffer cells embedded in the lining of the liver sinusoidal vasculatures by the complement-dependent and -independent pathways. The complement-dependent mode involves the activation of complement C3 at the bacterial surface, and the capture of the C3-opsonized bacteria by the CRIg and CR3 complement receptors on Kupffer cells. Cryo-electron microscopy analysis revealed a flexible structural framework for CRP's recognition of structurally diverse capsular polysaccharides. Because human CRP also possesses the broad capsule-binding activities, our findings provide a biological reason for the massive rise of plasma CRP during bacterial infections.

Cells monitor and dynamically regulate the lipid composition and biophysical properties of their plasma membrane (PM). The Target Of Rapamycin complex 2 (TORC2) is a protein kinase that acts as a central regulator of plasma membrane homeostasis, but the mechanisms by which it detects and reacts to membrane stresses are poorly understood. To address this knowledge gap, we characterized a family of amphiphilic molecules that physically perturb plasma membrane organization and in doing so inhibit TORC2 in yeast and mammalian cells. Using fluorescent reporters of various lipids in budding yeast, we show that exposure to these small molecules causes mobilization of PM ergosterol as well as inhibition of TORC2. TORC2 inhibition results in activation of the PM-ER sterol transporters Lam2 and Lam4 and the subsequent rapid removal of accessible ergosterol from the plasma membrane via PM-ER contact sites. This sequence of events, culminating in the reactivation of TORC2, is also observed with several other PM stresses, suggesting that TORC2 acts in a feedback loop to control active sterol levels at the plasma membrane to maintain its homeostasis.

Senescent cells drive age-related tissue dysfunction via the induction of a chronic senescence-associated secretory phenotype (SASP). The cyclin-dependent kinase inhibitors p21^Cip1 and p16^Ink4a have long served as markers of cellular senescence. However, their individual roles remain incompletely elucidated, particularly in vivo. Thus, we conducted a comprehensive examination of multiple single-cell RNA sequencing datasets spanning both murine and human tissues during aging. Our analysis revealed that p21^Cip1 and p16^Ink4a transcripts demonstrate significant heterogeneity across distinct cell types and tissues, frequently exhibiting a lack of co-expression. Moreover, we identified tissue-specific variations in SASP profiles linked to p21^Cip1 or p16^Ink4a expression. Using RNA velocity and pseudotime analyses, we discovered that p21+ and p16+ cells follow independent trajectory dynamics, with no evidence of direct transitions between these two states. Despite this heterogeneity, we identified a limited set of shared "core" SASP factors that may drive common senescence-related functions. Our study underscores the substantial diversity of cellular senescence and the SASP, emphasizing that these phenomena are inherently cell- and tissue-dependent.

During development, stem and progenitor cells divide and transition through multipotent states to generate the diverse cell types by undergoing defined changes in biomolecular composition, which underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, the cardiac and pharyngeal muscle programs are jointly primed in multipotent cardiopharyngeal progenitors, and segregate in distinct daughter cells only after cell division. Here, using the tunicate Ciona, we showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single-cell RNA-sequencing (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S, and G2 phases. We refer to this process as "transcriptome maturation", and identified candidate mature genes, including the Rho GAP-coding gene Depdc1b, which peaks in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and by enabling asymmetric cell division that influences subsequent fate decisions, illustrating the concept of "behavioral competence". We show that both classic regulatory circuits and coupling with the G1-S transition drive transcriptome maturation, ensuring the timely deployment of lineage-specific programs.

DNA replication is essential to life and ensures the accurate transmission of genetic information, which is significantly disturbed during cancer development and chemotherapy. While DNA replication is tightly controlled in time and space, methods to visualise and quantify replication dynamics within 3D human cells are lacking. Here, we introduce 3D-Spatial Assay for Replication Kinetics (3D-SPARK), an approach enabling nanoscale analysis of DNA synthesis dynamics in situ. 3D-SPARK integrates optimised nucleotide analogue pulse labelling with super-resolution microscopy to detect, classify, and quantify replication nanostructures in single cells. By combining immunofluorescence techniques with click chemistry-based nascent DNA labelling and transfection of fluorescent nucleotide derivatives, we map multi-colour DNA synthesis events in relation to established replication proteins, local RNA-protein condensates or large subnuclear domains. We demonstrate quantitative changes in size, relative abundance and spatial arrangement of nanoscale DNA synthesis events upon chemotherapeutic treatment, CDC6 oncogene expression and loss of chromatin organiser RIF1. The flexibility, precision and modular design of 3D-SPARK helps bridging the gap between spatial cell biology, genomics, and 2D fibre-based replication studies in health and disease.

Entry into and exit from cellular quiescence require dynamic adjustments in nutrient acquisition, yet the mechanisms by which quiescent cells downregulate amino acid (AA) transport remain poorly understood. Here we show that cells entering quiescence selectively target plasma membrane-resident amino acid transporters for endocytosis and lysosomal degradation. This process matches amino acid uptake with reduced translational demand and promotes survival during extended periods of quiescence. Mechanistically, we identify the α-arrestin TXNIP as a key regulator of this metabolic adaptation, since it mediates the endocytosis of the SLC7A5-SLC3A2 (LAT1-4F2hc) AA transporter complex in response to reduced AKT signaling. To promote transporter ubiquitination, TXNIP interacts with NEDD4L and other HECT-type ubiquitin ligases. Loss of TXNIP disrupts this regulation, resulting in dysregulated amino acid uptake, sustained mTORC1 signaling, and ultimately cell death under prolonged quiescence. The characterization of a novel TXNIP loss-of-function variant in a patient with a severe metabolic disease further supports its role in nutrient homeostasis and human health. Together, these findings highlight TXNIP's central role in controlling nutrient acquisition and metabolic plasticity with implications for quiescence biology and diseases.