BackgroundImaging-based spatial transcriptomics (ST) enables the quantification of gene expression at single-cell resolution while preserving spatial context, but its utility is limited by small gene panels and challenges in accurate cell segmentation. To address these limitations, we present a graph autoencoder framework that integrates subcellular transcript distribution patterns with cell-level gene expression profiles for enhanced cell clustering in imaging-based ST (SPICEiST).ResultsThe clustering performance of SPICEiST was systematically evaluated across several cancer datasets and gene panel sizes. The results demonstrate that SPICEiST consistently outperforms the conventional cell-level gene expression-based methods in distinguishing subtle differences in cell states, as measured by the number of cell clusters and clustering indices, such as the CHI and DBI. Moreover, the findings indicate that SPICEiST can further enhance the performance, even with advancements in cell segmentation, particularly for datasets with small gene panels. Overall, these improvements in cell clustering indices, CHI and DBI, were more pronounced in datasets with small gene panels of around 300 genes, in contrast to those with large panels containing over a thousand genes. Notably, SPICEiST also reveals more spatially intermixed and less compartmentalized cell clusters, a characteristic that better reflects the complex and heterogeneous nature of tumor microenvironments. This effect was especially evident in the datasets with large panels.ConclusionsThese findings highlight the value of leveraging subcellular transcript patterns to overcome the inherent limitations of imaging-based ST, particularly for small gene panels, and may provide new insights into tumor heterogeneity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s44342-025-00056-1.

Single-cell Raman imaging reveals fructose impairs brown adipocyte differentiation.

Correlative Imaging and super resolution microscopy studies reveal complexities in determining live-dead state of bacteria.

Ecotoxicological effects of polyethylene micro/nano-plastics and Cd on the physiological response, Cd migration, and their rhizosphere microbial community of Salix matsudana.

Copper but not manganese application reduces cadmium uptake and accumulation in bread wheat.

Elucidating the impact of trans-ned-19 on two-Pore channel 2 mutants of Dictyostelium: changes in intracellular calcium levels and subsequent effect on autophagic flux.

Understanding eukaryotic cell morphometry is fundamental to cell biology, as cells exhibit a broad range of sizes and shapes during processes such as senescence, cell death, mitosis, and migration. Dynamic changes in subcellular compartments and protein distribution also occur, impacting cytoplasmic and nuclear characteristics. Traditional measurement methods are often limited, highlighting the need for alternatives that comprehensively integrate data from both the cytosol and nuclei while tracking individual live cells over time. To address these limitations, we developed Cellular Morphometric Analysis (CellMorph), a novel tool designed to objectively assess multiple features of individual eukaryotic cells, including cell size, shape, cytosolic staining, and morphometry. CellMorph can analyze bright-field and fluorescent images, accommodating both nonstained cells and those expressing fluorescent reporters or chromogenic labels. We validated the tool using various cellular models and specific staining protocols that target fundamental processes such as apoptosis, autophagy, and senescence. CellMorph captures the intricate heterogeneity within cell populations by providing a multidimensional perspective on individual cellular features and their differential responses to various stresses. This capability to track phenotypic changes over time makes CellMorph particularly valuable for studying dynamic cellular responses. Detailed morphometric data are essential for investigating cellular behavior in pathogenic processes and responses to stressors, including therapies or environmental changes. By integrating multiple parameters, CellMorph represents a significant advancement in cell biology, offering researchers a powerful tool to explore the complexities of cellular morphometry effectively.

