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bioRxiv2025

Dendritic Polyglycerol Amine Substrate Extends the Viability of Mixed Glial Cultures for Repeated Isolation of Immature Oligodendrocyte Lineage Cells

Primary mixed glial cultures are key tools to isolate and study astrocytes, microglia and oligodendrocytes. Cell-substrate adhesion is critical for neural cell survival and differentiation. Cationic polymers like poly-D-lysine (PDL) are widely used to promote cell adhesion to cell culture substrates, however, PDL is not stable long-term, with cultured cells often detaching (peeling) after 2-3 weeks. Dendritic polyglycerol amine (dPGA) is a synthetic polycationic non-protein polymer biomimetic of poly-lysine that is highly resistant to degradation by cellular proteases. Substrates coated with dPGA promote cell adhesion and improve survival in long-term neuronal cultures. Here we assessed dPGA as a substrate coating to provide long-term support for mixed glial cultures. Oligodendrocyte precursor cells (OPCs) were isolated weekly by differential adhesion from cultures grown in T75 flasks with PDL or dPGA-coated substrates. Following two "shake-off" isolations, the cell layer in most PDL-coated flasks fully detached, rendering these flasks unusable for further culture. In contrast, dPGA-coated flasks consistently yielded cells for six or more sequential isolations over seven weeks in culture. dPGA-coated flasks produced more cells, a greater percentage of O4+ cells, and maintained similar proportions of OPCs and MBP-positive cells as when isolated from a PDL-coated substrate. dPGA is cyto-compatible, functionally superior, easy to use, low cost and a stable alternative to conventional cell substrate coatings. The enhanced long-term stability of mixed glial cultures grown on a dPGA substrate has the capacity to increase cellular yield, reduce animal use, and facilitate studies of oligodendrocyte cell biology.

neuroscience↗

bioRxiv2025

Growth Mechanics and the emergence of metabolic oscillations in growing cells

Unicellular organisms exhibit oscillations in metabolic activity and gene expression, even when growing in static media with no external dynamic stimuli. The cause and possible function of these endogenous oscillations are still not fully understood, with different hypotheses including its origin as a byproduct of misguided regulatory processes. To investigate whether such oscillations could be functional rather than incidental, we introduce Growth Mechanics (GM) as a general mathematical framework to study the dynamic resource allocation in models of whole self-replicating cells, built exclusively on the first principles of fitness maximization, mass conservation, nonlinear reaction kinetics, and constant cell density. Inspired by the physical theory of classical mechanics, we first find the simplest mathematical description the problem in terms of generalized coordinates, and then solve the optimal dynamical resource allocation for cells growing in any given medium using the Euler-Lagrange equation, resulting in analytical "equations of optimal motion" (EOM) that apply for growing cells in general. We then solve these equations numerically for simple growing cell models and show that, in general, oscillations are necessary to maximize cell fitness by getting more saturated reactions at the time they are most needed. We also show how the EOM predict the emergence of quasi-linear dependencies of the metabolic oscillation frequency and the ribosome protein allocation with the cell growth rate at different growth media, in line with the "growth laws" observed experimentally in microbes. This work contributes to the foundations of theoretical cell biology with general quantitative principles and a bridge to theoretical tools of physics.

biophysics↗

bioRxiv2025

Quantifying distribution shifts in single-cell data with scXMatch

A basic task that frequently arises when analyzing single-cell data is to assess if there is a global distribution shift between the data profiles of cells from two different conditions. Widely used approaches to address this task such as visual inspection of two-dimensional representations or clustering-based workflows lack a solid statistical underpinning and are notoriously unstable and prone to confirmation bias. To promote more rigorous analysis, we here present the scverse-compatible Python tool scXMatch. scXMatch is based on a non-parametric graph-based test to quantify distribution shifts in arbitrary data spaces for which a suitable distance measure is available. We evaluated scXMatch on single-cell gene expression, chromatin accessibility, and imaging-derived cell morphology data, showing that it can robustly detect distribution shifts for different types of single-cell data. scXMatch thus aims to set a new standard in the single-cell biology field, replacing easy-to-manipulate semi-manual distribution shift quantification workflows by principled statistical testing.

