Ringraziamo Gijs wuite, Erwin JG Peterman, e Peter Gross aiuto per la microfluidica e Alessia Tempestini di aiuto per la preparazione del campione. Questa ricerca è stata finanziata dal Settimo programma quadro dell&#39;Unione europea (7 ° PQ / 2007-2013), contratto di sovvenzione n ° 284.464 e dal Ministero italiano dell&#39;Istruzione, dell&#39;Università e della Ricerca FIRB 2011 RBAP11X42L006, Futuro in Ricerca 2013 RBFR13V4M2, e nel quadro di il Flagship Progetto NANOMAX.

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The authors have nothing to disclose.

Negli ultimi dieci anni, le tecniche di manipolazione e di imaging singole molecole hanno visto grandi progressi in termini di risoluzione spaziale e temporale. La combinazione di tecniche di manipolazione e di imaging è alla base di strumenti potenti che ora permettono il controllo delle condizioni meccaniche di un singolo polimero biologico, come DNA, RNA o filamenti del citoscheletro, e la localizzazione simultanea di singole proteine ​​interagenti con lo stesso polimero . Controllare le condizioni meccaniche de...

Tecniche di singola molecola (SM) hanno notevolmente sviluppato nel corso degli ultimi trenta anni per rispondere alla necessità di superare alcune delle limitazioni delle tradizionali, alla rinfusa misure soluzione 1-3. La manipolazione di molecole biologiche singoli ha creato la possibilità di misurare le proprietà meccaniche dei biopolimeri 4 e controllare i parametri meccanici di proteina-proteina 5 e le interazioni proteina-DNA 6,7. Fluorescenza SM, dall&#39;altro, rappresenta uno strumento estremamente versatile per studiare l&#39;attività della proteina in vitro e in vivo, che conduce alla possibilità di localizzazione e inseguimento singole molecole con precisione nanometrica. Attraverso il montaggio del punto-spread-funzione strumento all&#39;immagine SM, infatti, si può realizzare localizzazione con precisione in funzione principalmente segnale-rumore (SNR) e raggiungere un limite di circa un nanometro 8,9. Queste metodologie trovano potenteapplicazioni nello studio della dinamica delle proteine ​​motrici, nonché dei processi di diffusione sottostanti ricerche bersaglio di proteine ​​che legano il DNA. La capacità di determinare costanti di diffusione in funzione della sequenza di DNA, tempo di permanenza sul bersaglio e con precisione la misurazione della lunghezza del DNA esplorato durante gli eventi di diffusione monodimensionali, rappresentano un potente strumento per lo studio di interazioni proteina-DNA dinamiche di interazione e per la verifica dei meccanismi di specifica destinazione della ricerca. Recentemente, la combinazione di queste due tecniche ha prodotto una nuova generazione di apparati sperimentali 10-14 consentano la manipolazione simultanea di un substrato biologico (per esempio un filamento di actina o una molecola di DNA) e rilevamento / localizzazione di un partner interagenti enzima (per esempio miosina o una proteina di legame al DNA). I vantaggi di queste tecniche poggiano principalmente sulla possibilità di esercitare un controllo meccanico sul polimero intrappolato, così enabling lo studio delle dinamiche di interazione rispetto a forze o coppie. Inoltre, il metodo consente di misurare le reazioni biochimiche lontano dalla superficie, evitando una delle principali limitazioni dei metodi classici SM, cioè, la necessità di immobilizzazione delle molecole in esame su una superficie (vetrino o microsfere). La combinazione di due molecole singole tecniche è necessario superare diverse difficoltà tecniche, derivanti principalmente dai requisiti di stabilità meccanica e adeguata SNR (specialmente quando richiede la localizzazione con precisione nm) 15. In particolare, in caso di allacciamento di rilevamento della fluorescenza SM con pinzette ottiche, la riduzione del rumore e photobleaching da intrappolamento laser a infrarossi 16 e il controllo di buffer biochimici per l&#39;assemblaggio dei complessi biologici e le prestazioni delle misure sperimentali 11 sono di fondamentale importanza. Qui, descriviamo i metodi per l&#39;esecuzione di successomisurazioni in una configurazione a doppio localizzazione cattura / SM fluorescenza. La metodologia è illustrata con l&#39;esempio della proteina repressore lattosio (LacI) fluorescente (con Atto532) e rilevato come si lega ad una molecola di DNA (intrappolato tra due pinzette ottiche) contenente specifiche sequenze di legame Laci (ad esempio, operatori). Abbiamo dimostrato l&#39;efficacia del metodo di rilevazione di legame al DNA di LacI e diffusione lungo il suo profilo nel processo di ricerca di destinazione. Il metodo è applicabile a qualsiasi combinazione di sequenze di DNA e proteine ​​di legame al DNA, come pure ad altri sistemi (microtubuli o filamenti di actina e le proteine ​​motrici che interagiscono con essi).