FHF (fibroblast growth factor homologous factor) variants associate with arrhythmias. Although FHFs are best characterized as regulators of voltage-gated sodium channel (VGSC) gating, recent studies suggest broader, non-VGSC-related functions, including regulation of Cx43 (connexin 43) gap junctions and hemichannels, mechanisms that have generally been understudied or disregarded. We assessed cardiac conduction and cardiomyocyte action potentials in mice with constitutive cFgf13KO while targeting Cx43 gap junctions and hemichannels pharmacologically. We characterized FGF (fibroblast growth factor) 13 regulation of Cx43 abundance and subcellular distribution. With proximity labeling proteomics, we investigated novel candidate mechanisms underlying FGF13 regulation of Cx43. FGF13 ablation prolonged the QRS and QT intervals. Carbenoxolone, a Cx43 gap junction uncoupler, markedly prolonged the QRS duration, leading to conduction system block in cFgf13KO but not in wild-type mice. Optical mapping revealed markedly decreased conduction velocity during ventricular pacing. Microscopy revealed perturbed trafficking of Cx43, reduced localization in the intercalated disc, and suggested decreased membrane Cx43 but increased Cx43 hemichannels in cardiomyocytes from cFgf13KO mice. Resting membrane potential was depolarized, and action potential duration at 50% repolarization was prolonged in cFgf13KO cardiomyocytes. Both were restored toward wild-type values with Gap19 (a Cx43 hemichannel inhibitor), expression of FGF13, or expression of a mutant FGF13 incapable of binding to VGSCs, emphasizing VGSC-independent regulation by FGF13. To assess the functional impact of resting membrane potential depolarization, hearts were subjected to hypokalemia, which had no effect in wild-type hearts but fully rescued conduction velocity in cFgf13KO hearts. Proteomic analyses revealed candidate roles for FGF13 in the regulation of vesicular-mediated transport. FGF13 ablation destabilized microtubules and reduced the expression of tubulins and MAP4, the major cardiac microtubule regulator. FGF13 regulates microtubule-dependent trafficking and targeting of Cx43 and impacts cardiac impulse propagation via VGSC-independent mechanisms.

Abstract The precise control of subcellular distribution offers significant advantages for advancing the understanding of cellular biology and targeted theranostics strategies. Herein, we report anionic Hofmeister effect triggered allostery of pH‐responsive metal–organic cage MOC‐68, which regulates the subcellular distributions with the salting‐out MOC‐68‐BF 4 exhibiting preferential cell membrane anchoring, while the salting‐in MOC‐68‐Cl demonstrating more endocytosis for lysosome targeting. Owing to the integration of multi‐functionalities of protonating imidazole moieties, conformationally adaptive amine vertices, and microsecond luminescent lifetimes into a nanocage, these MOCs show pH‐responsive singlet oxygen ( 1 O 2 ) generation through energy transfer pathways, and achieve microenvironment‐programmed photodynamic precision by anchoring MOC‐68‐BF 4 to near‐neutral cell membranes whereas enriching MOC‐68‐Cl in acidic lysosomes, which drives spatially resolved 1 O 2 generation gradients via protonation‐optimized metal‐to‐ligand charge transfer. Moreover, MOC‐68‐Cl achieves superior photocytotoxicity through PANoptosis‐mediated immunogenic cell death, driving primary tumor ablation via localized 1 O 2 burst while systemically activating T cell‐dependent adaptive immunity to concurrently suppress tumor progression and inhibit lung metastasis. This work highlights the potential of anion‐mediated biological behaviors of MOCs, providing a way to advance nanocage biomedicine for future precision theranostic applications.

The precise control of subcellular distribution offers significant advantages for advancing the understanding of cellular biology and targeted theranostics strategies. Herein, we report anionic Hofmeister effect triggered allostery of pH-responsive metal-organic cage MOC-68, which regulates the subcellular distributions with the salting-out MOC-68-BF4 exhibiting preferential cell membrane anchoring, while the salting-in MOC-68-Cl demonstrating more endocytosis for lysosome targeting. Owing to the integration of multi-functionalities of protonating imidazole moieties, conformationally adaptive amine vertices, and microsecond luminescent lifetimes into a nanocage, these MOCs show pH-responsive singlet oxygen (1O2) generation through energy transfer pathways, and achieve microenvironment-programmed photodynamic precision by anchoring MOC-68-BF4 to near-neutral cell membranes whereas enriching MOC-68-Cl in acidic lysosomes, which drives spatially resolved 1O2 generation gradients via protonation-optimized metal-to-ligand charge transfer. Moreover, MOC-68-Cl achieves superior photocytotoxicity through PANoptosis-mediated immunogenic cell death, driving primary tumor ablation via localized 1O2 burst while systemically activating T cell-dependent adaptive immunity to concurrently suppress tumor progression and inhibit lung metastasis. This work highlights the potential of anion-mediated biological behaviors of MOCs, providing a way to advance nanocage biomedicine for future precision theranostic applications.