bioinformatics↗

bioRxiv2025

Multiparametric Correlative Topographical and Volumetric Fluorescence Microscopy

Live-cell imaging of cell surface topography and intracellular architecture is essential for understanding cellular function. However, conventional approaches often involve trade-offs between resolution, invasiveness, and volumetric coverage. Here, we present an integrated Scanning Ion Conductance Microscope and single-objective Oblique Plane Microscope (SICM-OPM) system that enables simultaneous non-contact topographical imaging and volumetric fluorescence imaging within the same live cell. Beyond correlative live imaging, the platform supports nanomechanical mapping with tens-of-nanometres resolution, fluorescence-guided localised molecular delivery via the SICM, and benefits from reduced photobleaching due to light-sheet excitation. We demonstrate this platforms capabilities by visualising imipramine-induced T-tubule remodelling in live cardiomyocytes, revealing subsurface detubulation while surface morphology remains preserved. Additionally, we show precision delivery of fluorescent cargos--including dextrans and -synuclein--into diatom and mammalian cells, alongside localised stiffness mapping to evaluate mechanical responses. We believe this technique opens new avenues for correlative structural, functional, and biophysical studies in live cells, with broad relevance to cell biology, neurodegeneration, and mechanobiology.

biophysics↗

bioRxiv2025

DIA-PASEF Proteomic Profiling Reveals MpkA-Dependent Iron Stress Responses and Siderophore Biosynthesis in Aspergillus nidulans

Filamentous faungi play essential roles in biotechnology as producers of valuable bioproducts and, conversely, as opportunistic pathogens. Aspergillus nidulans is a widely used model organism for fungal genetics and cell biology; however, comprehensive proteomic references for this species remain limited. In this study, we applied a data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF) approach to enable efficient and in-depth proteome profiling of A. nidulans. Leveraging ion mobility-based ion cloud information from DDA-PASEF experiments, we developed DIA-PASEF methods that identified 3,904 proteins across biological triplicates grown in rich medium. Compared to prior studies, this represents an increase in protein identifications of more than 140% and was achieved with more than five-fold reduction in analysis time. We employed this newly developed DIA-PASEF methodology to conduct both proteomic and phosphoproteomic analyses under iron-depleted conditions in an MpkA protein-kinase deficient mutant ({Delta}mpkA). The {Delta}mpkA strain exhibited expression of approximately 500 additional proteins and occupancy of over 1,800 additional phosphosites relative to a control. Differentially expressed and phosphorylated proteins increased by more than an order of magnitude in the {Delta}mpkA mutant across both iron-replete and iron-deplete conditions. Gene Ontology (GO) enrichment analysis revealed broader and distinct biological processes under iron-depleted conditions, highlighting adaptive responses specific to iron limitation and MAPK pathway disruption. This work establishes a high-coverage proteomic resource for A. nidulans and provides novel insights into fungal stress responses and signaling network perturbation. Importantly, high-throughput proteomic profiling reveals that limited iron availability and MAPK pathway disruption increases siderophore biosynthesis.

systems biology↗

bioRxiv2025

The RXLR-EER Motif Determines an Unconventional Secretion Pathway Associated with Extracellular Vesicle Production

Phytophthora infestans, the cause of potato late blight disease, delivers a suite of RXLR effectors into host plant cells to subvert immunity, whereas apoplastic effectors act extracellularly. Although the RXLR-EER motif in these effectors is critical for host translocation and is cleaved prior to secretion, the relevance of this processing is poorly understood. Prior evidence suggests RXLR effectors utilize a distinct, unconventional secretion pathway, raising the question of whether the RXLR-EER motif influences selection of the secretion route. Here, we combined genetic, molecular and cell biology approaches to investigate the secretion pathway of RXLR effectors. Confocal microscopy revealed that RXLR and apoplastic effectors localize to distinct vesicular compartments in cultured hyphae. Moreover, fusing the ER retention signal KDEL to RXLR effectors did not impair their secretion, in contrast to apoplastic effectors, which were retained in the endomembrane system, indicating that RXLR effectors bypass the canonical ER-to-Golgi pathway. Importantly, RXLR effectors associate with extracellular vesicles (EVs), whereas RXLR-EER motif mutants show reduced EV association and are rerouted through the ER-to-Golgi secretion pathway. These findings demonstrate that the RXLR-EER motif governs effector sorting into an unconventional, EV-linked secretion route. This study sheds light on the molecular basis of effector trafficking in P. infestans and underscores the potential role of EVs in delivering virulence factors during host colonization.

