Innate immune cells rapidly reprogram their metabolism upon activation, yet the metabolic basis of this flexibility in invertebrate systems remains largely unexplored. Here, we investigate the metabolic landscape of Drosophila larval hemocytes, functional analogs of vertebrate myeloid cells, across developmental stages, genotypes, and immune activation states, by combining metabolic flux measurements with single-cell transcriptomics. Under homeostatic conditions, hemocytes rely predominantly on mitochondrial oxidative phosphorylation for ATP production, with minimal glycolytic contribution. Immune activation, particularly lamellocyte differentiation, drives enhanced mitochondrial respiration and metabolic flexibility, accompanied by structural remodeling of the mitochondrial network. Mechanistically, functional lamellocytes require Drp1-mediated mitochondrial fission and utilize glucose and trehalose as primary carbon sources to sustain mitochondrial respiration, which is essential for effective immune responses. Overall, these findings establish that mitochondrial metabolic reprogramming is a conserved feature of innate immune activation in myeloid-like immune cells and reveal an evolutionarily ancient link between mitochondrial dynamics and immune cell activation, with implications for understanding metabolic regulation of innate immunity in invertebrate models and beyond. ### Competing Interest Statement The authors have declared no competing interest.
Agent-based modeling (ABM) is a computational method for predicting the emergent outcomes of interacting, autonomous individuals in a complex system. Here, ABM is used to simulate interactions between fibroblast and myofibroblast cells during idiopathic pulmonary fibrosis (IPF) in alveolar tissue microenvironments. These microenvironments are derived from histology of a healthy human lung sample and moderate- and severe-IPF lung samples. Fibroblast differentiation, cell migration, and collagen secretion in response to the spatial distribution of the cytokine transforming growth factor-beta are captured in the ABM using NetLogo software. Results are presented from one simulated year without treatment and with mechanisms representing treatment by pirfenidone and pentoxifylline, alone and in combination. A total of 180 in silico experiments are run, analyzed, and compared in a high-throughput workflow. The effects of the initial number of fibroblasts and treatment scenarios on various metrics related to collagen accumulation and collagen invasion into alveolar regions are determined. The ABM and the analysis files are shared to facilitate model reuse. By integrating computational modeling of IPF and therapeutics, this research aims to improve understanding of fibrosis progression and assess the efficacy of novel and existing treatments targeting different mechanisms to inform decision-making for IPF treatment. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, https://ror.org/021nxhr62, DMS-1929284 National Institute of General Medical Sciences, R35GM133763
As human space exploration expands to the Moon, Mars, and beyond, there is a growing need to study the effects of altered gravity on the microbial systems that we will bring with us for life support. Because spaceflight experiment opportunities are rare and resource-intensive, most space biology experiments are conducted using ground-based simulators. The most common microgravity simulator for microbial experiments, the rotating wall vessel, can approximate the low-shear and low-turbulence conditions that characterize microgravity. However, current designs do not allow for real-time measurement of growth or metabolic activity during rotation: experiments require destructive sampling or disruption of the microgravity simulation conditions. Here, we describe the development of an in situ spectroscopy system compatible with the Cell Spinpod rotating wall vessel, which enables measurement of both optical absorbance and fluorescence with high temporal resolution, producing growth curves similar to those from an off-the-shelf plate reader. These results are validated using two common microbial hosts: Escherichia coli and Saccharomyces cerevisiae. The Spinpod Optical System has the potential to diversify the types of microbiology experiments possible in simulated microgravity, allowing the measurement of not only growth curve parameters but also metabolic activity, gene expression, or community dynamics. It thus has the potential to improve the quality of experiments seeking to characterize microbial responses to spaceflight conditions. ### Competing Interest Statement The authors have declared no competing interest. National Aeronautics and Space Administration, https://ror.org/027ka1x80, 20-FG20_2-0015 Defense Advanced Research Projects Agency, https://ror.org/02caytj08, N660012324019
Collective cell migration drives tissue morphogenesis, repair and remodeling, and is often accompanied by transitions from solid-like to fluid-like states. While such tissue fluidization has been linked to physical parameters such as cell density, shape and activity, how it is actively regulated by mechano-chemical interplay remains unclear. Previous research has shown that transient attenuation of actomyosin contractility induces a transition from pulsatile, spatially confined motion to coherent, persistent long-range collective flow; however, the underlying cellular and signaling mechanisms remain unclear. Here we uncover the mechanistic basis by which transient perturbation of cell contractility reprograms the migration mode of confluent epithelial cells into a leader-like, fluidizing state, by combining kinase-reporter live imaging, force measurements and mathematical modeling. This transition arises from coordinated changes in cell morphology, mechanics, and signaling, including reduced cortical tension, enhanced cell-substrate adhesion and traction forces, and increased tissue deformability. At the signaling level, this process is accompanied by a rewiring of extracellular signal-regulated kinase (ERK)-mediated mechanotransduction toward a protrusion-coupled mode that sustains migration even under fully confluent conditions. Consistently, a multicellular computational model further demonstrates that protrusion-driven migration is sufficient to promote shape-velocity alignment and drive a transition from caged to flocking-like collective states. Together, our results identify transient mechanical relaxation as a trigger for an intrinsic leader-like state that fluidizes epithelial confluent tissues through coordinated remodeling of cytoskeletal, adhesive, and signaling systems. ### Competing Interest Statement The authors have declared no competing interest. Singapore Ministry of Education Academic Research Fund (AcRF) Tier 2, MOE-T2EP30223Β0010 National Research Foundation, Singapore (NRF) under its Mid-sized Grant, NRF-MSG-2023Β0001