Subcellular distribution-based reference-free cancer cell discrimination with a novel AIE cationic probe.

Dynamic remodeling of pectin and hemicellulose for contrastive cadmium accumulations with root cell walls in distinct Salvia miltiorrhiza ecotypes.

Regulation of K+-dependent Na+/Ca2+-exchanger subtype 4, NCKX4, by palmitoylation.

Heat stress exacerbated by global warming severely impairs the growth and tea quality of the tea plant (Camellia sinensis). Trehalose is pivotal for regulating plant growth and enhancing stress resistance. However, the molecular characteristics, expression patterns, and regulatory mechanisms of trehalose metabolism genes in tea plants under heat stress remain unclear. Therefore, this study conducted a comprehensive investigation of trehalose metabolism genes in the Tieguanyin tea plant genome. A total of 30 trehalose metabolism genes were identified, including 17 trehalose-6-phosphate synthase (CsTPS), 9 trehalose-6-phosphate phosphatase (CsTPP), and 4 trehalase (CsTRE) genes. These genes were characterized in terms of their chromosomal locations and gene structures; the encoded proteins were characterized in terms of their phylogenetic relationships, conserved motifs, functional domains, physicochemical properties, and subcellular distributions. The results showed that these genes exhibit family-specific structural and functional features, laying a foundation for further functional studies. Collinearity analysis identified 20 homologous gene pairs between tea plants and Arabidopsis thaliana, significantly more than the 3 pairs with Oryza sativa, suggesting a closer evolutionary relationship with A. thaliana. Additionally, five intraspecific duplicated gene pairs were identified, all with Ka/Ks values < 1, indicating they have undergone strong purifying selection during evolution, leading to functional stability. Cis-acting element analysis revealed abundant stress-responsive, light-responsive, and phytohormone-responsive elements in the promoter regions of these trehalose metabolism genes, indicating their potential involvement in tea plant stress resistance regulation. Differential expression analyses under heat stress with exogenous trehalose treatment (CK: control, T: water-sprayed heat stress, TT: 5.0 mM trehalose-sprayed heat stress) identified six differentially expressed genes (DEGs). We further analyzed the expression patterns of these DEGs. Specifically, CsTPS1, CsTPS5, and CsTPS12 were increasingly upregulated in CK, T, and TT, respectively, while CsTPP1 and CsTPP2 were upregulated in TT relative to T. Additionally, CsTRE1, CsTRE2, and CsTRE4 showed downregulation in TT compared to T, though they were not classified as DEGs. These findings indicate that exogenous trehalose application modulates trehalose metabolism by promoting CsTPS and CsTPP expression while inhibiting CsTRE expression, thereby increasing endogenous trehalose content in tea plants under heat stress. Yeast heat stress tolerance assays confirmed that CsTPS1, CsTPS5, CsTPS12, and CsTPP1 enhanced yeast survival at 38 °C, verifying their function in improving organismal heat stress tolerance. In conclusion, these results clarify the roles of trehalose metabolism genes in tea plants’ heat stress response, demonstrating that exogenous trehalose modulates their expression to increase endogenous trehalose levels. This study provides a theoretical foundation for exploring trehalose-mediated heat stress resistance mechanisms and improving tea plant stress tolerance via genetic engineering.

