Biology subjects

Wen, G.

Publications and source records attributed to Wen, G..

18 recordsLinked to original sources

bioRxiv2026

Structural cell biology by mega-expansion microscopy

Structural characterization of macromolecular assemblies within intact cells remains one of the central challenges in cell biology. While cryo-electron tomography provides unparalleled structural information, its applicability is limited by sample thickness, imaging throughput, and accessibility. Fluorescence microscopy offers molecular specificity and compatibility with intact biological specimens but has so far lacked the spatial resolution required to visualize cellular ultrastructure. Here we introduce Mega-expansion microscopy (Mega-ExM), a fluorescence imaging approach that enables structural visualization of whole cells using conventional confocal microscopes. Mega-ExM combines iterative hydrogel expansion with whole-proteome NHS-dye labeling and post-expansion immunostaining to achieve tunable expansion factors of up to [~]1,500-fold while preserving ultrastructure. At expansion factors of 40-260x, Mega-ExM resolves centrioles, mitochondrial cristae, protein-dense domains within mitochondrial cristae consistent with respiratory-chain supercomplexes, the synaptonemal complex, and nuclear pore complexes (NPCs) with high fidelity. Particle averaging of [~]200x expanded NPCs yields reconstructions with a structural resolution of [~]35 [A], approaching what cryo-electron tomography has achieved for selected protein assemblies. By combining molecular specificity, large imaging volumes, and nanoscale structural resolution on conventional fluorescence microscopes, Mega-ExM establishes a broadly accessible platform for in situ structural biology.

biophysics↗

bioRxiv2026

Physics-driven self-supervised learning for quantitative high-fidelity structured illumination microscopy

Structured illumination microscopy (SIM) enables rapid, long-term super-resolution (SR) imaging of live-cell dynamics. However, although current state-of-the-art (SOTA) SIM reconstruction methods achieve high-fidelity structural SR, they consistently lack reliable intensity quantification, restricting their use in quantitative biology. Here, we develop qHiFi-SIM, a physics-driven self-supervised learning framework for quantitative high-fidelity SR-SIM imaging. By leveraging the wide-field image as a physical intensity reference, our approach enables self-supervised training without reliance on SR data with ground-truth intensity. qHiFi-SIM achieves high structural fidelity (structural similarity, SSIM > 0.95) with a twofold resolution enhancement, while maintaining excellent intensity linearity (coefficient of determination, R{superscript 2} > 0.99). It also exhibits strong transferability across diverse SIM setups and typical samples, and is compatible with SOTA SIM algorithms, enabling direct quantitative correction of their intensity deviations without retraining. We demonstrate the unique advantages of qHiFi-SIM for live-cell quantitative visualization of mitochondrial structure, intensity, and membrane potential dynamics, as well as for high-fidelity SIM-FRET (Forster resonance energy transfer) functional imaging. We anticipate that qHiFi-SIM will serve as a practical tool for SR structural visualization and quantitative functional imaging in live cells.

Cell Biology↗

bioRxiv2026

PD1-induced Shp2 condensation organizes inhibitory signalosomes through selective substrate partitioning

The formation of microclusters is a hallmark of PD1 engagement with its ligands, yet the physical basis and functional significance of this phenomenon remain unclear. Here we show that ligand-bound PD1 licenses Shp2 self-association and liquid-liquid phase separation (LLPS), producing dynamic PD1:Shp2 condensates whose liquidity depends on Shp2 catalytic activity. Mutations that selectively disrupt Shp2 self-association weaken PD1 microcluster formation and impair PD1 inhibitory function. Mechanistically, PD1-induced Shp2 LLPS promotes the co-compartmentalization of signaling substrates such as CD3{zeta} and CD28, thereby facilitating its dephosphorylation. These findings identify Shp2 LLPS as an intrinsic organizing principle of the PD1 inhibitory pathway that links enzymatic activation, substrate selectivity and mesoscale assembly to suppress T cell activation.

biochemistry↗

bioRxiv2026

Tipping Points and Emergent Phases of Species Diversity in Mutualistic Ecological Networks under Global Warming