microbiology↗

bioRxiv2025

Is Language a Mechanical Signal? Cytoskeletal Responses to Speech in Yeast

What if vocal language were not only a medium for human communication but a vibrational force that leaves structural traces in living cells? This study explores how audible sound, particularly the structured elements of human speech, affects the cytoskeleton of Saccharomyces cerevisiae. Using a direct-contact acoustic system, we exposed yeast to distinct sound types: tonal vibrations, broadband noise, and consonant phonemes. Fluorescence microscopy revealed that tonal stimuli with coherent low-frequency patterns enhanced actin polymerization and shmoo formation, both markers of polarity and mating. In contrast, broadband noise disrupted actin integrity, while consonants produced no measurable effects. These results suggest that rhythmic continuity and spectral coherence, key features of speech, can modulate cytoskeletal organization in non-auditory cells. By reframing vocal language as mechanical input rather than semantic content, this study bridges microbial cell biology with acoustic ecology and proposes a new lens for exploring how human-generated soundscapes physically influence living systems.

microbiology↗

bioRxiv2025

The Functional Epididymal Amyloid Cystatin-Related Epididymal Spermatogenic (CRES) is a Component of the Mammalian Brain Extracellular Matrix

CRES is the defining member of a reproductive subgroup of family 2 cystatins of cysteine protease inhibitors. We previously showed that CRES and other subgroup members are part of a highly plastic amyloid-containing extracellular matrix (ECM) with host defense functions in the mouse epididymal lumen. Based on parallels between the epididymis and the brain, we hypothesized that CRES and CRES amyloids might also function within the brain including the ECM. Here we show that CRES is produced by hippocampal neurons and astrocytes in the male and female mouse and human brain. Further, approximately 50% of hippocampal astrocytes from aged mice, like the aged human donor samples, had significantly reduced levels of CRES compared to younger mice, suggesting an age-related decline in CRES could contribute to altered brain function. Immunofluorescence experiments showed CRES colocalized with the ECM markers phosphacan and wisteria floribunda agglutinin indicating that CRES is part of the ECM. CRES monomer and high molecular weight SDS-resistant forms were found in insoluble fractions of the hippocampus, cortex, cerebellum, and midbrain and bound to the protein aggregation disease (PAD) ligand, which preferentially binds amyloids but not protein monomers, suggesting a population of CRES exists in the brain as an amyloid structure. Collectively, our studies demonstrate that CRES/CRES amyloid is present in the mammalian brain and may contribute to ECM structure and function. Significance StatementWe previously established that the cystatin-related epididymal spermatogenic (CRES) protein is part of an amyloid-containing extracellular matrix (ECM) that protects the male germline in the epididymal lumen. Here we demonstrate that CRES is present within the mouse and human brain. Using cell biological and biochemical approaches, we show that CRES is found in hippocampal astrocytes and specific neuronal populations, including those that possess perineuronal nets, and colocalized with ECM markers suggesting it is part of the ECM. Biochemical analyses suggested a population of CRES is present as an ordered amyloid structure. Our studies reveal CRES is present in the male and female mammalian brain and may contribute to brain structure and function as a biological amyloid. Keyword: hippocampus, mouse, human, plasticity

neuroscience↗

bioRxiv2025

Nuclease-NTPase systems use shared molecular features to controlbacterial anti-phage defense

Bacteria encode an enormous diversity of defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a predicted nuclease and an accessory NTPase protein, suggesting additional classes of nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define shared features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid targeting for each system demonstrate protein-protein complex formation is a universal feature of nuclease-NTPase systems and explain patterns of phage targeting and susceptibility. We show that some nuclease-NTPase systems use highly degenerate recognition site preferences to enable exceptionally broad nucleic acid degradation. Our results uncover shared principles of anti-phage defense system function and provide a foundation to explain the widespread role of nuclease-NTPase systems in bacterial immunity.

