RESUMO
To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.
Assuntos
Proteômica , Imagem Individual de Molécula , DNA , Microscopia de Fluorescência/métodos , Neurônios , ProteínasRESUMO
Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.
Assuntos
Linfócitos T CD8-Positivos , Diferenciação Celular , Tolerância Imunológica , Biossíntese de Proteínas , Receptores de Antígenos de Linfócitos T , Linfócitos T CD8-Positivos/imunologia , Animais , Diferenciação Celular/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Tolerância Imunológica/imunologia , Biossíntese de Proteínas/imunologia , Transdução de Sinais/imunologia , Camundongos Endogâmicos C57BL , Autoantígenos/imunologiaRESUMO
Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches1-6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7-14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.
Assuntos
Antígenos CD20 , Células , DNA , Microscopia de Fluorescência , Disciplinas das Ciências Biológicas/instrumentação , Disciplinas das Ciências Biológicas/métodos , Disciplinas das Ciências Biológicas/normas , Imunoterapia , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Código de Barras de DNA Taxonômico , DNA/análise , DNA/química , Antígenos CD20/análise , Antígenos CD20/química , Células/efeitos dos fármacos , Células/metabolismoRESUMO
State-of-the-art super-resolution microscopy allows researchers to spatially resolve single proteins in dense clusters. However, accurate quantification of protein organization and stoichiometries requires a general method to evaluate absolute binder labeling efficiency, which is currently unavailable. Here we introduce a universally applicable approach that uses a reference tag fused to a target protein of interest. By attaching high-affinity binders, such as antibodies or nanobodies, to both the reference tag and the target protein, and then employing DNA-barcoded sequential super-resolution imaging, we can correlate the location of the reference tag with the target molecule binder. This approach facilitates the precise quantification of labeling efficiency at the single-protein level.
RESUMO
DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution fluorescence microscopy technique that achieves single-molecule 'blinking' by transient DNA hybridization. Despite blinking kinetics being largely independent of fluorescent dye choice, the dye employed substantially affects measurement quality. Thus far, there has been no systematic overview of dye performance for DNA-PAINT. Here we defined four key parameters characterizing performance: brightness, signal-to-background ratio, DNA-PAINT docking site damage and off-target signal. We then analyzed 18 fluorescent dyes in three spectral regions and examined them both in DNA origami nanostructures, establishing a reference standard, and in a cellular environment, targeting the nuclear pore complex protein Nup96. Finally, having identified several well-performing dyes for each excitation wavelength, we conducted simultaneous three-color DNA-PAINT combined with Exchange-PAINT to image six protein targets in neurons at ~16 nm resolution in less than 2 h. We thus provide guidelines for DNA-PAINT dye selection and evaluation and an overview of performances of commonly used dyes.
RESUMO
RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.
Assuntos
Grânulos de Ribonucleoproteínas Citoplasmáticas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Rotavirus/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Bovinos , Linhagem Celular , Grânulos de Ribonucleoproteínas Citoplasmáticas/efeitos dos fármacos , Grânulos de Ribonucleoproteínas Citoplasmáticas/ultraestrutura , Grânulos de Ribonucleoproteínas Citoplasmáticas/virologia , Regulação Viral da Expressão Gênica , Genes Reporter , Glicóis/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Haplorrinos , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração Osmolar , Fosforilação , Propilenoglicol/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Rotavirus/efeitos dos fármacos , Rotavirus/crescimento & desenvolvimento , Rotavirus/ultraestrutura , Transdução de Sinais , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.
Assuntos
Centrossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Microtúbulos/metabolismo , Neurogênese , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Movimento Celular , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Junções Intercelulares/metabolismo , Interfase , Ventrículos Laterais/anatomia & histologia , Glândulas Mamárias Animais/citologia , Camundongos , Tamanho do Órgão , Organoides/citologiaRESUMO
T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , DNA/imunologia , Complexo Principal de Histocompatibilidade/genética , Receptores de Antígenos de Linfócitos T/química , Animais , Células Apresentadoras de Antígenos/citologia , Linfócitos T CD4-Positivos/citologia , DNA/química , DNA/genética , Expressão Gênica , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ativação Linfocitária , Camundongos , Conformação de Ácido Nucleico , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Baço/citologia , Baço/imunologiaRESUMO
Super-resolution microscopy has revolutionized how researchers characterize samples in the life sciences in the last decades. Amongst methods employing single-molecule localization microscopy, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement method that uses the programmable and repetitive binding of dye-labeled DNA imager strands to their respective docking strands. Recently developed Peptide-PAINT replaces the interaction of oligonucleotides by short coiled-coil peptide sequences leading to an improved labeling scheme by reducing linkage errors to target proteins. However, only one coiled-coil pair is currently available for Peptide-PAINT, preventing multiplexed imaging. In this study, the initial Peptide-PAINT E/K coil is improved by modifying its length for optimized binding kinetics leading to improved localization precisions. Additionally, an orthogonal P3/P4 coil pair is introduced, enabling 2-plex Peptide-PAINT imaging and benchmarking its performance and orthogonality using single-molecule and DNA origami assays. Finally, the P3/P4 peptide pair is used to image the human epidermal growth factor receptors 2 (ErbB2/Her2) in 2D and 3D at the single receptor level using genetically encoded peptide tags.
