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We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane protrusions on the culture glass surface after lipopolysaccharide exposure. The concept of our work opens new possibilities for quantitative protein-specific PAINT using commercially available reagents.
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T cells coordinate intercellular communication through the meticulous regulation of cytokine secretion. Direct visualization of vesicular transport and intracellular distribution of cytokines provides valuable insights into the temporal and spatial mechanisms involved in regulation. Employing Jurkat E6-1 T cells and interleukin-2 (IL-2) as a model system, we investigated vesicular dynamics using single-particle tracking and the nanoscale distribution of intracellular IL-2 in fixed T cells using superresolution microscopy. Live-cell imaging revealed that in vitro activation resulted in increased vesicular dynamics. Direct stochastic optical reconstruction microscopy and 3D structured illumination microscopy revealed nanoscale clustering of IL-2. In vitro activation correlated with spatial accumulation of IL-2 nanoclusters into more pronounced and elongated clusters. These observations provide visual evidence that accelerated vesicular transport and spatial concatenation of IL-2 clusters at the nanoscale may constitute a potential mechanism for modulating cytokine release by Jurkat T cells.
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Interleucina-2 , Ativação Linfocitária , Linfócitos T , Humanos , Interleucina-2/metabolismo , Células Jurkat , Linfócitos T/metabolismo , Linfócitos T/citologiaRESUMO
We present a versatile single-molecule localization microscopy technique utilizing time-lapse imaging of single-antibody labeling. By performing single-molecule imaging in the subminute time scale and tuning the antibody concentration to create sparse single-molecule binding, we captured antibody labeling of subcellular targets to generate superresolution images. Single-antibody labeling enabled dual-target superresolution imaging using dye-conjugated monoclonal and polyclonal antibodies. We further demonstrate a dual-color strategy to increase the sample labeling density. Single-antibody labeling paves a new way to evaluate antibody binding for superresolution imaging in the native cellular environment.
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Anticorpos , Imagem Individual de Molécula , Microscopia de Fluorescência/métodos , Espaço Extracelular , Corantes Fluorescentes/químicaRESUMO
Dendritic cells (DCs) can infiltrate tight junctions of the epithelium to collect remote antigens during immune surveillance. While elongated membrane structures represent a plausible structure to perform this task, their functional mechanisms remain elusive owing to the lack of high-resolution characterizations in live DCs. Here, we developed fluorescent artificial antigens (FAAs) based on quantum dots coated with polyacrylic acid. Single-particle tracking of FAAs enables us to superresolve the membrane fiber network responsible for the antigen uptake. Using the DC2.4 cell line as a model system, we discovered the extensive membrane network approaching 200 µm in length with tunnel-like cavities about 150 nm in width. The membrane fiber network also contained heterogeneous circular migrasomes. Disconnecting the membrane network from the cell body decreased the intracellular FAA density. Our study enables mechanistic investigations of DC membrane networks and nanocarriers that target this mechanism.
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Células Dendríticas , Pontos Quânticos , Antígenos , Linhagem Celular , Vacinas SintéticasRESUMO
T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.
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Linfonodos/diagnóstico por imagem , Linfonodos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Citocromos c/imunologia , Diagnóstico por Imagem/métodos , Proteínas de Insetos/imunologia , Camundongos Transgênicos , Nanotecnologia/métodos , Peptídeos/imunologiaRESUMO
Natural proteins often rely on the disulfide bond to covalently link side chains. Here we genetically introduce a new type of covalent bond into proteins by enabling an unnatural amino acid to react with a proximal cysteine. We demonstrate the utility of this bond for enabling irreversible binding between an affibody and its protein substrate, capturing peptide-protein interactions in mammalian cells, and improving the photon output of fluorescent proteins.
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Fenilalanina/análogos & derivados , Engenharia de Proteínas/métodos , Proteínas/química , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Animais , Cisteína/química , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Fluorescência , Dados de Sequência Molecular , Mutação , Fenilalanina/química , Fótons , Conformação Proteica , Proteínas/genética , Proteínas/imunologia , Proteínas/metabolismo , Ratos , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Single-molecule super-resolution imaging is a new promising tool for investigation of sub-cellular structures. Concurrently, light-sheet microscopy, also known as selective plane illumination microscopy (SPIM), has gained rapid favor with the imaging community in developmental biology due to its fast speed, high contrast, deep penetration, and low phototoxicity. While nearly a dozen reviews thoroughly describe the development of light-sheet microscopy and its technological breakthroughs with a main focus on improving the 3D imaging speed of fish embryos, central nervous system, and other tissues, few have addressed the potential of combining light-sheet microscopy and localization-based super-resolution imaging to achieve sub-diffraction-limited resolution. Adapting light-sheet illumination for single-molecule imaging presents unique challenges for instrumentation and reconstruction algorithms. In this Minireview, we provide an overview of the recent developments that address these challenges. We compare different approaches in super-resolution and light-sheet imaging, address advantages and limitations in each approach, and outline future directions of this emerging field.