microbiology↗

bioRxiv2025

Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

Improved methods are needed to gain insights in how proteins exert their myriad roles in cells and organs. Multiplex in situ proximity ligation assay (misPLA), described herein, can provide a window into the functional states of proteins in cells and tissues by applying pairs of antibody-oligonucleotide conjugates to generate amplifiable DNA circles upon proximal binding. The analysis reveals interactions and modifications among sets of proteins, read out by recording the identity and location of the resulting localized DNA amplification products. We applied misPLA to both primary and cultured cells and to formalin-fixated paraffin-embedded (FFPE) tissues, to map dynamic changes in protein localizations, phosphorylations and interactions across surface markers, MAPK, immune-checkpoints, T- and B-cell receptors, and adhesion panels. Comparisons of single-plex versus nine-plex assays confirmed that misPLA maintains sensitivity and specificity while increasing throughput and spatial context. Across breast cancer, lymphomas and chronic myeloid leukemia (CML) misPLA uncovered shared and disease-specific signaling patterns, underscoring convergence of oncogenic networks. By preserving tissue architecture and enabling high-content functional spatial proteomics at single-cell resolution, misPLA offers a versatile platform for dissecting signaling heterogeneity, pathway crosstalk, and therapeutic responses, with broad applications in cell biology, biomarker discovery and in precision oncology.

molecular biology↗

bioRxiv2025

FLEXTAG: A Small and Self-renewable Protein Labeling System for Anti-fading Multi-color Super-resolution Imaging

Super-resolution fluorescence imaging enables visualization of subcellular structures and molecular interactions at the nanoscale, but its broader application has been hindered by long-standing limitations in current protein tagging systems, including rapid photobleaching, tag-induced artifacts, poor post-fixation labeling efficiency, and restricted multiplexing capability. Here, we present FLEXTAG (Fluorescent Labeling for Exchangeable, X-resilient Tagging in Advanced Generic Nanoscopy), a comprehensive protein labeling system comprising three orthogonal, ultrasmall (12-18 kDa), and self-renewable protein tags that collectively overcome these major limitations of existing tagging systems, enabling optimized multi-color super-resolution imaging. Through continuous exchange of organic fluorophores, FLEXTAG supports unprecedented durations of high-resolution imaging in both live and fixed cells with minimal photobleaching. It is compatible with major super-resolution modalities, such as SIM, STED, STORM, and PAINT, and is applicable to a wide range of subcellular targets. To further address fixation-induced labeling inefficiency and background fluorescence, we developed a novel protection-based fixation method and chemical blocking strategies that significantly preserve tag accessibility and enhance signal-to-noise ratio, improvements that are broadly applicable to other protein tagging systems. Altogether, FLEXTAG enables long-term tracking of dynamic behaviors and interactions of subcellular targets, as well as mapping of nanoscale protein organizations and cellular architecture, advancing both basic research and translational applications in cell biology.

bioengineering↗

bioRxiv2025

Systematic Characterization of Optical Aberrations Reveals Cryo-FLM Localization Fidelity

Cryo-correlative light and electron microscopy (cryo-CLEM) facilitates in situ imaging and structural analysis by combining the molecular specificity of fluorescence microscopy with the ultrastructural resolution of cryo-electron microscopy. By further combining single molecule localization with cryo-CLEM, molecular positions of individual emitters can be revealed in the context of the electron density map of a cell, providing unique insights to profound questions in cell biology and virology. However, cryogenic fluorescence light microscopy (cryo-FLM) suffers from severe and spatially heterogeneous optical aberrations that distort the point spread function, limiting the accuracy of molecular localizations as well as downstream cryo-transmission electron microscopy workflows. Here, we present a systematic and quantitative analysis of optical aberrations in a commercial cryo-FLM system, uncovering the sources of significant distortions such as system imperfections, refractive index mismatches, and sample-induced heterogeneities. These system and sample induced aberrations lead to localization errors up to 90 nm laterally and over 300 nm axially, challenging the feasibility of precise molecular positioning within the vitrified specimen. We demonstrate that these errors are partially mitigated by spatially matched or adaptive point spread function models pushing the error rate down to ten nanometers or less, offering practical guidance for aberration-aware cryo-FLM and cryo-CLEM strategies. Our findings highlight the necessity of accurate, in situ point spread function modeling to achieve nanometer-scale localization in cryo-FLM. The experimental pipeline developed in this work establishes a novel tool to assess optical performance in cryo-CLEM and cryogenic focused ion beam milling workflows as the field strives toward accurate and precise molecular localization.

