Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 939
Filtrar
1.
Nat Commun ; 12(1): 3407, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099685

RESUMO

Single-Molecule Localization Microscopy (SMLM) provides the ability to determine molecular organizations in cells at nanoscale resolution, but in complex biological tissues, where sample-induced aberrations hamper detection and localization, its application remains a challenge. Various adaptive optics approaches have been proposed to overcome these issues, but the exact performance of these methods has not been consistently established. Here we systematically compare the performance of existing methods using both simulations and experiments with standardized samples and find that they often provide limited correction or even introduce additional errors. Careful analysis of the reasons that underlie this limited success enabled us to develop an improved method, termed REALM (Robust and Effective Adaptive Optics in Localization Microscopy), which corrects aberrations of up to 1 rad RMS using 297 frames of blinking molecules to improve single-molecule localization. After its quantitative validation, we demonstrate that REALM enables to resolve the periodic organization of cytoskeletal spectrin of the axon initial segment even at 50 µm depth in brain tissue.


Assuntos
Encéfalo/patologia , Óptica e Fotônica/métodos , Imagem Individual de Molécula/métodos , Algoritmos , Animais , Células COS , Chlorocebus aethiops , Microscopia de Fluorescência/instrumentação , Ratos , Imagem Individual de Molécula/instrumentação , Software
2.
Nat Commun ; 12(1): 3791, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145284

RESUMO

Particle fusion for single molecule localization microscopy improves signal-to-noise ratio and overcomes underlabeling, but ignores structural heterogeneity or conformational variability. We present a-priori knowledge-free unsupervised classification of structurally different particles employing the Bhattacharya cost function as dissimilarity metric. We achieve 96% classification accuracy on mixtures of up to four different DNA-origami structures, detect rare classes of origami occuring at 2% rate, and capture variation in ellipticity of nuclear pore complexes.


Assuntos
DNA/química , Poro Nuclear/química , Conformação de Ácido Nucleico , Imagem Individual de Molécula/métodos , Nanoestruturas/química , Razão Sinal-Ruído
3.
Methods Mol Biol ; 2276: 153-163, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060039

RESUMO

The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400 ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.


Assuntos
Processamento Eletrônico de Dados/métodos , Microscopia de Fluorescência/métodos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Imagem Individual de Molécula/métodos , Animais , Metabolismo Energético , Potenciais da Membrana , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Ratos , Ratos Wistar
4.
Nat Protoc ; 16(7): 3695-3715, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099942

RESUMO

Super-resolution fluorescence imaging that surpasses the classical optical resolution limit is widely utilized for resolving the spatial organization of biological structures at molecular length scales. In one example, single-molecule localization microscopy, the lateral positions of single molecules can be determined more precisely than the diffraction limit if the camera collects individual photons separately. Using several schemes that introduce engineered optical aberrations in the imaging optics, super-resolution along the optical axis (perpendicular to the sample plane) has been achieved, and single-molecule localization microscopy has been successfully applied for the study of 3D biological structures. Nonetheless, the achievable axial localization accuracy is typically three to five times worse than the lateral localization accuracy. Only a few exceptional methods based on interferometry exist that reach nanometer 3D super-resolution, but they involve enormous technical complexity and restricted sample preparations that inhibit their widespread application. We developed metal-induced energy transfer imaging for localizing fluorophores along the axial direction with nanometer accuracy, using only a conventional fluorescence lifetime imaging microscope. In metal-induced energy transfer, experimentally measured fluorescence lifetime values increase linearly with axial distance in the range of 0-100 nm, making it possible to calculate their axial position using a theoretical model. If graphene is used instead of the metal (graphene-induced energy transfer), the same range of lifetime values occurs over a shorter axial distance (~25 nm), meaning that it is possible to get very accurate axial information at the scale of a membrane bilayer or a molecular complex in a membrane. Here, we provide a step-by-step protocol for metal- and graphene-induced energy transfer imaging in single molecules, supported lipid bilayer and live-cell membranes. Depending on the sample preparation time, the complete duration of the protocol is 1-3 d.