Global warming poses a severe threat to ecosystem resilience and biodiversity, potentially driving species loss, declining diversity, and ecosystem collapse. Accurately predicting tipping points of ecological resilience under global warming remains a fundamental challenge. Here, we develop a theoretical framework to identify climate-induced tipping points in mutualistic ecological networks, capturing critical transitions under warming scenarios. We show that rising temperatures, increasing intraspecific competition, and deteriorating mutualistic interaction can drive ecosystems through three distinct dynamical phases of species diversity: stable full coexistence, stable partial coexistence, and total extinction. Notably, ecosystems with low species abundances are particularly vulnerable, facing heightened risks of diversity loss and extinction. Moreover, the warming rate modulates these transitions, as slower warming grants endangered species a window of time within which to adjust and attenuate the loss of resilience. Applying this framework to assess various emission mitigation pathways, we find that "pollute first, clean up later" scenarios pose a threat to biodiversity, leading to irreversible biodiversity losses even after subsequent emission reductions. Our findings offer critical insights into the timing and mechanisms of ecological tipping points under climate change and underscore the urgency of proactive mitigation strategies.

ecology↗

bioRxiv2025

Migration patterns and hybridization within the Asian stonechat complex in response to a major geographical barrier

Long-distance avian migration is thought to be under strong natural selection. Facing geographical barriers, migrants display various patterns considered to be adaptive. For example, they may detour along either side around the barrier or cross it, requiring specialized behavioral adaptations. Variations within closely related taxa are excellent sources for understanding the evolutionary background of migration and how barriers are shaping migration routes. In Asia, some species are assumed to have a migratory divide in response to the major geographical barrier, the Qinghai-Tibet Plateau (QTP), including the stonechat taxa (Siberian Stonechat Saxicola maurus maurus and Amur Stonechat S. stejnegeri). As they detour along either side of the QTP, these taxa are believed to disfavor a crossing over the highland. However, the more southernly distributed Tibetan Stonechat (S. m. przewalskii) breeds on the QTP, suggesting adaptation to high elevation. To investigate migration patterns and the potentially associated genetic differences, we studied migration routes and population genetics of four populations around the assumed migratory divide in Russia and Mongolia, and of one from the QTP in China. Our results confirmed the existence of a migratory divide between maurus and stejnegeri, albeit with extensive hybridization. We observed both the hypothesized western and eastern routes, but also found individuals employing intermediate routes crossing the QTP, of which two-thirds were clear hybrids. Meanwhile, przewalskii followed a highland-crossing route and was genetically differentiated from maurus and stejnegeri. The diverse migration routes among Asian stonechats show differential responses towards the geographical barrier. The intermediate route may be associated with hybridization, and its conditional viability may facilitate gene flow between maurus and stejnegeri. The Asian stonechat complex thus offers great opportunities for novel research of the genetics and evolution of migration. The specific evolutionary background associated with inhabiting and crossing the QTP can offer new perspectives in this field. Teaser textMigratory divides can arise in birds because alternative routes around migratory barriers would select for behaviors to restrict hybridization. Hybrids of parental types that employ alternative routes are hypothesized to embark on intermediate routes that would expose them to suboptimal conditions, resulting in post-zygotic reproductive isolation. However, this hypothesis is challenged when a sister taxon actually breeds on the geographical barrier. This is the case in the Asian stonechat complex that breeds near or on the Qinghai-Tibet Plateau (QTP), the roof of the world. We demonstrated a migratory divide in central Siberia to Mongolia for race maurus and stejnegeri, yet showed also evidence for extensive hybridization. Hybrids migrated along a newly discovered intermediate route, seemingly viable and overlaps with the migration trajectory of race przewalskii over the eastern part of the QTP. The Asian migratory divide relative to the QTP thus provided new insights to the evolution of landbird migration.

evolutionary biology↗

bioRxiv2025

Aβ Antibodies Target Not Only Amyloid Plaques But Also Distinct Brain Cells And Vessels

