MAM, FS et MGM sont financés par niehs (Project 4, P42ES027704, PI, Rusyn). MAM, FS, RMM, MGM et PKS sont soutenus par le GCC Center for Advanced Microscopy and Image Informatics (RP170719) financé par le CPRIT. L’imagerie est également soutenue par le noyau de microscopie intégrée du Baylor College of Medicine avec le financement des NIH (DK56338 et CA125123), du CPRIT (RP150578), du Dan L. Duncan Comprehensive Cancer Center et du John S. Dunn Gulf Coast Consortium for Chemical Genomics.

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Les auteurs n’ont rien à divulguer.

La méthodologie smFISH décrite est basée sur les protocoles7,12,14publiés précédemment. Dans ce protocole, nous expliquons les étapes critiques qui ont été optimisées du laboratoire humide (y compris la densité d’ensemencement, le temps de fixation, la perméabilisation et la concentration de la sonde), à l’imagerie et à l’analyse d’images, fournissant un pipeline expérimental complet pour les laboratoires ...

La transcription et la traduction des gènes sont les deux processus majeurs impliqués dans le dogme central de la biologie, allant de la séquence d’ADN à l’ARN en passant par la protéine1. Dans cette étude, nous nous concentrons sur la production d’ARN messager (ARNm) pendant la transcription. La molécule d’ARN naissante comprend à la fois des introns non codants et des exons codants. Les introns sont épissés co-transcriptionnellement avant que l’ARNm ne se déplace dans le cytoplasme pour être traduit sur les ribosomes2,3.
Traditionnellement, la mesure de l’ARNm est effectuée dans des populations de cellules en vrac (des centaines à des millions) avec des tests classiques tels que la RT-qPCR ou le Northern blotting. Bien que très puissantes, ces méthodes sont limitées car elles ne fournissent pas d’informations sur la variation de cellule à cellule (hétérogénéité phénotypique), l’identification du nombre d’allèles transcriptionnellement actifs et, peut-être plus important encore, manquent d’informations spatiales. Plus récemment, les technologies à l’échelle du génome permettant le séquençage de l’ARN unicellulaire (RNAseq) ont commencé à combler le fossé entre les méthodes d’imagerie et le séquençage4. Cependant, RNAseq souffre d’une efficacité de détection relativement faible et les étapes de traitement entraînent une perte complète d’informationsspatiales 5. Bien que l’ARNAseq unicellulaire puisse fournir des informations sur l’hétérogénéité phénotypique au sein d’une population de cellules isogéniques, l’hybridation in situ par fluorescence d’ARN (ARN-FISH) peut faciliter une exploration plus complète de l’expression des gènes cibles au niveau spatial, et sur une base allèle par allèle6,7.
RNA FISH est une technique qui permet la détection et la localisation de molécules d’ARN cibles dans des cellules fixes. Contrairement aux méthodes laborieuses antérieures, RNA-FISH, actuellement à la pointe de la technologie, utilise des sondes d’acide nucléique disponibles dans le commerce qui sont complémentaires aux séquences d’ARN cibles, et ces sondes s’hybrident à leurs cibles par appariement de base Watson-Crick8. Les premières techniques d’hybridation in situ impliquaient un protocole similaire établi par Gall et Pardue en 1969, car un échantillon était traité avec des sondes d’acide nucléique qui s’hybridaient spécifiquement en un ARN cible9. À l’origine, le test était effectué à l’aide d’une détection radioactive ou colorimétrique; cependant, dans les années 1980, le développement de protocoles d’hybridation in situ par fluorescence (FISH) à l’ADN FISH et plus tard à l’ARN FISH a ouvert la voie à une détection plus sensible et au potentiel de multiplexage10,11.
D’autres développements dans l’ARN FISH ont conduit à la capacité de détecter et de quantifier des molécules d’ARN uniques (smFISH)12,13. La détection directe est une méthode dans laquelle les sondes elles-mêmes sont étiquetées avec des fluorophores. Un défi avec smFISH est qu’il est nécessaire d’avoir suffisamment de sondes / cibles hybridantes pour faciliter la détection des signaux fluorescents en tant que points distincts et limités par diffraction. Pour résoudre ce problème, on peut utiliser un ensemble de sondes d’oligonucléotides d’ADN simple brin courts complémentaires à diverses régions de l’ARN cible8,12,14. La liaison de plusieurs sondes augmente la densité locale des fluorophores, rendant l’ARN visible comme un point distinct de haute intensité par microscopie à fluorescence. Cette méthode est avantageuse car la liaison hors cible des oligonucléotides dans le pool d’ensembles de sondes peut être distinguée du signal spot d’ARN véritable en analysant quantitativement la taille et l’intensité des points pour différencier le signal réel et la liaison fausse aux oligonucléotides, facilitant ainsi une analyse plus précise en réduisant les faux positifs12.
Les méthodes alternatives pour détecter l’ARNm sont la méthode classique de traduction des entailles basée sur le clone BAC et le système d’ADN ramifié plus récemment développé. Dans la méthode de traduction par entaille clonée au BAC, l’ADN à étiqueter est entaillé enzymatiquement et un nouveau nucléotide est ajouté à l’extrémité 3' exposée. L’activité de l’ADN polymérase I ajoute un nucléotide marqué résultant en la sonde souhaitée15,16. La technique de l’ADN ramifié implique des sondes d’oligonucléotides qui s’hybrident par paires avec des cibles d’ARN et ces sondes sont amplifiées à l’aide de plusieurs molécules d’amplification du signal. Cette méthode peut améliorer considérablement le rapport signal/bruit, mais l’amplification peut fausser la quantification précise puisque les taches fluorescentes peuvent être très différentes en taille et en intensité17.
En tant que système modèle pour ce protocole, qui est pleinement utilisé dans le cadre de la participation au programme de recherche NIEHS Superfund pour quantifier les effets des perturbateurs endocriniens dans Galveston Bay / Houston Ship Channel, nous avons utilisé la lignée cellulaire MCF-7 du cancer du sein MCF-7 positive au récepteur des œstrogènes (ER) traitée pendant la nuit avec un agoniste ER, le 17β-estradiol (E2)7,18,19. E2 régule de nombreux gènes cibles ER, y compris le gène prototype ER-cible, GREB17,20,21,22. Le protocole smFISH décrit ici utilise un ensemble de deux ensembles de sondes séparés spectralement ciblant GREB1; l’un qui s’hybride en exons et l’autre en introns, permettant la mesure de l’ARN7mature et naissant. Nous utilisons ensuite la microscopie à épifluorescence couplée à la déconvolution pour imager des échantillons marqués smFISH, puis appliquons des routines d’analyse d’images pour quantifier le nombre d’allèles actifs et d’ARN matures par cellule.