Assuntos
Transferência de Energia , Grafite/química , Metais/química , Nanotecnologia/métodos , Imagem Individual de Molécula/métodos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Análise de Dados , Cães , Corantes Fluorescentes/química , Humanos , Imageamento Tridimensional , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Interface Usuário-Computador
5.
Methods Mol Biol ; 2277: 157-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080151

RESUMO

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.


Assuntos
Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia Confocal/métodos , Proteínas Mitocondriais/metabolismo , Bromouracila/análogos & derivados , Bromouracila/química , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , RNA Mitocondrial/química , Imagem Individual de Molécula/métodos , Uridina/análogos & derivados , Uridina/química
6.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064786

RESUMO

Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.


Assuntos
Fenômenos Fisiológicos Vegetais , Plantas/metabolismo , Imagem Individual de Molécula/métodos
7.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067933

RESUMO

Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 µs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the "ionic lock" between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.


Assuntos
Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/fisiologia , Imagem Individual de Molécula/métodos , Proteínas de Transporte/metabolismo , Cristalografia por Raios X/métodos , Ouro , Células HEK293 , Humanos , Íons/metabolismo , Ligantes , Nanotecnologia/métodos , Receptores de Serotonina/metabolismo , Receptores de Serotonina/fisiologia , Difração de Raios X/métodos , Raios X
8.
Nat Commun ; 12(1): 3515, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112774

RESUMO

MicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 µl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer.


Assuntos
Biomarcadores Tumorais/genética , MicroRNA Circulante/genética , Detecção Precoce de Câncer/métodos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias da Próstata/diagnóstico , Imagem Individual de Molécula/métodos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , MicroRNA Circulante/análise , MicroRNA Circulante/sangue , Detecção Precoce de Câncer/instrumentação , Fluorescência , Perfilação da Expressão Gênica , Humanos , Biópsia Líquida , Masculino , MicroRNAs/análise , MicroRNAs/sangue , MicroRNAs/genética , Nanoporos , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
9.
Methods Mol Biol ; 2274: 391-441, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050488

RESUMO

Single-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein-protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software, named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure ( https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer ).


Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Preparações Farmacêuticas/administração & dosagem , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Imagem Individual de Molécula/métodos , Software , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ligação Proteica , Fluxo de Trabalho
10.
Anal Bioanal Chem ; 413(18): 4645-4654, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34057558

RESUMO

Nucleic acid detection technology based on polymerase chain reaction (PCR) and antibody detection based on immunochromatography still have many problems such as false negatives for the diagnosis of coronavirus disease 2019 (COVID-19). Therefore, it is of great importance to develop new techniques to improve the diagnostic accuracy of COVID-19. We herein developed an ultrasensitive, rapid, and duplex digital enzyme-linked immunosorbent assay (dELISA) for simultaneous detection of spike (S-RBD) and nucleocapsid (N) proteins of SARS-CoV-2 based on a single molecule array. This assay effectively combines magnetic bead encoding technology and the ultrasensitive detection capability of a single molecule array. The detection strategies of S-RBD protein and N-protein exhibited wide response ranges of 0.34-1065 pg/mL and 0.183-338 pg/mL with detection limits of 20.6 fg/mL and 69.8 fg/mL, respectively. It is a highly specific method for the simultaneous detection of S-RBD protein and N-protein and has minimal interference from other blood proteins. Moreover, the spike assay showed a satisfactory and reproducible recovery rate for the detection of S-RBD protein and N-protein in serum samples. Overall, this work provides a highly sensitive method for the simultaneous detection of S-RBD protein and N-protein, which shows ultrasensitivity and high signal-to-noise ratio and contributes to improve the diagnosis accuracy of COVID-19.