Neutrophil extracellular traps (NETs) released by activated neutrophils in the tumor microenvironment has emerged as a pivotal mediator in promoting tumor metastasis. The alteration of the subcellular localization of neutrophil elastase (NE) is crucial for NETs formation. The majority of NE (≈80%) translocate from azurophilic granules to the nucleus, facilitating histone degradation and chromatin decondensation. A few NE are transported to the cell membrane, a unique feature of activated neutrophils that distinguishes them from other leukocyte subpopulations. To address NETs-mediated HCC metastasis, a peptidic nanomaterial (FTP-NPs) is developed that specifically binds NE on activated neutrophil membranes and undergoes in situ fibrillar transformation, forming NE-fibril clusters. These NE-fibril clusters deactivate NE by altering their conformation or binding mode. Subsequently, a series of feedback mechanisms is triggered, which regulates NE membrane concentration by promoting its transport to the membrane rather than the nucleus. The NE-fibril clusters can remain on the activated neutrophil membrane for an extended period, enabling continuous binding and deactivation of newly transported NE, thereby reversing the formation of NETs. Besides, the extracellular NE-fibril clusters also act as a physical barrier to prevent NETs from adhering to tumor cells, further disrupting the metastatic cascade. In vitro, in vivo, and single-cell RNA sequencing (scRNA-seq) data confirm that FTP-NPs significantly reduce NETs formation, reduce metastatic burden, and enhance antitumor immune response. Compared with commercial NE inhibitors, this strategy precisely and locally regulates NE subcellular distribution within neutrophils in tumor tissue, minimizing off-target effects and systemic toxicity. The NE-fibril clusters may establish an innovative therapeutic approach for NETs-mediated tumor metastasis.

The force-gated ion channel PIEZO1 confers mechanosensitivity to many cell types. While the structure and physiological roles of PIEZO1 are well-described, the subcellular distribution and the impact of the cellular microenvironment on PIEZO1 conformation and function are poorly understood. Here, using MINFLUX nanoscopy, we demonstrate that PIEZO1 channels accumulate in pit-shaped invaginations that are distinct from classical membrane invaginations such as clathrin-coated pits and caveolae, thereby possibly creating hotspots for mechanotransduction. Moreover, by measuring intramolecular distances in individual PIEZO1 channels with nanometer precision, we reveal subcellular compartment-specific differences in PIEZO1 conformation at rest and during activation that correlate with differences in PIEZO1 function and are possibly caused by differences in cytoskeletal architecture. Together, our data provide previously unrecognized insights into the complex interplay of forces that determine how PIEZO1 alters membrane shape and, vice versa, how the membrane together with the cytoskeleton affect the conformation and function of individual PIEZO1 channels.

Abstract Background and Aims Pathogenic variants of the polycystic kidney and hepatic disease 1 (PKHD1) gene alter epithelial water and ion transport, and lead to renal collecting duct dilatations and to liver cysts formation, hallmarks of autosomal recessive polycystic kidney disease (ARPKD). In the context of epithelial models using ARPKD genetics, loss of fibrocystin/polyductin (FPC) protein function results in a change from absorptive to secretory epithelial phenotype, thus reflecting one major pathophysiological mechanism for cyst growth. Our aim is to understand the molecular aspects of defective epithelial homeostasis by identifying channels that mediate aberrant electrolyte transport and promote cystic fluid accumulation. Here we employ a monolayered, three-dimensional (3D) epithelial model, allowing us to control cystogenic signals, determine channel distribution and test therapeutic targets. Method To model ARPKD conditions, Pkhd1 knockout clones were established using CRISPR/Cas9 in a principle-like Madin-Darby canine kidney (pl-MDCK) cell line. Single cells seeded into matrigel were allowed to form polarized spheroids with a lumen and stimulated by forskolin to enhance cAMP levels inducing secretory cystic epithelia. Measurement of cyst pressure and determination of the proportional lumen (i.e. ratio of lumen to spheroid area) confirmed enhanced water and ion transport across the epithelial barrier, as observed in ARPKD. The involvement of chloride channels in ARPKD disease progression was addressed by pharmacological inhibition, and subcellular distribution of water and ion channels was tested. Impact of FPC function was determined expressing functional domain constructs. Results Besides significantly enhanced cyst pressure, which is represented also by increased proportional lumina of pl-MDCK spheroids, FPC deficiency results in a prominent re-localization of the ion channels ENaC and CFTR from the apical to the basolateral membrane domain. In wild type cells, enhanced cAMP-levels mimic this phenotype and correlate with increased pressure values, whereas tight junctions (ZO-1) and the water channel aquaporin-2 remain apical in control and disease conditions. Both, treatment with selective chloride channel- inhibitors as well as controlled expression of the FPC cytoplasmic domain allowed us to suppressed cAMP-induced cyst formation in Pkhd1-KO cell lines, and controls. Conclusion A secretory phenotype of cyst-lining epithelia in disease conditions, as represented by enhanced cAMP-signals, mimics disturbed epithelial homeostasis in ARPKD and associates with altered ion channel distribution. Targeting of mis-localized ion channels represents a strategy to test for therapeutic options. Reconstitution of a functional c-terminal domain of FPC was sufficient to correct aberrant ion flux in our 3D in vitro model, suggesting that the cytoplasmic tail may function as a natural inhibitor of cyst growth and disease progression.