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			<td height="42" style="height:42px;width:216px;">17&#946; Estradiol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2257</td>
			<td style="width:167px;">CAS Number 50-28-2</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambion SSC (20X) Buffer, Rnase-free</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AM9770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning DMEM with L-Glutamine and 4.5 g/L Glucose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT10017CV</td>
			<td>Manufactured by Invitrogen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8417</td>
			<td>Without sodium pyruvate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran Sulfate, 50% Solution Sterile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3730-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline Ca++/Mg++</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21030CV</td>
			<td>Manufactured by Calbiochem</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol, 200 Proof (100%)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">07-678-005</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">353047</td>
			<td>Manufactured by Decon Laboratories</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum (FBS), 500 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-753-2978</td>
			<td>Manufactured by Gemini Bio Products</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope</td>
			<td style="width:71px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hyclone Water, Molecular Biology Grade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">SH3053802</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Immersion Oil 1.516</td>
			<td style="width:71px;">GE Healthcare Life Sciences</td>
			<td align="right" style="width:119px;">29162940</td>
			<td>Manufactured by GE Healthcare Bio-Science</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-glutamine Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MCF-7 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">HTB-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Grade Formamide</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">PAH5051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus 60x/1.42 NA Objective</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Parafilm</td>
			<td style="width:71px;">Bemis Company, Inc.</td>
			<td style="width:119px;">52858-076</td>
			<td>Distributed by VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicilin-Streptomycin Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT3001Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ribonucleoside Vanadyl Complex</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">101229-716</td>
			<td>Manufactured by New England Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNase Away</td>
			<td style="width:71px;">Ambion</td>
			<td align="right" style="width:119px;">9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium pyruvate, 100 mM</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">11-360-070</td>
			<td>Manufactured by Gibco</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thermo Scientific Glass Coverslips, #1.5</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-545-81P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100, 100 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td>Manufactured by Fisher BioReagents</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA Solution 0.25%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectashield Antifade Mounting Medium 10 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC9265087</td>
			<td>Manufactured by Vector Laboratories</td>
		</tr>
	</tbody>
</table>