bioinformatics↗

bioRxiv2025

Defined human tri-lineage brain microtissues

Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes in vitro, and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their in vivo environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic -synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.

neuroscience↗

bioRxiv2025

Absolute Membrane Potential Recording with ASAP-Type Genetically Encoded Voltage Indicators Using Fluorescence Lifetime Imaging

The electrical membrane voltage (Vm) characterizes the functional state of biological cells, thus requiring precise, non-invasive Vm-sensing techniques. While voltage-dependent fluorescence intensity changes from genetically encoded voltage indicators (GEVIs) indicate Vm changes, variability in sensor expression confound determination of absolute Vm. Fluorescence lifetime imaging microscopy (FLIM) promises a solution to this problem, as fluorescence lifetime is expected to be unaffected by sensor expression and excitation intensity. By examining ASAP1, ASAP3, JEDI-1P, rEstus, and rEstus-NI (G138N:T141I) with one-photon excited FLIM measurements, we demonstrate that all sensors display a voltage-dependent lifetime. With the highest lifetime change in the Vm range of -100 to 50 mV of about 730 ps, ASAP3 and rEstus-NI are preferred for FLIM recordings. At a physiologically relevant Vm of -30 mV, the voltage sensitivity of rEstus-NI (6.6 ps/mV) is 3.6 and 1.4 times greater than that of ASAP1 and rEstus, respectively. As a proof of concept, we successfully used rEstus-NI to estimate absolute resting Vm in HEK293T, A375 melanoma, and MCF7 breast cancer cells and quantified spontaneous Vm fluctuations in A375 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/669310v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1eaff8borg.highwire.dtl.DTLVardef@12f1a54org.highwire.dtl.DTLVardef@169ad83org.highwire.dtl.DTLVardef@fe9f44_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

A molecular glue: LecRKI.9 creates stiff plasma membrane cell wall connections

Water stress challenges plasma membrane - wall attachment. When plants are exposed to strong hyperosmotic stress, water exits the cell leading to plasmolysis, where the plasma membrane detaches partially from the cell wall and reveals discrete and persistent attachment sites in the form of Hechtian strands. Although these structures have been observed in several species for more than a century, the molecular bases behind plasma membrane - wall attachment remains elusive. Through a screen of candidate proteins, we reveal that the overexpression of two lectin receptor-like proteins (LecRK-I.9 and LecTM) increases the density of plasma membrane - wall connections in N. Benthamiana. The extracellular lectin domain of LecRK-I.9 was able to remain in the wall during plasmolysis. Conversely, deletion of the lectin domain in the LecRK-I.9 overexpressor restored the density of Hechtian strands to WT levels. In Arabidopsis, upon hyperosmotic stress, LecRK-I.9 formed largely immobile clusters, whereas lectin-deleted versions of LecRK-I.9 clusters were mobile. Cluster density correlated with predicted tensile stress levels and mechanical reinforcement in the wall before plasmolysis, consistent with a scenario in which attachment of the lectin domain also reflects the wall properties. Last, while overexpressing LecRK-I.9 conferred resistance to low water potential conditions, deletion of the lectin domain in the LecRK-I.9 overexpressor restored a WT response to water stress. Altogether, this demonstrates that the lectin domain of LecRK-I.9 creates persistent plasma membrane - wall attachment sites, with physiological relevance for plant resistance to water stress. SIGNIFICANCE STATEMENTAs observed since the mid-19th century, when placed in hyperosmotic conditions, plant cells undergo plasmolysis and form thin membraneous threads called Hechtian strands. These structures reflect the presence of persistent plasma membrane - cell wall attachment sites. Yet, the molecular components behind these sites are unknown. Through a candidate screen approach, we identify and demonstrate that the lectin domain of a receptor-like kinase provides such molecular glue. We also show that the clustering of the receptor is patterned and correlate with mechanically reinforced cell walls. Conversely, we find that the promotion of attachment increases resistance to water stress. This work revisits an old plant cell biology classic, and opens new avenues of research for plant adaptation to their environment.

