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Activation of innate immunity at the single-cell level is a heterogenous process, yet the origins of this variability – fundamentally linked to the control of immune responses – remain poorly understood. Here, we combine classical fluctuation tests with genomic and high-content microscopy approaches to investigate the heritability of single-cell gene-expression patterns in the evolutionarily conserved toll-like receptor (TLR) system. Using population-level and single-cell RNA-seq, we show that a subset of TLR4-dependent genes, approximately 7% overall and 15% among upregulated genes, including key cytokines and immune effectors, retained transcriptional memory across more than 25 cell divisions in clonal populations of immortalised murine macrophages. High-content microscopy of selected immune-relevant proteins from these RNA-seq analyses, including the TNF-alfa and IL1-beta cytokines, across thousands of clonal populations for up to ~10 cell divisions, revealed that transcriptional heritability remains strong over multiple generations but is ultimately transient and shaped by environmental and population context. Among these, CD36, a scavenger receptor involved in bacterial recognition, showed strong heritability, and we demonstrated that CD36-positive clones were more susceptible to Listeria monocytogenes infection, directly linking transcriptional heritability to infection outcome. Together, these findings demonstrate widespread, long-term TLR-mediated transcriptional heritability and provide a conceptual framework for understanding how variability in heritable transcriptional traits shapes immune function.

Imaging Methods: fluorescence microscopy

Organisms: Mus musculus

Optical multifrequency time-harmonic elastography (OMTHE) was used for rapid mechanical characterization of extra-cellular matrix-derived collagen networks at micrometer resolution. OMTHE was optimized for point-wise shear wave excitation in small sample volumes and compared to tabletop magnetic resonance elastography (ttMRE) and optical intensity changes. Dynamic stiffening due to the fluid-gel transition during collagen polymerization and chemical crosslinking using glutaraldehyde was tracked by shear waves speed (SWS) at vibration frequencies between 3 and 10 kHz and frame rates up to 4 kHz. During collagen polymerization, after an initial lag phase, SWS increased on average 6 ± 3 min earlier than optical density, suggesting that a load-bearing percolating fiber network was established before fibril thickening enhanced light scattering. In contrast, chemical crosslinking showed a lag-free, diffusion-driven SWS increase from 1.7 ± 0.4 m/s to 2.5 ± 0.5 m/s, matching the relative SWS change from ground-truth ttMRE. In conclusion, OMTHE provides a unique research tool that quantifies biomechanical property changes in small biological samples with spatiotemporal resolutions of micrometers and seconds.

Imaging Methods: bright-field microscopy

We present cloneXplorer, a fully automated live cell analysis and clone picking platform that uses time lapse monitoring of cell proliferation, cytokine secretion, and surface marker expression in ~100,000 single cell co-cultures to identify and isolate desired cells for clonal expansion or sequence analysis. We employ this platform in a cell line development workflow, in which single cells are selected from a heterogeneous source population based on cell proliferation rate and a fluorescent reporter, and are successfully expanded into monoclonal cell lines in >95% of attempts. Next, we devise a TCR epitope matching workflow by engineering Jurkat cells to express NFAT-GFP, CD8, and a TCR that recognizes a specific antigen. When Jurkat cells are co-cultured with a K562 library of 100 antigen presenting cells (APC), we show that the molecules recovered from microwells with positive GFP expression have the correct antigen in ~80% of attempts, determined by sequencing. To monitor immune activation in mouse and human primary samples, we implement a fluorescent sandwich assay to detect IFN-γ secretion in individual co-cultures. Finally, we combine these capabilities in a proof-of-concept demonstration, which uses IFN-γ secretion and the presence of CD8 surface markers as hierarchical gates to isolate and expand single T cells, and we verify their antigen-specificity by tetramer staining. Together, these results showcase potential applications of the cloneXplorer platform in cell line development and immune discovery applications.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens Mus musculus

This study integrates temporal image analysis, automated watering and weighing, and Bayesian growth modeling to dissect how 47 diverse maize inbred lines respond to drought, heat, and their combination. The results demonstrate genotype-specific effects, and that the combined stress is more closely related to heat than drought stress responses. A follow-up experiment also demonstrates that early stress impacts days to anthesis and plant area long after the stress has concluded. These findings underscore the need to evaluate maize diversity under multifactorial stress regimes to accurately identify resilient genotypes for environment-adaptive breeding.