single cell analysis of transcriptionally active alleles by single molecule fish

La transcription des gènes est un processus essentiel en biologie cellulaire et permet aux cellules d’interpréter et de répondre aux signaux internes et externes. Les méthodes traditionnelles de population en vrac (Transfert de Northern, PCR et ARSEq) qui mesurent les niveaux d’ARNm n’ont pas la capacité de fournir des informations sur la variation des réponses d’une cellule à l’autre. Une visualisation et une quantification précises d’une seule cellule et d’une seule cellule sont possibles grâce à l’hybridation in situ par fluorescence d’ARN à molécule unique (smFISH). ARN-FISH est réalisé en hybridant des ARN cibles avec des sondes oligonucléotidiques étiquetées. Ceux-ci peuvent être imagés dans des modalités de débit moyen / élevé et, grâce à des pipelines d’analyse d’images, fournir des données quantitatives sur les ARN matures et naissants, le tout au niveau de la cellule unique. Les étapes de fixation, de perméabilisation, d’hybridation et d’imagerie ont été optimisées en laboratoire pendant de nombreuses années à l’aide du système de modèle décrit ici, ce qui se traduit par une analyse unicellulaire réussie et robuste de l’étiquetage smFISH. L’objectif principal de la préparation et du traitement des échantillons est de produire des images de haute qualité caractérisées par un rapport signal/bruit élevé afin de réduire les faux positifs et de fournir des données plus précises. Ici, nous présentons un protocole décrivant le pipeline de la préparation des échantillons à l’analyse des données en conjonction avec des suggestions et des étapes d’optimisation à adapter à des échantillons spécifiques.

Pour analyser les réponses hormonales via smFISH, à titre d’exemple, nous avons choisi le modèle de récepteur d’œstrogène (ER) utilisé dans les essais à haut débit pour déterminer la présence de perturbateurs endocriniens dans les catastrophes environnementales dans le cadre de la participation au programme de recherche NIEHS Superfund7. Dans cette expérience, les cellules adhérentes du cancer du sein MCF-7 ont été traitées avec l’agoniste ER 17β-estradiol (E2, 10 nM) ou vé...

Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

L’hybridation in situ par fluorescence d’ARN à molécule unique (smFISH) est une méthode permettant de quantifier avec précision les niveaux et la localisation d’ARN spécifiques au niveau de la cellule unique. Ici, nous rapportons nos protocoles de laboratoire validés pour le traitement au banc humide, l’imagerie et l’analyse d’images pour la quantification unicellulaire d’ARN spécifiques.

Analyse unicellulaire d’allèles transcriptionnellement actifs par molécule unique FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

single-cell-analysis-transcriptionally-active-alleles-single-molecule

Research

JoVE Journal

Biochemistry

3.1K vues.  GCC Center for Advanced Microscopy and Image Informatics. L’hybridation in situ par fluorescence d’ARN à molécule unique (smFISH) est une méthode permettant de quantifier avec précision les niveaux et la localisation d’ARN spécifiques au niveau de la cellule unique. Ici, nous rapportons nos protocoles de laboratoire validés pour le traitement au banc humide, l’imagerie et l’analyse d’images pour la quantification unicellulaire d’ARN spécifiques.

Vidéo: Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Manipulation de molécules simples de G-quadruplexes par pinces magnétiques

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Recherche

Biochimie

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

Immobilisation covalente de protéines pour la spectroscopie de Force seule molécule

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Échelle du protéome Quantification d’homogénéité à l’échelle de la molécule unique d’étiquetage

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Conjugués de BODIPY conventionnels pour la microscopie à super-résolution à cellules vives et le suivi à molécule unique

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Un essai in vitro d’imagerie à molécule unique pour l’analyse de la cinétique de traduction cap-dépendante

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

Analyse du temps de séjour d’une molécule unique du clivage de l’ADN médié par l’endonucléase de restriction

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

Étude de transfert d’énergie de fluorescence à molécule unique de synthèse de protéines ribosomes

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Analyse à molécule unique de moteurs de la famille Sf9 superprocessive kinésine-3 purifiée

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Diffusion et assemblage d’une molécule unique sur des membranes lipidiques encombrées de polymères

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

<meta charset="utf8"></head><body>Reconstitution in situ de nucléosomes pour l’étude d’une molécule unique

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

<meta charset="utf8"></head><body>Pleins feux sur l’auteur : Reconstitution efficace des nucléosomes pour les techniques à molécule unique

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Assemblage de nucléosomes in situ pour la microscopie à force corrélative et à fluorescence d’une molécule unique

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...