plant biology↗

bioRxiv2025

Let there be multifunctionality: Uncovering the criticality zoo of the AC-DCgenetic circuit

Gene regulatory networks (GRNs) govern processes such as cell fate, patterning, and adaptation. While multistability and oscillations are both common GRN dynamics in cell biology, they are typically studied and engineered in isolation. Here, we challenge this separation using the AC-DC circuit, a minimal three- gene network that merges the classical toggle switch and repressilator. Using a thermodynamic formalism and Bayesian inference, we show that even a single-inducer version of the circuit can display diverse mul-tifunctional dynamics, including the coexistence of oscillations and multistability. In addition we explore robustness, classify emergent behaviours, and analyse critical slowing down and regime transitions. Re-markably, the AC-DC circuit can produce more than 30 topologically distinct bifurcation diagrams, chal-lenging the classical view that network topology rigidly constrains dynamical outcomes. This flexibility enables synthetic capabilities that couple hysteresis with oscillations, critical slowing down, and reversibility. By uncovering the hidden potential of minimal genetic circuits and outlining design principles for their implementation, this work opens new directions for harnessing emergent complexity using the basic building blocks of life.

systems biology↗

bioRxiv2025

In situ architecture of the endosymbiont Wolbachia pipientis

Hidden within host cells, the endosymbiont Wolbachia pipientis is the most prevalent bacterial infection in the animal kingdom. Scientific breakthroughs over the past century yielded fundamental mechanisms by which Wolbachia controls arthropod reproduction to shape dynamic ecological and evolutionary trajectories. However, the structure and spatial organization of symbiont machineries that underpin intracellular colonization and orchestrate maternal inheritance remain unknown. Here, we used cryo-electron tomography to directly image the nanoscale architecture of bacterial tools deployed for host manipulation and germline transmission. We discovered that Wolbachia assembles multiple structures at the host-endosymbiont interface including a filamentous ladder-like framework hypothesized to serve as a specialized motility mechanism that enables bacterial translocation to specific host cell compartments during embryogenesis and somatic tissue dissemination. In addition, we present the first in situ structure of the Rickettsiales vir homolog type IV secretion system (rvh T4SS). We provide evidence that the rvh T4SS nanomachine exhibits architectural similarities to the pED208-encoded T4SS apparatus including the biogenesis of rigid conjugative pili extending hundreds of nanometers beyond the bacterial cell surface. Coupled with integrative structural modeling, we demonstrate that in contrast to canonical T4SS architectures, the -proteobacterial T4SS outer membrane complex assembles a periplasmic baseplate structure predicted to comprise VirB9 oligomers complexed with cognate VirB10 subunits that form extended antennae projections surrounding the translocation channel pore. Collectively, these studies provide an unprecedented view into Wolbachia structural cell biology and unveil the molecular blueprints for architectural paradigms that reinforce ancient host-microbe symbioses.

microbiology↗

bioRxiv2025

Generative semantic multiplexing (SemaPlex) for accessible and scalable multiplexed fluorescence imaging

Multiplexed fluorescence imaging enhances spatially-resolved interrogation of complex, multi-molecular cell processes that are insufficiently sampled using standard 4-5 plex imaging. To improve accessibility and scalability for multiplexed imaging, we demonstrate generative Semantic Multiplexing (SemaPlex); a simple experimental and deep learning strategy for amplifying marker plexity several-fold by semantically unmixing multiple markers combined per imaging channel. We first characterise key determinants of SemaPlex performance, achieving precise computational multiplexing of 2-to-8 markers synthetically mixed in one channel, facilitating enhanced cell phenotype classification. We then demonstrate practical SemaPlex application, acquiring 10 markers over 4 channels (3*3-plex+1) to efficiently emulate real multiplexed labelling. This permitted accurate reconstruction of quantitative single-cell phenotypic manifolds delineating cell-cycle and mitotic dynamics, with internally validated error-detection. Finally, we exemplify use of semantic guides; additional input channels that significantly enhance multiplexing fidelity. SemaPlex makes multiple-fold increases in fluorescence imaging-plexity accessible, scalable and customisable; democratising multiplexed imaging-based interrogation of complex cell biology.

systems biology↗