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Luz , Microscopia/métodosRESUMO
PURPOSE: Many peripheral and cutaneous T-cell lymphoma (CTCL) subtypes are poorly responsive to conventional chemotherapeutic agents and associated with dismal outcomes. The zinc finger transcription factor GATA-3 and the transcriptional program it instigates are oncogenic and highly expressed in various T-cell neoplasms. Posttranslational acetylation regulates GATA-3 DNA binding and target gene expression. Given the widespread use of histone deacetylase inhibitors (HDACi) in relapsed/refractory CTCL, we sought to examine the extent to which these agents attenuate the transcriptional landscape in these lymphomas. EXPERIMENTAL DESIGN: Integrated GATA-3 chromatin immunoprecipitation sequencing and RNA sequencing analyses were performed in complementary cell line models and primary CTCL specimens treated with clinically available HDACi. RESULTS: We observed that exposure to clinically available HDACi led to significant transcriptional reprogramming and increased GATA-3 acetylation. HDACi-dependent GATA-3 acetylation significantly impaired both its ability to bind DNA and transcriptionally regulate its target genes, thus leading to significant transcriptional reprogramming in HDACi-treated CTCL. CONCLUSIONS: Beyond shedding new light on the mechanism of action associated with HDACi in CTCL, these findings have significant implications for their use, both as single agents and in combination with other novel agents, in GATA-3-driven lymphoproliferative neoplasms.
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Linfoma Cutâneo de Células T , Neoplasias Cutâneas , Humanos , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/genética , Acetilação , Inibidores de Histona Desacetilases/farmacologia , DNA , Transcrição GênicaRESUMO
We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.
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In single-molecule localization microscopy (SMLM), immunofluorescence (IF) staining affects the quality of the reconstructed superresolution images. However, optimizing IF staining remains challenging because IF staining is a one-step, irreversible process. Sample labeling through reversible binding presents an alternative strategy, but such techniques require significant technological advancements to enhance the dissociation of labels without sacrificing their binding specificity. In this article, we introduce time-lapse imaging of single-antibody labeling. Our versatile technique utilizes commercially available dye-conjugated antibodies. The method controls the antibody concentrations to capture single-antibody labeling of subcellular targets, thereby achieving SMLM through the labeling process. We further demonstrate dual-color single-antibody labeling to enhance the sample labeling density. The new approach allows the evaluation of antibody binding at the single-antibody level and within the cellular environment. This comprehensive guide offers step-by-step instructions for time-lapse imaging of single-antibody labeling experiments and enables the application of the single-antibody labeling technique to a wide range of targets. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for single-antibody labeling Basic Protocol 2: Data acquisition for single-molecule localization microscopy Alternate Protocol: Dual-color single-antibody labeling using OptoSplit II equation Basic Protocol 3: Image analysis.
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Processamento de Imagem Assistida por Computador , Imagem Individual de Molécula , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo , Imagem Individual de Molécula/métodos , Coloração e RotulagemRESUMO
Quantum dot (QD)-organic dye couple chromophores are topical due to their applications in biology, catalysis, and energy. The maximization of energy transfer efficiency can be guided by the underlying Förster or Dexter mechanisms; however, the impact of fluorescence intermittency must also be considered. Here we demonstrate that the average ⟨ton⟩ and ⟨toff⟩ times of dye acceptors in coupled QD-dye chromophores are substantially affected by the donors' blinking behavior. With regard to biological imaging, this effect beneficially minimizes the photobleaching of the acceptor dye. The implications for alternative energy are less encouraging as the acceptors' capacity to store energy, using ⟨ton⟩/⟨toff⟩ as a metric, was reduced by as much as â¼95%. These detrimental effects can be mitigated by suppressing QD blinking via surface treatment. This study also demonstrates several instances of the nonconformity of QD blinking dynamics to a power law distribution, as a robust examination of the off times reveals log-normal behavior that is consistent with the Albery model.