Assuntos
COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Imagem Individual de Molécula/métodos , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Proteínas do Nucleocapsídeo de Coronavírus/genética , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Imunoensaio/métodos , Magnetismo , Microesferas , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética
11.
Nat Commun ; 12(1): 3158, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039974

RESUMO

The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.


Assuntos
Microscopia Intravital/métodos , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos , Transcrição Genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Serina/metabolismo , Análise Espaço-Temporal , Imagem com Lapso de Tempo
12.
Nat Commun ; 12(1): 2847, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990554

RESUMO

Single molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data.


Assuntos
Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Algoritmos , Linhagem Celular , Simulação por Computador , DNA/química , DNA/ultraestrutura , Humanos , Imageamento Tridimensional , Conformação Molecular , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Razão Sinal-Ruído , Imagem Individual de Molécula/estatística & dados numéricos
13.
Nat Commun ; 12(1): 3077, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031402

RESUMO

Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and ß2-spectrin in neurons. ASTER's sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.


Assuntos
Iluminação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Algoritmos , Animais , Células COS , Chlorocebus aethiops , Lasers , Luz , Microtúbulos , Reprodutibilidade dos Testes
14.
Nat Commun ; 12(1): 2510, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947854

RESUMO

Cell-extracellular matrix sensing plays a crucial role in cellular behavior and leads to the formation of a macromolecular protein complex called the focal adhesion. Despite their importance in cellular decision making, relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here, we combine cRGD-ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling. We uncover that integrin receptor clustering is governed by a non-random organization with complexes spaced at 20-30 nm distances. The DNA-based tension sensor and analysis framework provide powerful tools to study a multitude of receptor-ligand interactions where forces are involved in ligand-receptor binding.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , DNA/metabolismo , Nanotecnologia/métodos , Imagem Individual de Molécula/métodos , Actinas/química , Actinas/ultraestrutura , Adesão Celular , Análise por Conglomerados , Citoesqueleto/ultraestrutura , DNA/química , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Humanos , Ligantes , Ligação Proteica , Imagem Individual de Molécula/instrumentação , Propriedades de Superfície , Talina/genética , Talina/metabolismo
15.
Nat Commun ; 12(1): 2502, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947864

RESUMO

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Antígenos de Histocompatibilidade/química , Receptores de Antígenos de Linfócitos T/química , Imagem Individual de Molécula/métodos , Anticorpos de Cadeia Única/química , Animais , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/imunologia , Citocromos c/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Antígenos de Histocompatibilidade/imunologia , Cinética , Ligantes , Bicamadas Lipídicas/química , Camundongos , Peptídeos/química , Receptores de Antígenos de Linfócitos T/imunologia , Imagem Individual de Molécula/instrumentação , Anticorpos de Cadeia Única/imunologia , Análise Espaço-Temporal
16.
Nat Commun ; 12(1): 2525, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953191

RESUMO

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.


Assuntos
Replicação do DNA/fisiologia , DNA/química , Quadruplex G , Imagem Individual de Molécula/métodos , Animais , Biofísica , Linhagem Celular , Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Instabilidade Genômica , Humanos , Proteínas Recombinantes , Células Sf9
17.
Methods Mol Biol ; 2281: 49-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847951

RESUMO

Recent single-molecule studies have demonstrated that the composition of multi-protein complexes can strike a balance between stability and dynamics. Proteins can dynamically exchange in and out of the complex depending on their concentration in solution. These exchange dynamics are a key determinant of the molecular pathways available to multi-protein complexes. It is therefore important that we develop robust and reproducible assays to study protein exchange. Using DNA replication as an example, we describe three single-molecule fluorescence assays used to study protein exchange dynamics. In the chase exchange assay, fluorescently labeled proteins are challenged by unlabeled proteins, where exchange results in the disappearance of the fluorescence signal. In the FRAP exchange assay, fluorescently labeled proteins are photobleached before exchange is measured by an increase in fluorescence as non-bleached proteins exchange into the complex. Finally, in the two-color exchange assay, proteins are labeled with two different fluorophores and exchange is visualized by detecting changes in color. All three assays compliment in their ability to elucidate the dynamic behavior of proteins in large biological systems.