BackgroundAntibodies against amyloid-{beta} (A{beta}) are the only therapies capable of slowing cognitive decline and reducing A{beta} plaque burden in Alzheimers disease (AD). Yet the precise sites where antibodies engage A{beta} in the brain, and the mechanisms that drive A{beta} clearance, are not fully defined. Defining the A{beta} antibody engagement with A{beta} in brain is essential to understand how immunotherapy can be beneficial for AD. MethodsWe administered the well-establsihed N-terminal A{beta} antibody 6E10 via intrahippocampal (IH), cisterna magna (CM), or intraperitoneal (IP) injection in AD mouse models. N-terminal A{beta} antibodies were seen as most effective in AD mouse models. Antibody 6E10 was not only assessed in A{beta} plaques, but also examined in association with diverse brain cells and vascular compartments. Glymphatic dynamics were evaluated following A{beta} antibody treatment. ResultsAs expected, A{beta} antibody 6E10 bound to A{beta} plaques but remarkably also localized to vulnerable neurons, such as hippocampal CA1 pyramidal cells, as well as microglia, astrocytes, oligodendrocytes, perivascular macrophages (PVMs), and blood vessels. Glymphatic function showed no significant alterations after antibody administration. ConclusionsA{beta} antibodies distribute not only to amyloid plaques but also to neurons, glial cells, and blood vessels. This study provides detailed localizations of antibody in the AD brain, offering valuable insights into cellular targets and spatial dynamics of A{beta} antibody-based immunotherapy.

neuroscience↗

bioRxiv2024

Discovery of novel 1,4-dicarbonylthiosemicarbazides as DNA gyrase inhibitors for the treatment of MRSA infection

Antibiotic resistance has become a serious threat to public health, thus novel antibiotics are urgently needed to combat drug-resistant bacteria including MRSA (methicillin-resistant S. aureus). The 1,4-dicarbonylthiosemicarbazide is an interesting chemotype that could exhibit antibacterial activity. However, the currently available compounds are not as potent as clinical antibiotics. Herein, we adopted the computer-aided drug design strategy, substructure search, to retrieve antibacterial 1,4-dicarbonylthiosemicarbazide derivatives, and identified compound B5 (Specs ID: AG-690/15432331) from the Specs chemical library that exhibited moderate activity (minimum inhibitory concentration (MIC): 6.25 {micro}g/mL) against S. aureus (ATCC 29213). Based on that compound, we further designed and synthesized 45 derivatives, and evaluated their antibacterial activity. Eight derivatives were more potent than or equivalent to vancomycin (MIC: 1.56 {micro}g/mL). We compared the three most potent ones for their cytotoxicity to HepG2 and HUVEC cells and selected compound 1b as our lead compound for comprehensive biological evaluation. As a result, compound 1b exhibited a bacteriostatic mode, and was active against a panel of gram-positive bacteria strains, metabolically stable, and effective to protect the mice from MRSA infection. More importantly, we applied 2D similarity calculation and reverse docking to predict potential targets of compound 1b. Through experimental validation and molecular dynamics simulation, we were able to confirm that compound 1b inhibited S. aureus DNA gyrase (IC50: 1.81 {micro}M) and DNA supercoiling, potentially by binding to the ATPase domain, where ASP81, GLU58 and GLN91 formed key hydrogen bonds. Taken together, we have discovered a new class of DNA gyrase inhibitors represented by compound 1b for the treatment of MRSA infection, through the design, synthesis, and biological evaluation of novel 1,4-dicarbonylthiosemicarbazides. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/611349v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@549dadorg.highwire.dtl.DTLVardef@13bff64org.highwire.dtl.DTLVardef@1fc2c00org.highwire.dtl.DTLVardef@acee76_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAn interesting 1,4-dicarbonylthiosemicarbazide derivative B5 with moderate antibacterial activity for S. aureus was discovered by substructure search. C_LIO_LIBased on B5, 45 new 1,4-dicarbonylthiosemicarbazides were designed, synthesized and biologically evaluated, which led to the discovery of a promising lead compound 1b. C_LIO_LICompound 1b exhibited good antibacterial activity for a panel of gram-positive bacteria strains, and was effective to protect mice from MRSA infection. C_LIO_LIThe 1,4-dicarbonylthiosemicarbazides represented by compound 1b were discovered as a new class of DNA gyrase inhibitors, by target prediction and experimental validation. C_LI

pharmacology and toxicology↗

bioRxiv2024

Flat-field superresolution structured illumination microscopy with joint temporal-spatial light modulation