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Pontos Quânticos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes/química , Pontos Quânticos/químicaRESUMO
Antibody-antigen interactions represent one of the most exploited biomolecular interactions in experimental biology. While numerous techniques harnessed immobilized antibodies for nanoscale fluorescence imaging, few utilized their reversible binding kinetics. Here, we investigated noncovalent interactions of the monoclonal hemagglutinin (HA) epitope tag antibody, 12CA5, in the fixed cellular environment. We observed that the use of a chaotropic agent, potassium thiocyanate (KSCN), promoted the dissociation of the 12CA5 antibody fragment (Fab), which already displayed faster dissociation compared to its immunoglobulin G (IgG) counterpart. Molecular dynamic simulations revealed notable root-mean-square deviations and destabilizations in the presence of KSCN, while the hydrogen-bonding network remained primarily unaffected at the antigen-binding site. The reversible interactions enabled us to achieve a superresolution molecular census of local populations of 3xHA tagged microtubule fibers with improved molecular quantification consistency compared to single-molecule localization microscopy (SMLM) techniques utilizing standard immunofluorescence staining for sample labeling. Our technique, termed superresolution census of molecular epitope tags (SR-COMET), highlights the utilization of reversible antibody-antigen interactions for SMLM-based quantitative superresolution imaging.
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Anticorpos Monoclonais , Hemaglutininas , Fragmentos de Imunoglobulinas , Censos , Epitopos/química , AntígenosRESUMO
The spatial organization of T cell receptors (TCRs) correlates with membrane-associated signal amplification, dispersion, and regulation during T cell activation. Despite its potential clinical importance, quantitative analysis of the spatial arrangement of TCRs from standard fluorescence images remains difficult. Here, we report Statistical Classification Analyses of Membrane Protein Images or SCAMPI as a technique capable of analyzing the spatial arrangement of TCRs on the plasma membrane of T cells. We leveraged medical image analysis techniques that utilize pixel-based values. We transformed grayscale pixel values from fluorescence images of TCRs into estimated model parameters of partial differential equations. The estimated model parameters enabled an accurate classification using linear discrimination techniques, including Fisher Linear Discriminant (FLD) and Logistic Regression (LR). In a proof-of-principle study, we modeled and discriminated images of fluorescently tagged TCRs from Jurkat T cells on uncoated cover glass surfaces (Null) or coated cover glass surfaces with either positively charged poly-L-lysine (PLL) or TCR cross-linking anti-CD3 antibodies (OKT3). Using 80 training images and 20 test images per class, our statistical technique achieved 85% discrimination accuracy for both OKT3 versus PLL and OKT3 versus Null conditions. The run time of image data download, model construction, and image discrimination was 21.89 s on a laptop computer, comprised of 20.43 s for image data download, 1.30 s on the FLD-SCAMPI analysis, and 0.16 s on the LR-SCAMPI analysis. SCAMPI represents an alternative approach to morphology-based qualifications for discriminating complex patterns of membrane proteins conditioned on a small sample size and fast runtime. The technique paves pathways to characterize various physiological and pathological conditions using the spatial organization of TCRs from patient T cells.
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Processamento de Imagem Assistida por Computador/métodos , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Análise por Conglomerados , Análise Discriminante , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Microscopia de Fluorescência , Modelos Estatísticos , Probabilidade , Análise de Regressão , Estatística como Assunto , Linfócitos T/imunologiaRESUMO
We report single-particle characterization of membrane-penetrating semiconductor quantum dots (QDs) in T cell lymphocytes. We functionalized water-soluble CdSe/CdZnS QDs with a cell-penetrating peptide composed of an Asp-Ser-Ser (DSS) repeat sequence. DSS and peptide-free control QDs displayed concentration-dependent internalization. Intensity profiles from single-particle imaging revealed a propensity of DSS-QDs to maintain a monomeric state in the T cell cytosol, whereas control QDs formed pronounced clusters. Single-particle tracking showed a direct correlation between individual QD clusters' mobility and aggregation state. A significant portion of control QDs colocalized with an endosome marker inside the T cells, while the percentage of DSS-QDs colocalized dropped to 9%. Endocytosis inhibition abrogated the internalization of control QDs, while DSS-QD internalization only mildly decreased, suggesting an alternative cell-entry mechanism. Using 3D single-particle tracking, we captured the rapid membrane-penetrating activity of a DSS-QD. The ability to characterize membrane penetrating activities in live T cells creates inroads for the optimization of gene therapy and drug delivery through the use of novel nanomaterials.
