Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 78
Filtrar
1.
Nat Commun ; 11(1): 4384, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873796

RESUMO

The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , DNA/química , Nanoestruturas/química , Imagem Individual de Molécula/instrumentação , Biomarcadores/análise , Proteína C-Reativa/análise , Desenho de Equipamento , Humanos , Limite de Detecção , Nanotecnologia/métodos
2.
PLoS One ; 15(9): e0238470, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32911526

RESUMO

We report a powerful method for capturing the time-resolved concentration profiles, liquid swelling and surface phenomena during the absorption of methane (CH4) in still liquid ethanol (C2D6O) and n-decane (n-C10D22) and at high spatial resolution (pixel size 21.07 µm) using neutron imaging. Absorption of supercritical methane was followed at two temperatures and two pressures of methane, namely 7.0, 37.8 °C and 80, 120 bar. Fick's second law, which was used in the liquid-fixed coordinates, enabled for an adequate parameterization of the observed concentration profiles and liquid levels using simple analytical expressions. For both studied liquids, anomalously slow diffusion was observed in the initial stage of the absorption experiment. This was ascribed to the slow formation of the surface excess on the interface, time constant ranged 130-275 s. The axial symmetry of the cell allowed for the tomographic reconstructions of the profiles of the menisci. Based on these profiles, contact angle and surface tension were evaluated using the Young-Laplace equation. Overall, neutron imaging made it possible to capture time- and space-resolved information from which the methane concentration, liquid level and meniscus shape under high-pressure conditions inside a cylindrical titanium vessel were quantitatively derived. Multiple characteristics of ethanol, a methane hydrate inhibitor, and n-decane, a model constituent of crude oil, were thus measured for the first time under industrially relevant conditions in a one-pot experiment.


Assuntos
Metano/química , Imagem Individual de Molécula/métodos , Alcanos/química , Difusão , Etanol/química , Modelos Estatísticos , Nêutrons , Petróleo , Fenômenos Físicos , Imagem Individual de Molécula/instrumentação , Temperatura , Água
3.
Nat Commun ; 11(1): 3781, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728047

RESUMO

Nanomechanical mass spectrometry has proven to be well suited for the analysis of high mass species such as viruses. Still, the use of one-dimensional devices such as vibrating beams forces a trade-off between analysis time and mass resolution. Complex readout schemes are also required to simultaneously monitor multiple resonance modes, which degrades resolution. These issues restrict nanomechanical MS to specific species. We demonstrate here single-particle mass spectrometry with nano-optomechanical resonators fabricated with a Very Large Scale Integration process. The unique motion sensitivity of optomechanics allows designs that are impervious to particle position, stiffness or shape, opening the way to the analysis of large aspect ratio biological objects of great significance such as viruses with a tail or fibrils. Compared to top-down beam resonators with electrical read-out and state-of-the-art mass resolution, we show a three-fold improvement in capture area with no resolution degradation, despite the use of a single resonance mode.


Assuntos
Espectrometria de Massas/métodos , Nanotecnologia/métodos , Dispositivos Ópticos , Imagem Individual de Molécula/métodos , Amiloide/química , Desenho de Equipamento , Espectrometria de Massas/instrumentação , Nanopartículas/química , Nanotecnologia/instrumentação , Imagem Individual de Molécula/instrumentação , Vírus/química
4.
Nat Commun ; 11(1): 3607, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32680983

RESUMO

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm2/s. In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.


Assuntos
DNA/química , Proteínas/química , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/instrumentação
5.
PLoS One ; 15(5): e0232540, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374767

RESUMO

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution.


Assuntos
Apoferritinas/química , Apoferritinas/ultraestrutura , Microscopia Crioeletrônica/métodos , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica/instrumentação , Cristalografia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Subunidades Proteicas , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Eletricidade Estática
6.
Methods Mol Biol ; 2055: 399-412, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31502162

RESUMO

Single-molecule array (Simoa) technology enables ultrasensitive protein detection that is suited to the development of peripheral blood-based assays for assessing immuno-oncology responses. We adapted a panel of Simoa assays to measure systemic cytokine levels from plasma and characterized physiologic variation in healthy individuals and preanalytic variation arising from processing and handling of patient samples. Insights from these preclinical studies led us to a well-defined set of Simoa assay conditions, a specimen processing protocol, and a data processing approach that we describe here. Simoa enables accurate quantitation of soluble immune signaling molecules in an unprecedented femtomolar range, opening up the potential for liquid biopsy-type approaches in immuno-oncology. We are using the method described here to distinguish PD-1 inhibitor nonresponders as early as after one dose after therapy and envision applications in characterizing PD-1 inhibitor resistance and detection of immune-related adverse effects.