AbstractSuper-resolution structured illumination microscope (SR-SIM) has been established as a powerful tool for visualizing subcellular dynamics and studying organelle interactions in live cells. However, the interfering Gaussian beams result in a limited and nonuniform field of view (FOV) which hinders its application for large whole-cell dynamics and pathological sample imaging. Here, we proposed a joint spatial-temporal light modulation (JSTLM) method to reshape the excitation light field into flat-field structured illumination without disturbing the interfering fringes. Our flat-field structured illumination microscopy (flat-field SIM) improves the uniformity across the whole FOV significantly, hence enabling SR image stitching. Skeleton dynamics and vesicle transportation in and between whole cells were visualized by flat-field SIM. With the stitching of multi-FOV flat-field SIM images, millimeter-sized SR images can be obtained which provides the possibility for cell heterogeneity studies and pathological diagnoses. The JSTLM method can be further incorporated with regions of interest to reduce unnecessary photodamage to live cells during multicolor imaging. ContributionsY.L. and X.H.C. conceived and designed the idea. Y.L., S.M.L., X.J., and G.W. built the SIM setup. Y.L. performed the data acquisitions. Y.L. and X.H.C. conducted the optical wave simulation. J.Z. prepared the cell samples. Y.T. and L.B.W. performed the image analyses. Y.L. prepared the illustrations. X.H.C. and J.G. supervised the project. Y.L. and H.L. wrote the manuscript.

cell biology↗

bioRxiv2023

Castration Model Illuminates Sex Differences in Healthy Aging: Insights from Metabolome and Transcriptome Analyses

BackgroundSex, as a critical biological variable, has historically been underappreciated, despite the pervasive influence of sexual dimorphism across physiological and pathological processes. A significant obstacle to advancing sex-biased biological research is the absence of an effective animal model. In recent years, castration has emerged as a potential model for elucidating sex-based differences in the context of healthy aging, where it has been shown to equalize lifespan and growth trajectories in genetically diverse mice. However, the molecular shifts induced by castration in common laboratory models, such as C57BL/6 mice, and the broader applicability of this model to other sex-related biological contexts remain largely unexplored. MethodsWe employed multi-omics and observational analyses to investigate the molecular changes associated with sex and sex hormones following castration. We analyzed serum, kidney, and liver samples from 12-week-old and 18-month-old castrated male C57BL/6 mice, alongside intact male and female counterparts. The castration model was further applied to assess differences in cisplatin-induced toxicity and age-related cognitive decline in comparison to unaltered controls. ResultsLC-MS/MS metabolomics revealed that castrated males exhibited substantial alterations in steroid hormone levels and increased concentrations of antioxidant compounds, such as taurine, despite identical diets. Integrated metabolome-transcriptome analysis confirmed distinct patterns of lipid peroxidation and oxidative stress across sham-operated female, male, and castrated male mice. Histopathological evaluations following cisplatin treatment and aging-related behavioral tests demonstrated the models utility in investigating sex-dependent drug toxicity and cognitive decline. These findings underscored the critical role of sex hormones in modulating both toxicity defense mechanisms and cognitive performance. ConclusionThis study provides a systematic multi-omics spectrum on the castration model and demonstrates its capacity to feminize metabolic and transcriptomic profiles, establishing it as a valuable tool for exploring sex hormone-driven biological differences. Our findings lay the groundwork for further mechanistic studies and broaden the potential applications of the castration model in diverse biomedical research domains.

systems biology↗

bioRxiv2023

BacScan: An Unbiased and Genome-Wide Approach to Identify Bacterial Highly Immunogenic Proteins

Bacterial pathogens are the second leading cause of death worldwide. However, the development of bacterial vaccines has been challenged by the presence of multiple serotypes and the lack of cross-protection between serotypes. Therefore, there is an urgent need to identify protective antigens conserved across serotypes in order to develop a broadly protective vaccine. Here, we have developed an unbiased and genome-wide technique, BacScan, which uses bacterial-specific serum to rapidly identify highly conserved immunogenic proteins by combining phage display, immunoprecipitation, and next-generation sequencing. As a proof of concept, we identified 19 highly immunogenic proteins from Streptococcus suis core proteins. Immunoreactivity analysis of mouse, pig, and human sera indicated that 2 proteins could be the potential targets for the development of serological diagnostics. Eight proteins provided 20%-100% protection against S. suis challenge in immunized animals, indicating the potential vaccine targets. BacScan can be applied to any bacterial pathogen and has the potential to accelerate the development of a broadly protective bacterial vaccine. TeaserA novel method to identify the highly conserved immunogenic bacterial proteins as targets for the development a broadly protective bacterial vaccine.