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Preparações Farmacêuticas , Pontos Quânticos , Citosol , Imunoterapia , Linfócitos TRESUMO
Transient receptor potential melastatin 7 (TRPM7) contributes to a variety of physiological and pathological processes in many tissues and cells. With a widespread distribution in the nervous system, TRPM7 is involved in animal behaviors and neuronal death induced by ischemia. However, the physiological role of TRPM7 in central nervous system (CNS) neuron remains unclear. Here, we identify endocytic defects in neuroendocrine cells and neurons from TRPM7 knockout (KO) mice, indicating a role of TRPM7 in synaptic vesicle endocytosis. Our experiments further pinpoint the importance of TRPM7 as an ion channel in synaptic vesicle endocytosis. Ca2+ imaging detects a defect in presynaptic Ca2+ dynamics in TRPM7 KO neuron, suggesting an importance of Ca2+ influx via TRPM7 in synaptic vesicle endocytosis. Moreover, the short-term depression is enhanced in both excitatory and inhibitory synaptic transmissions from TRPM7 KO mice. Taken together, our data suggests that Ca2+ influx via TRPM7 may be critical for short-term plasticity of synaptic strength by regulating synaptic vesicle endocytosis in neurons.
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Endocitose , Inibição Neural , Plasticidade Neuronal , Neurônios/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Células Cromafins/metabolismo , Potenciais Pós-Sinápticos Excitadores , Feminino , Células HEK293 , Humanos , Potenciais Pós-Sinápticos Inibidores , Cinética , Masculino , Camundongos Knockout , Vesículas Sinápticas/genética , Canais de Cátion TRPM/genéticaRESUMO
We report a molecular-docking and virtual-screening-based identification and characterization of interactions of lead molecules with exoribonuclease (ExoN) enzyme in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). From previously identified DEDDh/DEEDh subfamily nuclease inhibitors, our results revealed strong binding of pontacyl violet 6R (PV6R) at the catalytic active site of ExoN. The binding was found to be stabilized via two hydrogen bonds and hydrophobic interactions. Molecular dynamics simulations further confirmed the stability of PV6R at the active site showing a shift in ligand to reach a more stabilized binding. Using PV6R as the lead molecule, we employed virtual screening to identify potential molecular candidates that form strong interactions at the ExoN active site. Our study paves ways for evaluating the ExoN as a novel drug target for antiviral treatment against SARS-CoV-2.
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Synergistic developments in advanced fluorescent imaging and labeling techniques enable direct visualization of the chromatin structure and dynamics at the nanoscale level and in live cells. Super-resolution imaging encompasses a class of constantly evolving techniques that break the diffraction limit of fluorescence microscopy. Structured illumination microscopy provides a twofold resolution improvement and can readily achieve live multicolor imaging using conventional fluorophores. Single-molecule localization microscopy increases the spatial resolution by approximately 10-fold at the expense of slower acquisition speed. Stimulated emission-depletion microscopy generates a roughly fivefold resolution improvement with an imaging speed proportional to the scanning area. In parallel, advanced labeling strategies have been developed to "light up" global and sequence-specific DNA regions. DNA binding dyes have been exploited to achieve high labeling densities in single-molecule localization microscopy and enhance contrast in correlated light and electron microscopy. New-generation Oligopaint utilizes bioinformatics analyses to optimize the design of fluorescence in situ hybridization probes. Through sequential and combinatorial labeling, direct characterization of the DNA domain volume and length as well as the spatial organization of distinct topologically associated domains has been reported. In live cells, locus-specific labeling has been achieved by either inserting artificial loci next to the gene of interest, such as the repressor-operator array systems, or utilizing genome editing tools, including zinc finer proteins, transcription activator-like effectors, and the clustered regularly interspaced short palindromic repeats systems. Combined with single-molecule tracking, these labeling techniques enable direct visualization of intra- and inter-chromatin interactions. This article is categorized under: Laboratory Methods and Technologies > Imaging.
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Cromatina/química , DNA/química , Cromatina/metabolismo , DNA/metabolismo , Corantes Fluorescentes/química , Edição de Genes , Humanos , Hibridização in Situ Fluorescente , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
Metformin is the front-line treatment for type 2 diabetes worldwide. It acts via effects on glucose and lipid metabolism in metabolic tissues, leading to enhanced insulin sensitivity. Despite significant effort, the molecular basis for metformin response remains poorly understood, with a limited number of specific biochemical pathways studied to date. To broaden our understanding of hepatic metformin response, we combine phospho-protein enrichment in tissue from genetically engineered mice with a quantitative proteomics platform to enable the discovery and quantification of basophilic kinase substrates in vivo. We define proteins whose binding to 14-3-3 are acutely regulated by metformin treatment and/or loss of the serine/threonine kinase, LKB1. Inducible binding of 250 proteins following metformin treatment is observed, 44% of which proteins bind in a manner requiring LKB1. Beyond AMPK, metformin activates protein kinase D and MAPKAPK2 in an LKB1-independent manner, revealing additional kinases that may mediate aspects of metformin response. Deeper analysis uncovered substrates of AMPK in endocytosis and calcium homeostasis.