Organisms: Zea mays

The main source of circulating erythropoietin (Epo) in the adult are kidney Norn cells, a recently identified interstitial cell type capable of becoming renal Epo-producing (REP) cells following a local decrease in tissue oxygenation. REP cells are restricted to small clusters in the cortico-medullary border region, suggesting that their microenvironment is relevant for cell differentiation and/or proper regulation of Epo production. Possibly for the same reason, REP cells cease to produce Epo in injured kidneys, which is rapidly reverted by stabilizers of the hypoxia-inducible factor (HIF). To shed new light on the mechanisms governing Epo production, we combined spatial transcriptomics, mRNA-FISH and sequential immunofluorescence, enabling the characterization of the direct neighbourhood of active REP cells. While in the hypoxic mouse kidney REP cells were closest to proximal tubule (PT) segments (S) S1 to S2/3 and endothelial cells, Epo was reinduced by HIF-stabilizers in injured kidneys in the vicinity of damaged PT cells that expressed high levels of injury markers. In contrast, the Norn and endothelial cell profiles remained normal. The REP cell microenvironment switched from pathways involved in energy metabolism under hypoxic conditions to inflammatory and fibrotic pathways under injury conditions. In summary, these data demonstrate that in the diseased kidney HIF-stabilizers reinduce Epo expression in REP cells with a metabolically inactive PT neighbourhood, consistent with a causal role of tubular cells during the loss of Epo expression.

Imaging Methods: fluorescence microscopy

Organisms: Mus musculus

PLA (Proximity Ligation Assay) and Immunoflourescence(IF) Images

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

Microscopy dataset underlying the results presented in the article "E. coli prepares for starvation by dramatically remodeling its proteome in the first hours after loss of nutrients".

Imaging Methods: fluorescence microscopy phase contrast microscopy

Organisms: Escherichia coli

Lunaphore COMET (cyclic immunofluorescent) data of the liver, together with IMC from adjacent tissue slices.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

Raw two-channel TIRF microscopy images (488 nm and 640 nm) generated for particle detection and ROI-based colocalization analysis. The dataset includes experimental and control image sets acquired under identical imaging conditions. Images were analyzed using custom ImageJ (Fiji) macros to identify particles in each channel and quantify spatial co-occurrence based on ROI overlap.

Imaging Methods: fluorescence microscopy

Organisms: Mus musculus

Raw data corresponding to the publication, "Cell surface localisation of GPI-anchored receptors in Trypanosoma brucei", published in eLife (https://doi.org/10.7554/eLife.107191.1)

Organisms: Trypanosoma brucei brucei

Parkinson’s disease is considered biologically a neuronal alpha-synuclein disease, largely ignoring the more widespread alpha-synuclein deposition that occurs in astrocytes. Recent single cell transcriptomics have identified early astrocytic differences in both Parkinson’s disease and mouse models with an increase in reactive astrocytes associated with proteostasis. To identify whether astrocytes accumulate alpha-synuclein before or after neurons, the present study histologically assessed astrocytes and alpha-synuclein accumulation in the M83 A53T transgenic mouse model of Parkinson’s disease prior to significant neuronal alpha-synuclein accumulation. The brains of M83 A53T transgenic (n=5) and wild-type (n=4) mice were perfusion fixed and serial sections of the midbrain and striatum processed for multiplex labelling. Digital images were captured from standardised sampling regions and astrocyte quantitation performed using QuPath software. Multivariate linear region models with Turkey posthoc tests were used to evaluate the effects of genotype on regional astrocyte morphology and numbers. The density of astrocytes within the substantia nigra pars compacta was approximately 30% greater compared with other sampled regions (P<0.005). Small aggregates of alpha-synuclein were observed in astrocytic processes, including in wild-type mice where a quarter of all astrocytes had an obvious alpha-synuclein aggregate. Compared to wild-type, A53T transgenic astrocytes had significantly enlarged somas (P<0.001) with more processes (P<0.001) consistent with a reactive phenotype. The expression of vascular endothelial growth factor A was present in analysed astrocytes, but not the synthesising enzyme for vitamin D CYP27B1. The A53T transgenic mice had more than double the numbers of astrocytes (P<0.001) and 2.5 times more astrocytes with alpha-synuclein aggregates compared to wild-type mice (P<0.001). These data suggest that -synuclein is normally cleared by astrocytes and that the substantia nigra pars compacta requires more astrocytic support than other midbrain dopaminergic regions or the striatum. This adds another vulnerability factor to those already known for the substantia nigra. In the A53T transgenic mouse model, astrocytes have an early upregulation of their clearance of alpha-synuclein aggregates. While speculative, a loss of this ability to take up alpha-synuclein in these regions may precipitate the selective neuronal degeneration and pathologies observed in Parkinson’s disease. As we move to a biological definition for this disease, understanding this early role of astrocytes needs to be considered further.

Imaging Methods: confocal microscopy

Organisms: Mus musculus

Understanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) visualize either specific labels or anatomical ultrastructure but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical imaging method that combines ~16-24-fold linear expansion with fluorescent pan-stainings of proteins and lipids (providing EM-like ultrastructural context), and immunolabeling (for molecular imaging). We demonstrate the versatility of this approach by imaging synaptic and cell-specific antibodies in the ultrastructural 3D context of pre and postsynaptic densities, neuropil nanoarchitecture, and cellular organelles in dissociated neuron cultures and mouse brain tissue sections. Furthermore, we demonstrate tracing of neuronal circuitry from pan-ExM-t image volumes, suggesting that any laboratory with access to a confocal microscope can now localize specific molecules within nanoscale cellular and circuit contexts.

Imaging Methods: spinning disk confocal microscopy

Organisms: Mus musculus