Assuntos
DNA/genética , Corantes Fluorescentes/química , Complexos Multiproteicos/ultraestrutura , Imagem Individual de Molécula/métodos , Replicação do DNA , Recuperação de Fluorescência Após Fotodegradação , Cinética , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência , Complexos Multiproteicos/química , Software
18.
Methods Mol Biol ; 2281: 135-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847956

RESUMO

Bacterial RecA and eukaryotic Rad51 are recombinases indispensable for DNA homologous recombination and repair of double-stranded DNA breaks. Understanding the functions and biophysical properties of the DNA recombinases benefits the research in human medicine such as cancer biology. Single-molecule techniques provide the mechanistic details of complex biological reactions. Tethered particle motion (TPM) experiment is a simple and multiplex single-molecule tool to monitor DNA-protein interactions. We have developed a single-molecule TPM assay to study DNA recombinase filament assembly and disassembly on individual DNA molecules in real time. Characterization of the temporal change of the Brownian motion of DNA tethers during recombinase assembly and disassembly in real time allows the determination of multiple kinetic parameters of nucleation rate, extension rate, dissociation rate, and length of the recombinase-DNA filament.


Assuntos
DNA/metabolismo , Recombinases/metabolismo , Imagem Individual de Molécula/métodos , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga
19.
Methods Mol Biol ; 2281: 193-207, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847959

RESUMO

Homologous recombination (HR) is a highly conserved DNA repair pathway required for the accurate repair of DNA double-stranded breaks. DNA recombination is catalyzed by the RecA/Rad51 family of proteins, which are conserved from bacteria to humans. The key intermediate catalyzing DNA recombination is the presynaptic complex (PSC), which is a helical filament comprised of Rad51-bound single-stranded DNA (ssDNA). Multiple cellular factors either promote or downregulate PSC activity, and a fine balance between such regulators is required for the proper regulation of HR and maintenance of genomic integrity. However, dissecting the complex mechanisms regulating PSC activity has been a challenge using traditional ensemble methods due to the transient and dynamic nature of recombination intermediates. We have developed a single-molecule assay called ssDNA curtains that allows us to visualize individual DNA intermediates in real-time, using total internal reflection microscopy (TIRFM). This assay has allowed us to study many aspects of HR regulation that involve complex and heterogenous reaction intermediates. Here we describe the procedure for a basic ssDNA curtain assay to study PSC filament dynamics, and explain how to process and analyze the resulting data.


Assuntos
DNA de Cadeia Simples/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , DNA Fúngico/metabolismo , Regulação da Expressão Gênica , Recombinação Homóloga , Microscopia de Interferência , Proteínas de Saccharomyces cerevisiae/genética
20.
Biochem Biophys Res Commun ; 556: 59-64, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33839415

RESUMO

Acquiring events massively from single-molecule force spectroscopy (SMFS) experiments, which is crucial for revealing important biophysical information, is usually not straightforward. A significant amount of human labor is usually required to identify events in the measured force spectrum during measuring or before performing further data analysis. This prevents the experiment from being done in a fully-automated manner or scaling with the throughput of the measuring setup. In this work, we attempt to tackle this problem with a deep learning approach. A deep neural network model is developed to infer the occurrence of the events using the data stream from the measuring setup. We demonstrated that the proposed method could achieve high accuracy with force spectrums of a variety of samples from both optical tweezers and AFMs by learning from user-given samples instead of complicated manual algorithm designing or parameter tuning. Furthermore, we found that the trained model can be used to perform event detection on datasets measured from a different optical tweezer setup, showing the potential of being leveraged in more complex deep learning schemes.


Assuntos
Aprendizado Profundo , Imagem Individual de Molécula/métodos , Automação , Microscopia de Força Atômica , Pinças Ópticas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...