Neurons, as long-lived non-dividing cells with complex morphology, depend on a highly elaborate secretory trafficking system which enables a constant turnover of proteins and membranes. Previously, it was shown that simplified, Golgi-related structures called Golgi satellites (GS) are present in the dendrites of primary hippocampal neurons. These organelles are distinct from the somatic Golgi complex and are involved in de novo glycosylation and local forward trafficking of membrane proteins. However, the question of whether GS are also targeted to the axons of principal neurons remained unanswered. In this study, we investigated the subcellular distribution of GS in adult hippocampal neurons. Our findings showed that GS are present all along the axon, extending to the distal tips of the growth cone. Similar to dendritic GS, the axonal organelles are labeled by the same GS markers and are capable of mature glycosylation. Live imaging experiments revealed the presence of both mobile and immobile GS in the axon, and that the switch between active transport and stalling of GS was modulated by neuronal firing. We found that GS frequently pause at en passant synapses and remain stationary for longer time periods at activated pre-synaptic boutons. This behavior is dependent on the actin cytoskeleton and the actin-based motor protein myosin VI. Overall, our study demonstrates that neuronal activity can dynamically regulate the positioning of GS in the axon, shedding light on the intricate mechanisms underlying organelle trafficking in neurons.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05896-2.

Fluorescent biomarkers are widely used in cell biology to study gene expression and protein localization. Translational fusions, where a fluorescent protein is directly linked to a protein of interest, allow researchers to monitor subcellular distribution, while transcriptional fusions are used to assess promoter activity. Despite their extensive application, the potential physiological impact of these fluorescent tags on host cells remains largely overlooked. In this study, we investigated how the Green Fluorescent Protein (GFP) influences stress responses in the yeast Saccharomyces cerevisiae. We generated translational fusions of GFP with two proteins: Pab1p, a key component of stress granules, and Sur7p, a membrane-associated protein involved in the organization of Can1-enriched plasma membrane domains. These targets were selected because the cellular behavior of S. cerevisiae under varying heat and oxidative stress conditions remains incompletely understood. Our main findings indicate that the Pab1p-GFP fusion confers increased resistance to heat shock compared to the wild-type strain. Furthermore, strains expressing GFP-tagged proteins displayed altered cultivability under oxidative stress, suggesting that the presence of GFP can modulate the cellular stress response. In silico structural analysis confirmed that GFP fusion does not alter the overall 3D structure or function of the tagged proteins. This suggests that the observed phenotypic differences are likely due to the intrinsic properties of GFP, particularly its known ability to scavenge reactive oxygen species (ROS). These results highlight an important consideration for researchers using fluorescent tags: while GFP is generally considered a neutral reporter, it can influence cellular behaviour under stress, potentially affecting the interpretation of experimental outcomes.

A common mechanism by which cancer cells acquire resistance to chemotherapeutics is through the overexpression of efflux pumps, enabling the removal of cytotoxic agents, such as anthracycline drugs. However, platinum anticancer agents that crosslink DNA and interact with proteins are poor efflux pump substrates. Here, we design dual warhead drug conjugates by tethering a platinum pharmacophore to the doxorubicin backbone. These drug conjugates retain the anticancer activity of anthracyclines and exhibit the ability to both circumvent drug efflux and delay the acquisition of drug resistance. In vivo experiments demonstrate that such drug conjugates extend survival in a preclinicalorganoid-based model of metastatic colon cancerin mice. Mechanistic studies indicate that these drug conjugates overcome resistance through covalent platinum-protein interactions, leading to significantly improved drug retention and alteration of subcellular drug distribution. This application of platinum offers many opportunities to confront issues related to chemoresistance and alternative pathways for augmenting conventional chemotherapeutics.