Assuntos
Citocinas/sangue , Neoplasias/imunologia , Imagem Individual de Molécula/instrumentação , Biomarcadores Tumorais/sangue , Humanos , Imunoterapia , Neoplasias/sangue , Análise Serial de Proteínas/instrumentação
7.
J Phys Chem Lett ; 10(24): 7672-7677, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31763851

RESUMO

Current submillisecond time-resolved broad-band infrared spectroscopy, one of the most frequently used techniques for studying structure-function relationships in life sciences, is typically limited to fast-cycling reactions that can be repeated thousands of times with high frequency. Notably, a majority of chemical and biological processes do not comply with this requirement. For example, the activation of vertebrate rhodopsin, a prototype of many protein receptors in biological organisms that mediate basic functions of life, including vision, smell, and taste, is irreversible. Here we present a dispersive single-shot Féry spectrometer setup that extends such spectroscopy to irreversible and slow-cycling systems by exploiting the unique properties of brilliant synchrotron infrared light combined with an advanced focal plane detector array embedded in a dispersive optical concept. We demonstrate our single-shot method on microbial actinorhodopsin with a slow photocycle and on vertebrate rhodopsin with irreversible activation.


Assuntos
Rodopsina/química , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Espectrofotometria Infravermelho/instrumentação , Espectrofotometria Infravermelho/métodos , Cinética , Luz , Processos Fotoquímicos , Conformação Proteica
8.
Nat Commun ; 10(1): 5318, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754221

RESUMO

Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of SRS microscopy is limited to ~10 mM for endogenous biomolecules. Electronic pre-resonant SRS allows detection of sub-micromolar chromophores. However, label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10-30 cm2 sr-1) remains unreachable. Here, we demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation stress. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical systems.


Assuntos
Lasers , Microscopia Óptica não Linear/métodos , Imagem Individual de Molécula/métodos , Análise Espectral Raman/métodos , Staphylococcus aureus/metabolismo , Adenina/metabolismo , Elétrons , Isótopos de Nitrogênio/metabolismo , Reprodutibilidade dos Testes , Imagem Individual de Molécula/instrumentação , Estresse Fisiológico
9.
Annu Rev Virol ; 6(1): 161-176, 2019 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-31567066

RESUMO

Until recently X-ray crystallography has been the standard technique for virus structure determinations. Available X-ray sources have continuously improved over the decades, leading to the realization of X-ray free-electron lasers (XFELs). They provide high-intensity femtosecond X-ray pulses, which allow for new kinds of experiments by making use of the diffraction-before-destruction principle. By overcoming classical dose constraints, they at least in principle allow researchers to perform X-ray virus structure determination for single particles at room temperature. Simultaneously, the availability of XFELs led to the development of the method of serial femtosecond crystallography, where a crystal structure is determined from the measurement of hundreds to thousands of microcrystals. In the case of virus crystallography this method does not require freezing of the crystals and allows researchers to perform experiments under non-equilibrium conditions (e.g., by laser-induced temperature jumps or rapid chemical mixing), which is currently not possible with electron microscopy.


Assuntos
Cristalografia/métodos , Elétrons , Lasers , Imagem Molecular/métodos , Vírus/química , Cristalografia/instrumentação , Imagem Molecular/instrumentação , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Vírus/ultraestrutura , Raios X
10.
Nat Commun ; 10(1): 3400, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363088

RESUMO

Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. Here, we employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. We employ the versatility of DII-state SMLM to resolve the nanoscopic spatial regulation and dynamics of single fatty acid analogs (FAas) and lipid droplets (LDs) in living yeast and mammalian cells with two colors. In fed cells, FAas localize to the endoplasmic reticulum and LDs of ~125 nm diameter. Upon fasting, however, FAas form dense, non-LD clusters of ~100 nm diameter at the plasma membrane and transition from free diffusion to confined immobilization. Our reported SMLM capability of conventional BODIPY conjugates is further demonstrated by imaging lysosomes in mammalian cells and enables simple and versatile live-cell imaging of sub-cellular structures at the nanoscale.