Mechanical forces transmitted through focal adhesions regulate cell behavior and disease progression, yet remain difficult to quantify at the molecular level. Genetically encoded FRET-based tension probes enable measurements of piconewton-scale forces across specific proteins in living cells, but their quantitative interpretation is highly sensitive to probe design and measurement modality. Here, we systematically compared vinculin tension sensors under identical experimental conditions, evaluating unloaded reference constructs, fluorophore pairs, mechanical sensor modules, and circularly permuted variants. Unloaded controls established a common no-force baseline and validated force-dependent readout. Among the fluorophore pairs tested, the green-red combination Clover-mScarlet-I yielded a higher unloaded FRET efficiency and hence a broader measurable dynamic range. Comparison of six mechanical sensor modules identified the binary-response sensors FL and CC-S2 as the most responsive, showing the largest force-dependent FRET changes and broadest FRET distributions. At the sub-focal adhesion level, CC-S2 reported the steepest proximal-to-distal tension gradient, indicating that vinculin tension increases sharply along peripheral adhesions and exceeds 10 piconewton. Circular permutation experiments revealed that fluorophore orientation has a strong, module-dependent influence on the measured FRET readout. Together, these results establish a comparative framework for interpreting FLIM-based vinculin tension measurements and provide practical design principles for selecting and engineering molecular tension probes. ### Competing Interest Statement The authors have declared no competing interest. Research Foundation - Flanders, https://ror.org/03qtxy027, G0C2422N, G0A8L24N, G0B9922N, 1S95125N KU Leuven, C14/22/085
The biological significance of non-coding RNAs has been increasingly appreciated as their roles in various cellular processes are uncovered. However, single-cell transcriptomic profiling of human samples has focused primarily on protein-coding genes by targeting polyadenylated RNA transcripts, leaving the expression patterns of non-coding RNA underexplored. Here, we expand Tabula Sapiens to the non-coding transcriptome with single-cell and single-nucleus total RNA sequencing across 22 human organs and tissues. By simultaneously profiling both polyadenylated and non-polyadenylated transcripts, the resulting dataset enables joint analysis of the protein-coding and non-coding transcriptomes at single-cell and subcellular resolution. Using these data, we assessed the cell type specificity of non-coding genes and found that a greater proportion of non-coding genes are differentially expressed by single cell types compared to protein-coding genes. We then compared single-cell and single-nucleus data from the same samples to infer subcellular localization patterns, revealing cell type-dependent nuclear and cytoplasmic enrichment of specific non-coding RNAs. Next, we showed that tRNA repertoires are cell type-specific and that this specificity is not simply explained by differences in codon usage across cell types. Finally, we characterized dynamic expression patterns of non-coding RNAs across the cell cycle and senescence-associated cell states, identifying non-coding genes with putative roles in cell division and growth arrest. Our work establishes a resource for investigating the landscape of non-coding RNAs across a diverse set of human tissues and cell types. ### Competing Interest Statement The authors have declared no competing interest.
The vertebrate forebrain exhibits striking diversity in anatomical architecture, yet is built from deeply conserved gene regulatory programs and cell types that underlie shared neural functions and behaviors. Understanding how these conserved cellular programs are maintained and modified across ~500 million years of vertebrate evolution requires systematic cross-species single-cell comparisons, a challenge compounded by complex gene evolutionary histories that constrain joint analyses to shared one-to-one orthologs. Here we derive evolutionarily informed gene sets from a global homology graph and represent cells in a shared, interpretable gene-set feature space. Applying this framework to forebrain profiles from eleven vertebrate species, from sea lamprey to human, we construct a unified cross-vertebrate cell atlas. We find that conserved transcriptional programs define stable cell-type identities across vertebrates, with evolutionary divergence occurring predominantly within, rather than between, cell types. Gene-set conservation scales with evolutionary age, whereas lineage-modified programs reflect coordinated clade-level remodeling. Radial glia exhibit a conserved fate bifurcation into neurogenic and gliogenic trajectories with lineage-dependent modulation of transcriptional dynamics. Finally, human neuropsychiatric GWAS signals map onto conserved neural substrates across vertebrates. Together, our results demonstrate that vertebrate forebrain evolution proceeds through lineage-specific tuning of deeply conserved transcriptional programs embedded within stable cellular architectures. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, 1R01GM144560-01
Rxiv mechanobio