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:203px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td style="width:151px;">Comments/Description</td>
			<td style="width:203px;">Web Address</td>
		</tr>
		<tr height="114">
			<td height="114" style="height:115px;">elastomeric isolators</td>
			<td style="width:141px;">Newport</td>
			<td style="width:167px;">Newdamp</td>
			<td style="width:151px;">Choose the appropriate Newdamp elastomer depending on the microscope weight and resonance frequencies</td>
			<td><a href="http://www.newport.com/">http://www.newport.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical isolator</td>
			<td style="width:141px;">Optics for Research</td>
			<td style="width:167px;">IO-3-YAG-VHP</td>
			<td></td>
			<td><a href="http://www.ofr.com/">http://www.ofr.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nd:YAG laser, 1064 nm wavelength</td>
			<td style="width:141px;">Spectra-Physics</td>
			<td style="width:167px;">Millennia IR</td>
			<td></td>
			<td><a href="http://www.newport.com/">http://www.newport.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acousto-optic deflectors (AODs)</td>
			<td style="width:141px;">A&#38;A optoelectronic</td>
			<td style="width:167px;">DTS-XY 250</td>
			<td></td>
			<td><a href="http://www.aaoptoelectronic.com/">http://www.aaoptoelectronic.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Direct Digital Synthesizers</td>
			<td style="width:141px;">Analog Devices</td>
			<td style="width:167px;"></td>
			<td></td>
			<td><a href="http://www.analog.com/">http://www.analog.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">quadrant detector photodiodes</td>
			<td style="width:141px;">OSI optoelectronics</td>
			<td style="width:167px;">SPOT-15-YAG</td>
			<td></td>
			<td><a href="http://www.osioptoelectronics.com/">http://www.osioptoelectronics.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">DIO and FPGA board</td>
			<td style="width:141px;">National Instruments</td>
			<td style="width:167px;">NI-PCI-7830R</td>
			<td></td>
			<td><a href="http://www.ni.com/">http://www.ni.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Halogen lamp</td>
			<td style="width:141px;">Schott</td>
			<td style="width:167px;">KL 1500 LCD</td>
			<td></td>
			<td><a href="http://www.schott.com/">http://www.schott.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Condenser</td>
			<td style="width:141px;">Olympus</td>
			<td style="width:167px;">U-AAC 1.4NA Aplanat Apchromat</td>
			<td></td>
			<td><a href="http://www.olympus-global.com/en/">http://www.olympus-global.com/en/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">Objective</td>
			<td style="width:141px;">Nikon</td>
			<td style="width:167px;">CFI Plan Apochromat 60x 1.2NA water immersion</td>
			<td></td>
			<td><a href="http://www.nikoninstruments.com/">http://www.nikoninstruments.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">532 nm laser</td>
			<td style="width:141px;">Coherent</td>
			<td style="width:167px;">Sapphire</td>
			<td></td>
			<td><a href="http://www.coherent.com/">http://www.coherent.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CCD 200X and 2000X</td>
			<td style="width:141px;">Hamamatsu&#160;</td>
			<td style="width:167px;">XC-ST70 CE</td>
			<td></td>
			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">electron-multiplied CCD</td>
			<td style="width:141px;">Hamamatsu&#160;</td>
			<td style="width:167px;">C9100-13</td>
			<td></td>
			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">piezo stage with nm-accuracy</td>
			<td style="width:141px;">Physik Instrumente</td>
			<td style="width:167px;">P-527.2CL&#160;</td>
			<td></td>
			<td><a href="http://www.physikinstrumente.com/">http://www.physikinstrumente.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Emission Filter</td>
			<td style="width:141px;">Chroma Technologies</td>
			<td style="width:167px;">600/100m</td>
			<td></td>
			<td><a href="http://www.chroma.com/">http://www.chroma.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silica beads (1.54 mm)</td>
			<td style="width:141px;">Bangs Laboratories</td>
			<td style="width:167px;">SS04N/5303</td>
			<td></td>
			<td><a href="http://www.bangslabs.com/">http://www.bangslabs.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum (BSA)</td>
			<td style="width:141px;">Sigma Aldrich</td>
			<td style="width:167px;">B4287</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">pentyl acetate&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">46022</td>
			<td style="width:151px;">Flammable liquid and vapour (No 1272/2008)</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">nitrocellulose</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">N8267-5EA</td>
			<td style="width:151px;">Flammable solid&#160; (No 1272/2008)</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">heat block</td>
			<td style="width:141px;">MPM Instruments Srl</td>
			<td style="width:167px;">M502-HBD</td>
			<td style="width:151px;">with 2 removable blocks; preheated at 120&#176; C</td>
			<td><a href="http://www.mpminstruments.com/">http://www.mpminstruments.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoPort assemblies</td>
			<td style="width:141px;">Upchurch Scientific Inc.</td>
			<td style="width:167px;">N-333</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">polyetheretherketone tubing&#160;</td>
			<td style="width:141px;">Upchurch Scientific Inc.</td>
			<td style="width:167px;">1535</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
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		<tr height="84">
			<td height="84" style="height:84px;width:203px;">home-made metallic holder for the assembly of the flow-chamber pressure reservoir made of Plexiglass</td>
			<td style="width:141px;"></td>
			<td style="width:167px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">luer lock-tip syringes 2.5 mL</td>
			<td style="width:141px;">Terumo</td>
			<td style="width:167px;">SS 02LZ1</td>
			<td></td>
			<td><a href="http://www.terumomedical.com/">http://www.terumomedical.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">shut-off valves&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">P-732</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
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		<tr height="42">
			<td height="42" style="height:42px;">flangeless fittings&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">LT-111</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">fluorinated ethylene propylene tubing&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">1549</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:203px;">two computer-controlled solenoid valves</td>
			<td style="width:141px;">Clippard, Cincinnati, USA</td>
			<td style="width:167px;">ET-2-H-M5</td>
			<td></td>
			<td><a href="http://www.clippard.com/">http://www.clippard.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pressure transducer</td>
			<td style="width:141px;">Druck LTD</td>
			<td style="width:167px;">PTX 1400</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">biotin-14-dCTP&#160;</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:167px;">19518-018</td>
			<td></td>
			<td><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Terminal deoxynucleotidyl Transferase (TdT)</td>
			<td style="width:141px;">Thermoscientific</td>
			<td style="width:167px;">EP0161</td>
			<td></td>
			<td><a href="http://www.thermoscientificbio.com/">http://www.thermoscientificbio.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATTO532 maleimide</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">68499</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="157">
			<td height="157" style="height:157px;width:203px;">N,N-dimethylformamide (DMF)&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">227056</td>
			<td style="width:151px;">Combustible Liquid, Harmful by skin absorption., Irritant, Teratogen. H226; H303; H312; H316; H319; H331; H360; P201; P261; P280;P305; P351; P338; P311</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP)&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">C4706</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">L-Glutathione reduced (GSH)</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">G4251</td>
			<td style="width:151px;">Acute toxicity, Oral (Category 5), H303</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:203px;">Amicon Ultra-15, PLQK Ultracel-PL Membrane, 10&#160;kDa cutoff spin concentrators</td>
			<td style="width:141px;">Merck Millipore</td>
			<td style="width:167px;">UFC901024</td>
			<td></td>
			<td><a href="http://www.merckmillipore.it/">http://www.merckmillipore.it/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">streptavidin-coated polystyrene beads 1,87 &#181;m</td>
			<td style="width:141px;">Spherotech, Inc.</td>
			<td style="width:167px;">SVP-15-5</td>
			<td></td>
			<td><a href="http://www.spherotech.com/">http://www.spherotech.com/</a></td>
		</tr>
	</tbody>
</table>