Assuntos
DNA , Oligonucleotídeos , Humanos , Microscopia de Fluorescência/métodos , DNA/química , Oligonucleotídeos/química , Nanotecnologia/métodos , PeptídeosRESUMO
DNA-PAINT's imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging.
Assuntos
DNA/química , Imagem Molecular/métodos , Anticorpos , Sítios de Ligação , Linhagem Celular , Humanos , Imageamento Tridimensional , Microscopia de Fluorescência/métodos , Microtúbulos , Nanoestruturas , Nanotecnologia/métodos , VimentinaRESUMO
MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.
Assuntos
DNA/metabolismo , DNA/ultraestrutura , Iluminação/métodos , Microscopia de Fluorescência/métodos , Modelos Teóricos , Nanoestruturas/ultraestrutura , Imagem Individual de Molécula/métodos , Animais , Humanos , Iluminação/instrumentação , Nanotecnologia/métodos , FótonsRESUMO
Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.
Assuntos
Mecanotransdução Celular , Nanotecnologia/métodos , Análise de Célula Única , Animais , Fenômenos Biomecânicos , Plaquetas/fisiologia , Fibroblastos/fisiologia , Humanos , Camundongos , Nanotecnologia/instrumentaçãoRESUMO
Nucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair 'voxels' that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.
Assuntos
Algoritmos , DNA/química , DNA/síntese química , Nanoestruturas/química , Nanotecnologia , Conformação de Ácido Nucleico , Animais , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Nucleotídeos/química , Rotação , Análise de Sequência de DNA , UrsidaeRESUMO
Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach "DNA-PAINT" robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by "Exchange-PAINT", which requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure, we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3-6 nm) and multicolor registration of dual- and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.
Assuntos
Nanoestruturas , Imagem Individual de Molécula , DNA/química , Microscopia de Fluorescência/métodos , Oligonucleotídeos/química , Imagem Individual de Molécula/métodosRESUMO
DNA points accumulation in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement super-resolution technique. However, image acquisition is slow compared to most other approaches. Here, we overcome this limitation by designing optimized DNA sequences and buffer conditions. We demonstrate our approach in vitro with DNA origami and in situ using cell samples, and achieve an order of magnitude faster imaging speeds without compromising image quality or spatial resolution. This improvement now makes DNA-PAINT applicable to high-throughput studies.
Assuntos
DNA/química , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Animais , Sequência de Bases , Soluções Tampão , Células COS , Chlorocebus aethiops , Células HeLa , HumanosRESUMO
Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.
Assuntos
DNA , Imagem Individual de MoléculaRESUMO
Methods that fuse multiple localization microscopy images of a single structure can improve signal-to-noise ratio and resolution, but they generally suffer from template bias or sensitivity to registration errors. We present a template-free particle-fusion approach based on an all-to-all registration that provides robustness against individual misregistrations and underlabeling. We achieved 3.3-nm Fourier ring correlation (FRC) image resolution by fusing 383 DNA origami nanostructures with 80% labeling density, and 5.0-nm resolution for structures with 30% labeling density.
Assuntos
DNA/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Nanoestruturas/química , Imagem Individual de Molécula/métodos , Humanos , Razão Sinal-RuídoRESUMO
Although current implementations of super-resolution microscopy are technically approaching true molecular-scale resolution, this has not translated to imaging of biological specimens, because of the large size of conventional affinity reagents. Here we introduce slow off-rate modified aptamers (SOMAmers) as small and specific labeling reagents for use with DNA points accumulation in nanoscale topography (DNA-PAINT). To demonstrate the achievable resolution, specificity, and multiplexing capability of SOMAmers, we labeled and imaged both transmembrane and intracellular targets in fixed and live cells.
Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas de Fluorescência Verde/química , Limite de Detecção , Microscopia de Fluorescência/métodosRESUMO
MOTIVATION: Classification of images is an essential task in higher-level analysis of biological data. By bypassing the diffraction limit of light, super-resolution microscopy opened up a new way to look at molecular details using light microscopy, producing large amounts of data with exquisite spatial detail. Statistical exploration of data usually needs initial classification, which is up to now often performed manually. RESULTS: We introduce nanoTRON, an interactive open-source tool, which allows super-resolution data classification based on image recognition. It extends the software package Picasso with the first deep learning tool with a graphic user interface. AVAILABILITY AND IMPLEMENTATION: nanoTRON is written in Python and freely available under the MIT license as a part of the software collection Picasso on GitHub (http://www.github.com/jungmannlab/picasso). All raw data can be obtained from the authors upon reasonable request. CONTACT: jungmann@biochem.mpg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Microscopia , SoftwareRESUMO
Improving labeling probes for state-of-the-art super-resolution microscopy is becoming of major importance. However, there is currently a lack of tools to quantitatively evaluate probe performance regarding efficiency, precision, and achievable resolution in an unbiased yet modular fashion. Herein, we introduce designer DNA origami structures combined with DNA-PAINT to overcome this issue and evaluate labeling efficiency, precision, and quantification using antibodies and nanobodies as exemplary labeling probes. Whereas current assessment of binders is mostly qualitative, e. g. based on an expected staining pattern, we herein present a quantitative analysis platform of the antigen labeling efficiency and achievable resolution, allowing researchers to choose the best performing binder. The platform can furthermore be readily adapted for discovery and precise quantification of a large variety of additional labeling probes.