microbiology↗

bioRxiv2023

Trifunctional linkers enable improved visualization of actin by expansion microscopy

Expansion Microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed, however, with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by [~]10 times. We demonstrate the potential of trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10x ExM of actin labeled with TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases where the concentration of target-bound monomers is insufficient for high-quality ExM.

biochemistry↗

bioRxiv2022

Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed by computational detection tools. During the last decade, a plethora of such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools were used and detected over 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were empirically validated using three orthogonal methods. Generally, tool-specific precision values are high and similar (median of 98.8%, 96.3%, and 95.5% for qPCR, RNase R, and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant tool differentiators. Furthermore, we demonstrate the complementarity of tools through the increase in detection sensitivity by considering the union of highly-precise tool combinations while keeping the number of false discoveries low. Finally, based on the benchmarking results, recommendations are put forward for circRNA detection and validation.

genomics↗

bioRxiv2022

Rapid Development of a Mucosal Nanoparticle Flu Vaccine by Genetic Engineering of Bacteriophage T4 using CRISPR-Cas

Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry sites are considered to be the most effective way to prevent infection. A universal platform that can be customized for development of mucosal vaccines against any given pathogen is therefore highly desired. Here, we demonstrate an efficient approach to develop nasal mucosal vaccines through genetic engineering of T4 phage to generate antigen-decorated nanoparticles. The antigen coding sequence was inserted into T4 genome in-frame at the C terminus of Soc (small outer capsid protein) using the CRISPR-Cas phage editing technology. During the propagation of recombinant T4 phages in E. coli, the Soc-antigen fusion proteins self-assemble on T4 capsids to form antigen-decorated nanoparticles that have intrinsic adjuvant activity and mucosal adhesive property. As a proof of concept, we showed that intranasal immunization with Flu viral M2e-decorated T4 nanoparticles efficiently induced local mucosal as well as systemic immune responses and provided complete protections against divergent influenza viruses in a mouse model. Potentially, our platform can be customized for any respiratory pathogen to rapidly generate mucosal vaccines against future emerging epidemics and pandemics.

immunology↗

bioRxiv2021

Bacteriophage T4 Vaccine Platform for Next-generation Influenza Vaccine Development

Developing influenza vaccines that protect against a broad range of viruses is a public health priority, and several conserved viral proteins or domains have been identified as promising targets for such vaccine development. However, none of the targets is immunogenic, and vaccine platforms that can incorporate multiple antigens with enhanced immunogenicity are desperately needed. In this study, we provided proof-of-concept for the development of next-generation influenza vaccine using T4 phage virus-like particle (VLP) platform. With extracellular domain of influenza matrix protein 2 (M2e) as a readout, we showed that more than 1,280 M2e molecules can be assembled on a 120x90 nanometer phage capsid to form T4-M2e VLPs, which are highly immunogenic and induced complete protection against influenza virus challenge without any addition adjuvant. Potentially, additional conserved antigens or molecular adjuvants could be incorporated into the T4-M2e VLPs to customize influenza vaccines to address different issues. All the components of T4 VLP vaccines can be mass-produced in E. coli in a short time, therefore, providing a rapid approach to deal with the potential influenza pandemic.

microbiology↗

bioRxiv2020

Parietal cell-Specific SLC26A9 Deletion induces spontaneous Gastric Carcinogenesis in Mice

Previous study showed that Slc26a9 loss impairs parietal cell function and survival. We investigated whether Slc26a9 loss causes spontaneous gastric carcinogenesis in mice and plays a role in the development and progression in human gastric cancer (GC). Gastric histopathology and potential molecular mechanism were explored in Slc26a9 knockout mice and wild-type littermates as well as Slc26a9fl/fl/Atp4b-Cre and Slc26a9fl/fl mice from 8 days to 18 months by histological and immunohistochemical analyses, quantitative PCR, in situ hybridization, and RNA microarray analysis, respectively. We demonstrated that loss of parietal cell expression of Slc26a9 is the key event to induce spontaneous gastric carcinogenesis in mice, and clarified the sequence of events leading to malignant transformation, including Slc26a9 deficiency in parietal cells resulted in dysregulated differentiation of stem cells in an inflammatory environment, activated Wnt signaling pathway to induce gastric epithelia cell hyperproliferation and apoptosis inhibition, as well as spontaneous epithelial to mesenchymal transition-induced cancer stem cell phenotypes. Downregulation of SLC26A9 correlated with GC patients short survival. Graphical AbstractLoss of parietal cell expression of Slc26a9 is the key event to induce spontaneous gastric carcinogenesis in transgenic mice. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/316398v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@33a2dcorg.highwire.dtl.DTLVardef@94ce8org.highwire.dtl.DTLVardef@9851eforg.highwire.dtl.DTLVardef@2b5fc3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