Assuntos
Compostos de Boro/química , Rastreamento de Células/métodos , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , Linhagem Celular Tumoral , Rastreamento de Células/instrumentação , Células/química , Células/citologia , Células/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/instrumentação
11.
Nat Commun ; 10(1): 3552, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391532

RESUMO

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes , Microscopia/métodos , Plasmídeos/genética , Imagem Individual de Molécula/métodos , Proteína 9 Associada à CRISPR/genética , Dosagem de Genes , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia/instrumentação , Modelos Genéticos , Método de Monte Carlo , Motivos de Nucleotídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula/instrumentação , Fatores de Tempo
12.
Nat Commun ; 10(1): 3712, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420559

RESUMO

Nanopore-based single nanoparticle detection has recently emerged as a vibrant research field with numerous high-impact applications. Here, we introduce a programmable optofluidic chip for nanopore-based particle analysis: feedback-controlled selective delivery of a desired number of biomolecules and integration of optical detection techniques on nanopore-selected particles. We demonstrate the feedback-controlled introduction of individual biomolecules, including 70S ribosomes, DNAs and proteins into a fluidic channel where the voltage across the nanopore is turned off after a user-defined number of single molecular insertions. Delivery rates of hundreds/min with programmable off-times of the pore are demonstrated using individual 70S ribosomes. We then use real-time analysis of the translocation signal for selective voltage gating of specific particles from a mixture, enabling selection of DNAs from a DNA-ribosome mixture. Furthermore, we report optical detection of nanopore-selected DNA molecules. These capabilities point the way towards a powerful research tool for high-throughput single-molecule analysis on a chip.


Assuntos
Dispositivos Lab-On-A-Chip , Nanoporos , Dispositivos Ópticos , Imagem Individual de Molécula/instrumentação , DNA , Escherichia coli , Ribossomos
13.
Methods Mol Biol ; 2027: 171-180, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31309481

RESUMO

Single-molecule techniques such as fluorescence-based methods offer superior sensitivity in biosensing. By direct coupling of analyte recognition and signal amplification, a new sensing strategy, single-molecule mechanochemical sensing, has demonstrated high signal-to-noise ratio in the detection of chemicals and biochemicals. However, parallel sensing is limited. In this strategy, DNA origami nanoassemblies with different sensing probes are used as templates for multiplexing tasks. Assisted by the mechanochemical reporting units, these templates are capable of simultaneous detection of biological samples such as platelet-derived growth factor (PDGF) and DNA fragments in microfluidic channels. The origami-based strategy therefore increases multitasking compatibility of the mechanochemical sensing.


Assuntos
Técnicas Biossensoriais/instrumentação , DNA/química , Dispositivos Lab-On-A-Chip , Nanoestruturas/química , Imagem Individual de Molécula/instrumentação , Técnicas Biossensoriais/métodos , Fenômenos Mecânicos , Conformação de Ácido Nucleico , Fator de Crescimento Derivado de Plaquetas/análise , Imagem Individual de Molécula/métodos
14.
J Vis Exp ; (145)2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30933057

RESUMO

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.


Assuntos
Nanotecnologia , Imagem Individual de Molécula/instrumentação , Espectroscopia de Ressonância Magnética , Movimento (Física)
15.
Adv Mater ; 31(23): e1900422, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30941823

RESUMO

Plasmonic and nanopore sensors have separately received much attention for achieving single-molecule precision. A plasmonic "hotspot" confines and enhances optical excitation at the nanometer length scale sufficient to optically detect surface-analyte interactions. A nanopore biosensor actively funnels and threads analytes through a molecular-scale aperture, wherein they are interrogated by electrical or optical means. Recently, solid-state plasmonic and nanopore structures have been integrated within monolithic devices that address fundamental challenges in each of the individual sensing methods and offer complimentary improvements in overall single-molecule sensitivity, detection rates, dwell time and scalability. Here, the physical phenomena and sensing principles of plasmonic and nanopore sensing are summarized to highlight the novel complementarity in dovetailing these techniques for vastly improved single-molecule sensing. A literature review of recent plasmonic nanopore devices is then presented to delineate methods for solid-state fabrication of a range of hybrid device formats, evaluate the progress and challenges in the detection of unlabeled and labeled analyte, and assess the impact and utility of localized plasmonic heating. Finally, future directions and applications inspired by the present state of the art are discussed.


Assuntos
Técnicas Biossensoriais/métodos , Metais/química , Nanoporos , Imagem Individual de Molécula/métodos , Técnicas Biossensoriais/instrumentação , Campos Eletromagnéticos , Cinética , Ácidos Nucleicos/análise , Polímeros/química , Proteínas/análise , Imagem Individual de Molécula/instrumentação , Espectrometria de Fluorescência , Análise Espectral Raman , Propriedades de Superfície
16.
J Vis Exp ; (146)2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-31009011

RESUMO

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.