combining single-molecule manipulation and imaging for the study of protein-dna interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

In un esperimento riuscito, uno (o più) proteine ​​marcate sottoposti vincolante / non vincolante diffusione e / o monodimensionale lungo la molecola di DNA (Figura 3A). La localizzazione delle proteine ​​lungo la molecola di DNA consente la quantificazione dei parametri cinetici in funzione della sequenza di DNA. Quando vengono applicate le condizioni di buffer causano diffusione 1D, è possibile seguire traiettorie proteici, e determinare, per esempio, il coefficiente di diffusione D 1D....

Watch this Scientific Journal Video about Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions at JoVE.com

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Qui si descrive la strumentazione e metodi per la rilevazione di molecole proteiche singolo fluorescenza marcata che interagiscono con una singola molecola di DNA sospeso tra due microsfere otticamente intrappolati.

Combinando singola molecola di manipolazione e di imaging per lo studio delle interazioni proteina-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

combining-single-molecule-manipulation-imaging-for-study-protein-dna

Research

JoVE Journal

Biology

11.5K Views.  University of Florence.  Qui si descrive la strumentazione e metodi per la rilevazione di molecole proteiche singolo fluorescenza marcata che interagiscono con una singola molecola di DNA sospeso tra due microsfere otticamente intrappolati. 

Video: Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Ricerca

Biologia

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated modello animale Stimoli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

L&#39;isolamento e la manipolazione genetica di adulti miociti cardiaci per confocale Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Singola molecola Imaging dei trasporti nucleari

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Singola molecola di imaging del regolamento Gene<em&gt; In vivo</em&gt; Utilizzo di attivazione Cotranslational da Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

Il metodo ChIP-exo: Identificazione interazioni proteina-DNA con Vicino paia di basi di precisione

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Studio delle interazioni proteina-proteina nella ricerca di autofagia

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

Combinazione di attuatore di forza magnetica 3D e imaging a fluorescenza multifunzionale per studiare la meccanobiologia del nucleo

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...