HiFi-SIM: reconstructing high-fidelity structured illumination microscope images

Structured illumination microscopy (SIM) has been a widely-used super-resolution (SR) fluorescence microscopy technique, but artifacts often appear in reconstructed SR images which reduce its fidelity and might cause misinterpretation of biological structures. We present HiFi-SIM, a high-fidelity SIM reconstruction algorithm, by engineering the effective point spread function (PSF) into an ideal form. HiFi-SIM can effectively reduce commonly-seen artifacts without loss of fine structures and improve the axial sectioning. Since results of HiFi-SIM are not sensitive to used PSF and reconstruction parameters, it lowers the requirements for dedicated PSF calibration and complicated parameter adjustment, thus promoting SIM as a daily imaging tool.

biophysics↗

bioRxiv2020

Astrocytes express DMT1 and transferrin receptors, which transport iron thus activating Ca2+ signalling: possible role in neuroprotection against iron overload?

ABSTRACTIron is the fundamental element for numerous physiological functions. Reduced ferrous (Fe2+) and oxidized ferric (Fe3+) are the two ionized iron states in the living organisms. In the cell membrane, divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of Fe2+, whereas transferrin receptors (TFR) carry transferrin (TF)-bound Fe3+. In this study we performed, for the first time, detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Using qPCR and immunocytochemistry we identified DMT1 and TFR in astrocytes in primary cultures, in acutely isolated astrocytes and in brain tissue preparations; in situ both DMT1 and TFR are concentrated in astroglial perivascular endfeet. Administration of Fe2+ or Fe3+ in low μM concentrations evoked Ca2+ signals in astrocytes in vitro and in vivo. Iron ions triggered increase in [Ca2+]i by acting through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibited astroglial Na+-K+-ATPase (NKA), which led to an elevation in cytoplasmic Na+ concentration (as measured by SBFI probe), thus reversing Na+/Ca2+ exchanger (NCX) thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulated phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus trigering InsP3 receptor-mediated Ca2+ release from the endoplasmic reticulum. Iron-induced Ca2+ signals promote astroglial release of arachidonic acid and prostaglandin E2 cytokines by activating cytosolic phospholipase A2 (cPLA2) and NF-κB signalling cascade. In summary, these findings reveal new mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload, in response to which astrocytes actively accumulate excessive iron and activate neuroprotective pathways.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Evaluation of direct grafting strategies in Expansion Microscopy

High resolution fluorescence microscopy is a key tool in the elucidation of biological fine-structure, providing insights into the distribution and interactions of biomolecular systems down to the nanometer scale. Expansion microscopy is a recently developed approach to achieving nanoscale resolution in optical imaging. In the experiment, biological samples are embedded in a hydrogel, which is isotropicaly swollen. This physically pulls labels apart, allowing more of them to be resolved. However, in the gelation and swelling process, two factors combine to reduce the signal in the final image; signal dilution and the polymerization reaction, which can damage some fluorophores. Here, we show a chemical linking approach that allows covalent grafting of biomolecular target and reporter in expansion microscopy. Through the combination of a targeting ligand, a reporter moiety and a polymerizable group in a single linker, complex constructs can be prepared in a single, labelling step. We show application of this new series of molecules in the targeting of the cell cytoskeleton, a first example of lipid membranes in expansion microscopy; direct immunostaining with primary and secondary antibodies, and direct grafting of ISH probes and signal amplification initiators (HCR and RollFISH). Our probes allow direct, multiplexed targeting of the cellular blueprint and enable a range of novel imaging approaches in combination with expansion microscopy.

biophysics↗