Assuntos
Microscopia/instrumentação , Imagem Individual de Molécula/instrumentação , Lentes , Óxidos , Semicondutores
17.
Methods Enzymol ; 619: 225-247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30910022

RESUMO

The ubiquitin-proteasome system (UPS) contributes to changes in cell state and homeostatic maintenance in humans by modulating the stability of about a third of human proteins. For example, cell-cycle regulation requires a central ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), which starts a ubiquitination cascade leading to the degradation of multiple targets. This targeted degradation is mediated by the 26S proteasome, a 2.5-MDa protein complex, which recognizes and degrades ubiquitinated proteins at rates partially controlled by the variations in ubiquitin chain topology. Substrate selectivity of ubiquitin ligases such as the APC/C and of the 26S proteasome from pools of near-identical targets reflects highly regulated kinetic mechanisms. Single-molecule techniques are powerful tools that allow distinction between differential substrate affinities and identification of reaction intermediates in complex mixtures. Here we describe fluorescence-based single-molecule imaging of in vitro ubiquitination reactions catalyzed by the APC/C and ubiquitin-dependent degradation reactions catalyzed by the 26S proteasome.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Imagem Óptica/métodos , Complexo de Endopeptidases do Proteassoma/metabolismo , Imagem Individual de Molécula/métodos , Ubiquitinação , Animais , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Desenho de Equipamento , Humanos , Imagem Óptica/instrumentação , Estabilidade Proteica , Proteólise , Imagem Individual de Molécula/instrumentação
18.
Nat Protoc ; 14(4): 1130-1168, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30903110

RESUMO

Among the different developed solid-state nanopores, nanopores constructed in a monolayer of molybdenum disulfide (MoS2) stand out as powerful devices for single-molecule analysis or osmotic power generation. Because the ionic current through a nanopore is inversely proportional to the thickness of the pore, ultrathin membranes have the advantage of providing relatively high ionic currents at very small pore sizes. This increases the signal generated during translocation of biomolecules and improves the nanopores' efficiency when used for desalination or reverse electrodialysis applications. The atomic thickness of MoS2 nanopores approaches the inter-base distance of DNA, creating a potential candidate for DNA sequencing. In terms of geometry, MoS2 nanopores have a well-defined vertical profile due to their atomic thickness, which eliminates any unwanted effects associated with uneven pore profiles observed in other materials. This protocol details all the necessary procedures for the fabrication of solid-state devices. We discuss different methods for transfer of monolayer MoS2, different approaches for the creation of nanopores, their applicability in detecting DNA translocations and the analysis of translocation data through open-source programming packages. We present anticipated results through the application of our nanopores in DNA translocations and osmotic power generation. The procedure comprises four parts: fabrication of devices (2-3 d), transfer of MoS2 and cleaning procedure (24 h), the creation of nanopores within MoS2 (30 min) and performing DNA translocations (2-3 h). We anticipate that our protocol will enable large-scale manufacturing of single-molecule-analysis devices as well as next-generation DNA sequencing.


Assuntos
Dissulfetos/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microtecnologia/métodos , Molibdênio/química , Nanoporos/ultraestrutura , Nanotecnologia/métodos , DNA/análise , DNA/genética , Diálise/instrumentação , Diálise/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Microtecnologia/instrumentação , Nanotecnologia/instrumentação , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos
19.
J Cell Sci ; 132(4)2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30659118

RESUMO

The actin cytoskeleton is subjected to dynamic mechanical forces over time and the history of force loading may serve as mechanical preconditioning. While the actin cytoskeleton is known to be mechanosensitive, the mechanisms underlying force regulation of actin dynamics still need to be elucidated. Here, we investigated actin depolymerization under a range of dynamic tensile forces using atomic force microscopy. Mechanical loading by cyclic tensile forces induced significantly enhanced bond lifetimes and different force-loading histories resulted in different dissociation kinetics in G-actin-G-actin and G-actin-F-actin interactions. Actin subunits at the two ends of filaments formed bonds with distinct kinetics under dynamic force, with cyclic mechanical reinforcement more effective at the pointed end compared to that at the barbed end. Our data demonstrate force-history dependent reinforcement in actin-actin bonds and polarity of the actin depolymerization kinetics under cyclic tensile forces. These properties of actin may be important clues to understanding regulatory mechanisms underlying actin-dependent mechanotransduction and mechanosensitive cytoskeletal dynamics.This article has an associated First Person interview with the first author of the paper.


Assuntos
Actinas/química , Proteínas Aviárias/química , Proteína de Capeamento de Actina CapZ/química , Mecanotransdução Celular , Imagem Individual de Molécula/métodos , Tropomodulina/química , Citoesqueleto de Actina , Actinas/genética , Actinas/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Galinhas , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Microscopia de Força Atômica , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/instrumentação , Estresse Mecânico , Tropomodulina/genética , Tropomodulina/metabolismo
20.
Nat Commun ; 10(1): 272, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30655518

RESUMO

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein's binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/métodos , Thermus thermophilus/metabolismo , Teorema de Bayes , Sítios de Ligação , DNA de Cadeia Simples/metabolismo , Cinética , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Ligação Proteica , Imagem Individual de Molécula/instrumentação , Software
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA