We thank Gijs Wuite, Erwin J.G.&#160;Peterman, and Peter Gross for help with the microfluidics and Alessia Tempestini for help with sample preparation. This research was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n&#176; 284464 and from the Italian Ministry for Education, University and Research FIRB 2011 RBAP11X42L006, Futuro in Ricerca 2013 RBFR13V4M2, and in the framework of the Flagship Project NANOMAX.

1. Optical Tweezers Setup with Nanometer Stability
The experimental setup must provide two optical tweezers with pointing stability at the nanometer level and intensity fluctuations of the trapping laser below 1%. Combination of these conditions will assure nanometer stability of the dumbbell under typical tension (1 pN - few tens of pN), trap stiffness (0.1 pN/nm) and measurement bandwidth (image acquisition rate 20 sec-1). A scheme of the experimental setup is depicted in Figure&#160;1.
<ol>
	<li>Optical tweezers design and construction15,17:
	<ol>
		<li>Mount the experimental apparatus on an optical table with active isolators to reduce mechanical vibrations. Mount the microscope structure on elastomeric isolators to absorb acoustic noise and mechanical resonances of the microscope structure.</li>
		<li>Insert an optical isolator in the path of the trapping laser, near the laser source, to reduce random amplitude fluctuations due to optical feedback.</li>
		<li>Reduce the path length of the trapping laser as much as possible and enclose the whole path in a sealed box to reduce laser pointing fluctuations due to air currents and turbulence.</li>
		<li>Create double optical tweezers by splitting a single near-infrared laser source (Nd:YAG laser, 1,064 nm wavelength) into two beams with orthogonal polarizations. Do not use time-shared traps because they cause oscillation of the dumbbell under tension12.</li>
		<li>Use acousto-optic deflectors (AODs) driven by Direct Digital Synthesizers (DDSs) to allow fine movements of (at least) one trap and precise regulation of DNA tension. DDSs allow a better trap stability than Voltage-Controlled Oscillators (VCOs).</li>
		<li>Insert two quadrant detector photodiodes (QDPs) in the back focal plane of the condenser to detect the position of the trapped beads with sub-nm accuracy.</li>
	</ol>
	</li>
	<li>Check that intensity fluctuations of the trapping laser at the microscope entrance are below 1% by using a photodiode with a bandwidth larger than the rate of image acquisition.</li>
	<li>Check pointing stability of the two trapping lasers:
	<ol>
		<li>Prepare silica beads in phosphate buffer: Dilute 20 &#956;l of silica beads (1.54 &#956;m, 10% solids) into 1 ml of acetone, sonicate 30 sec, vortex briefly, and centrifuge 2 min at 19,000 x g. Resuspend in 1 ml acetone and repeat wash. Resuspend in 1 ml of 50 mM phosphate buffer, wash 2 times, and finally resuspend in 100 &#956;l of 50 mM phosphate buffer.</li>
		<li>Calibrate optical tweezers with silica beads: prepare a flow cell by attaching a coverslip to a microscope slide using double-sticky tape (about 60 &#956;m thick). Flow 1 mg/ml BSA and wait 3 min. Flow silica beads diluted 1/1,000 in phosphate buffer and seal the flow chamber with silicon grease. Trap one bead in each trap and calibrate the traps using the power spectrum method 18.</li>
		<li>Prepare silica beads in pentyl acetate and nitrocellulose: Dilute 20 &#956;l of silica beads (1.54 &#956;m, 10% solids) into 1 ml of acetone, sonicate 30 sec, vortex briefly, and centrifuge 2 min at 19,000 x g. Resuspend in 1 ml acetone and repeat wash. Resuspend in 1 ml of pentyl acetate and wash 2 times. Finally resuspend them in pentyl acetate with 0.1% nitrocellulose dissolved in pentyl acetate.</li>
		<li>Spread 2 &#956;l of 1.54 &#956;m diameter silica beads dissolved in pentyl acetate + 0.1% nitrocellulose onto the surface of a coverslip (24 x 24 mm) using a second coverslip (24 x 60 mm). Attach the coverslip to a microscope slide by using two pieces of double-sided tape to create a flow cell. Fill the flow-cell with 50 mM phosphate buffer and seal it with silicon grease.</li>
		<li>Image one silica bead in brightfield microscopy at 2,000X magnification. The field of view should be 7 x 5 &#956;m acquired at 768 x 576 pixel, so that the bead image has a diameter of 190 pixels. Compensate thermal drifts with a feedback software that, as in ref. 17, calculates x-y position from the image centroid and z from the diffraction rings and corrects drifts moving a piezo stage with nm-accuracy or better.</li>
		<li>Overlap the center of left trap with the bead center (the x-y signal levels from the QDP must be the same measured during the calibration) and measure the position noise and its standard deviation from the QDP. Repeat the measurement for the right trap.</li>
	</ol>
	</li>
</ol>
2. Single Molecule Localization with Nanometer Accuracy
<ol>
	<li>Fluorescence microscopy design and construction15,19
	<ol>
		<li>Use a high quantum efficiency and electron-multiplied CCD to reach the high signal-to-noise ratios necessary for nanometer localization accuracy.</li>
		<li>Choose optics to get an image pixel size ~80 nm (for example, an image magnification ~200X with a camera pixel size of 16 &#956;m). This is sufficiently small to neglect localization error due to pixelation8, but sufficiently large to collect a considerable number of photons per pixel.</li>
		<li>As excitation source, use laser light that is unpolarized (by passing through a non-polarization-maintaining optical fiber) or circularly polarized (by passing through a &#955;/4 waveplate) to maximize excitation of single chromophores, regardless of their orientation.</li>
		<li>Choose emission bandpass filters that efficiently cut-off both excitation and trapping laser lights. Note: In our experiments we labeled our protein with Atto532 dye and used a bandpass emission filter centered at 600 nm with 100 nm bandwidth.</li>
	</ol>
	</li>
</ol>
3. Microfluidics
To achieve a precise control of buffer exchange and &#39;dumbbell&#39; assembly, i.e., the anchoring of a single DNA molecule between two optically trapped microspheres, a custom-built laminar multichannel flow system must be developed. This is composed by a flow chamber, a pressure-reservoir and a pressure control system (Figure&#160;2).
<ol>
	<li>Flow cell preparation 
	NOTE: The flow chamber used for single-molecule experiments on protein-DNA interaction has preferably 4 inlets and 1 outlet, to allow up to 4 parallel buffer flows, each one containing one of the different components required for the experiment: beads, DNA, fluorescently-labeled protein, and imaging buffer. Separation of the components in the flow-channels allows efficient assembly of the dumbbell. Moreover, the imaging channel is useful to avoid background fluorescence from diffusing proteins and attain the high signal-to-noise ratio required for the localization of the DNA-binding protein with a precision of the order of few nanometers.
	<ol>
		<li>Drill 1 mm diameter holes in the microscope slide (76 x 76 x 1 mm) with a diamond-tip, in order to create 4 inlets and one outlet.</li>
		<li>Clean the microscope slides with ethanol and center the port with the O-ring inserted and the adhesive ring in the slide surrounding the access holes. It is fundamental to wear gloves during this step to avoid fingerprints, which would disturb the gluing process.</li>
		<li>Clamp the NanoPorts to the slide, and place it in the oven preheated at 180 &#176;C for 1 hr to allow complete attachment.</li>
		<li>Draw the outline pattern of the flow cell on a piece of paper. The channels should be ~3 mm wide and match the holes position on the microscope slide.</li>
		<li>Place the drawing on top of two pieces of Parafilm, and with a scalpel make sharp and continuous cuts along the drawn outline. Remove any protuberance formed. Discard the lower Parafilm layer, which is just used to guarantee a clean support.</li>
		<li>Place the microscope slide containing the attached NanoPorts upside down in the metallic support.</li>
		<li>Carefully align the Parafilm chamber outline with the holes, making sure that the parafilm does not obtrude the inlets and outlet of the chamber.</li>
		<li>Cover with a cleaned microscope coverslip (62 x 56 x 0.15 mm) and carefully remove excess Parafilm surrounding the chamber.</li>
		<li>Place two heat blocks, previously heated at 120 &#176;C, on top of the flow cell to apply constant pressure on it. Let the Parafilm melt for approximately 25 min.</li>
	</ol>
	</li>
	<li>Pressure reservoir and buffer containers 
	The pressure reservoir should be made of Plexiglas (or similar) and allow accommodation of six buffer containers. The possibility of having at least two extra buffer containers improves the instrument flexibility. Note that transparent containers and tubes considerably assist in flow-system handling and troubleshooting of trapped air bubbles. Sterile and disposable luer lock-tip syringes (2.5 ml), from which the plungers have been previously removed, are good buffer containers and allow an easy connection with the inlet tubing. Make sure that the total fluid volume is small compared with the air volume inside the pressure reservoir, a condition essential to obtain smooth and stable flow.
	<ol>
		<li>Connect shut-off valves at the exit of the buffer containers for turning the buffer flow on or off during the experiments.</li>
		<li>Mount the flow cell on the microscope stage. Connect the shut-off valves to the flow chamber inlets with polyetheretherketone tubing (inner diameter ~150 &#181;m) and flangeless fittings. Add ~3 cm of fluorinated ethylene propylene tubing (inner diameter ~500 &#181;m) as a linkage between the tubing and the NanoPort assemblies of the flow cell. This step is essential to reduce the buffer flow at the chamber entrance to avoid breakage of the flow cell or NanoPort detachment from the glass. The use of large inner diameter tubing alone, on the other hand, would require huge volumes of biological samples.</li>
		<li>Connect the flow chamber outlet to an open Falcon tube at atmospheric pressure. This tube serves as disposal for the buffer flowing out of the flow chamber.</li>
	</ol>
	</li>
	<li>Pressure control system
	<ol>
		<li>Build a pressure control system capable of finely adjusting the air pressure inside the pressure reservoir. This can be done by using two computer-controlled solenoid valves, one connected to a +0.5 atm pressure source (compressor) and the second one to atmospheric pressure. Solenoid valves are repeatedly opened for approximately 7 msec to increase or decrease the pressure in the line until the desired value is reached. NOTE: this approach was adapted from Carlos Bustamante20, and it yields a smoother flow than using a stepping motor syringe pump21.</li>
		<li>Add a pressure transducer controlled by a custom-written software to monitor the effective pressure exerted on the pressure reservoir. Typical working pressures are on the order of 20 mBar for dumbbell assembly and 200 mBar for washing the flow system components.</li>
	</ol>
	</li>
</ol>
4. DNA Tailing with Biotinylated Deoxycytidine Triphosphate (biotin-dCTP)
The DNA molecule should be longer than 1 &#956;m to facilitate its extension under flow and anchoring to the trapped beads. Moreover, a long DNA will prevent the IR trapping light from illuminating the DNA binding protein. This is of special relevance because absorption of IR light from an excited chromophore results in increased photobleaching. NOTE: In the present study, the DNA molecule was 3.7 &#956;m long. The molecule contained three copies of the primary operator O1 and one copy of each of the two auxiliary operators, O2 and O3. These sequences have been inserted in the middle of the molecule, thus resulting approximately equidistant from the trapped beads and IR laser beams.
<ol>
	<li>Mix 1 pmol of linear DNA 3&#8217;-ends with 60 pmol of biotin-14-dCTP, 4 &#956;l of 5x Terminal deoxynucleotidyl Transferase (TdT) buffer (1&#160;M potassium cacodylate, 125&#160;mM Tris, 0.05%&#160;(v/v) Triton X-100, 5&#160;mM CoCl2 (pH&#160;7.2 at 25&#176;C)) and 1.5 &#956;l of TdT and ddH2O to a total volume of 20 &#956;l. Incubate the mixture at 37 &#176;C for 15 min. This will result in the addition of up to 60 nucleotides per DNA extremity. Note that DNA 3&#8217;-overhangs are tailed with higher efficiency than recessed or blunt ends.</li>
	<li>Purify tailed DNA with spin columns or phenol/chloroform extraction and subsequent ethanol precipitation to remove CoCl2 from the TdT reaction buffer.</li>
	<li>After purification, DNA can be stored at 4 &#176;C for several weeks or at -20 &#176;C for longer periods but avoid repeated thawing and refreezing.</li>
</ol>
5. Labeling Protein Thiol Groups with ATTO532
Labeling through modification of cysteine residues must not alter the protein activity. To overcome this problem, we used a LacI mutant, LacIQ231C, which carries just one cysteine per monomer at position 231. This mutant preserves the same characteristics of the wild type and chemical modifications at position 231 have been shown to not interfere with protein stability 22. NOTE: We labeled the protein with ATTO532 maleimide.
<ol>
	<li>Ensure that the protein is dissolved in a buffer with a pH ranging from 7.0 to 7.5 and with a concentration ranging from 2 to 10 mg/ml. NOTE: in these experiments, LacI was dissolved in potassium phosphate 0.3 M, 5% glucose, pH 7.5 (buffer A).</li>
	<li>Dissolve Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) to a final concentration of 100 mg/ml in H2O. TCEP solution must be prepared fresh prior to use.</li>
	<li>Mix 100 &#956;l of the protein with 3 &#956;l TCEP and vortex carefully.</li>
	<li>Dissolve the dye in N,N-dimethylformamide (DMF) to a final concentration of 10 mg/ml working at low light condition. Vortex until completely dissolved.</li>
	<li>Reaction mix: Add 33 &#956;l of dye to the protein+TCEP and vortex carefully. Fast spin to collect as much solution as possible.</li>
	<li>Incubate the reaction mix for 2 hr in a shaker (8,000 rpm) at 20 &#176;C (or at 4 &#176;C O/N), protected from light.</li>
	<li>Dissolve L-Glutathione reduced (GSH) to a final concentration of 100 mg/ml in H2O. Add 3 &#956;l to the reaction mixture and incubate on the shaker at 8,000 rpm for 15 min. GSH will stop the reaction by blocking the reactivity of thiol groups.</li>
	<li>Purify the labeled protein using standard gel filtration columns, dialysis or spin concentrators. NOTE: In these experiments, labeled LacI was purified using 10&#160;kDa cutoff spin concentrators.
	<ol>
		<li>Dilute the reaction mixture with up to 12 ml of buffer A, apply it to the concentrator device, and centrifuge it at 8,000 x g, 1 hr, 4 &#176;C. The labeled LacI is thus concentrated to a final volume of about 500 &#956;l and the excess dye passes the membrane in the flow-through. Discard the flow-through and repeat step 5.8.1 at least three times.</li>
	</ol>
	</li>
	<li>Divide the labeled protein into small aliquots and store at -80 &#176;C. Avoid repeated freezing and thawing.</li>
</ol>
6. Combined Optical Trapping and Single-molecule Fluorescence Imaging Experiments
<ol>
	<li>Add 1 ml of buffer A to the buffer containers and apply 200 mBar pressure to wash the connecting tubes and flow cell. To maximize diffusion of the binding protein on DNA, here and in the following steps, buffer A may be replaced with a low ionic strength buffer. NOTE: In these experiments, we used TBE 0.5x.</li>
	<li>Check for the presence of air-bubbles, which might disrupt the flow smoothness. A gentle flick on the outlet tube will help to remove any remaining bubbles.</li>
	<li>Decrease the pressure to 20 mBar and verify that all channels and tubes are clear from air bubbles and the buffer flows with similar velocities in all channels.</li>
	<li>Prepare the samples to a final volume of 300 &#181;l: streptavidin-coated polystyrene beads 1.87 &#181;m; 20 pM of linear DNA, biotinylated on both extremities according to section 4; 300 pM of LacI tetramer labeled with ATTO532 according to section 5.</li>
	<li>Add each sample to one of the syringe containers in the following order: beads in the first channel, DNA in the second, third channel with the imaging buffer only (buffer A + oxygen scavenger system) and the labeled protein in the fourth channel.</li>
	<li>Turn the flow on at 200 mBar for 3 min to let the samples arrive inside the flow channel. Then reduce the pressure to 20 mBar.</li>
	<li>While imaging the first channel under bright field illumination, switch on the two optical tweezers and catch a polystyrene bead in each trap.</li>
	<li>Move quickly to the second channel to avoid that other beads fall into the traps. Note that the arrival in the DNA channel is easily noticeable by the end of the bead flow.</li>
	<li>Using one AOD, move one trap (bead) back and forth in the proximity of the other bead to allow the attachment of the flow-extended DNA in between the two optically trapped beads. When a DNA dumbbell is formed, the static bead starts following the movement of the bead that is actively moved back and forth by the trap.</li>
	<li>Move quickly to the buffer channel and turn off the buffer flow. Perform a force-extension curve analysis4,23-25 to verify the presence of a single DNA molecule. If more than one molecule is present (i.e., the enthalpic, raising phase of the force-extension curve occurs at a length shorter than the DNA contour length), discard the dumbbell and start again from step 6.7.</li>
	<li>Once a single DNA dumbbell is obtained, turn on the flow again and move the dumbbell to the protein channel. Switch to wide field fluorescence microscopy to monitor the interaction of the labeled-LacI with DNA. Turn off the flow and start acquisition.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+precise+algorithm+based+on+maximum+radial+symmetry+for+single+molecule+localization.">Fast and precise algorithm based on maximum radial symmetry for single molecule localization.</a> Opt Lett. 37, 2481-2483 (2012).</li><li>Parthasarathy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+accurate+particle+tracking+by+calculation+of+radial+symmetry+centers.">Rapid, accurate particle tracking by calculation of radial symmetry centers.</a> Nat Methods. 9, 724-726 (2012).</li></ol>

The authors have nothing to disclose.

In the last decade, single molecule manipulation and imaging techniques have seen great progress in terms of spatial and temporal resolution. The combination of manipulation and imaging techniques is at the base of powerful instruments that now allow the control of the mechanical conditions of a single biological polymer, such as DNA, RNA or cytoskeletal filaments, and the simultaneous localization of single proteins interacting with the same polymer. Controlling the mechanical conditions of the trapped polymer is of par...

Single molecule (SM) techniques have greatly developed over the past thirty years to respond to the need of overcoming some of the limitations of traditional, bulk solution measurements 1-3. The manipulation of single biological molecules has created the opportunity to measure mechanical properties of biopolymers4 and control the mechanical parameters of protein-protein5 and protein-DNA interactions6,7. SM fluorescence detection, on the other hand, represents an incredibly versatile tool for studying protein activity in vitro and in vivo, leading to the possibility of localizing and tracking single molecules with nanometer precision. Through fitting of the instrument point-spread-function to the SM image, in fact, one can accomplish localization with a precision depending mainly on signal-to-noise ratio (SNR) and reaching a limit of about one nanometer8,9. These methodologies find powerful applications in the study of the dynamics of motor proteins, as well as of the diffusion processes underlying target search in DNA-binding proteins. The capability of determining diffusion constants as a function of the DNA sequence, residence time on the target and accurately measuring the DNA length explored during one-dimensional diffusion events, represent a powerful tool for the study of protein-DNA interaction dynamics and for the investigation of the mechanisms of specific target search.
Recently, the combination of these two techniques has produced a new generation of experimental setups10-14 enabling the simultaneous manipulation of a biological substrate (for example an actin filament or a DNA molecule) and detection/localization of an interacting partner enzyme (for example myosin or a DNA-binding protein). The advantages of these techniques mainly rest on the possibility of exerting mechanical control over the trapped polymer, thus enabling the study of interaction dynamics versus forces or torques. Also, the methodology allows measuring biochemical reactions far from the surface, avoiding one of the main limitations of classic SM methods, i.e., the need for immobilization of the molecules under study on a surface (glass slide or microspheres).
The combination of two single molecules techniques requires overcoming several technical difficulties, mainly arising from the requirements of mechanical stability and adequate SNR (especially when requiring localization with nm precision)15. Particularly, when coupling SM fluorescence detection with optical tweezers, the reduction of noise and photobleaching from the trapping infrared lasers16 and the control of biochemical buffers for assembly of the biological complexes and performance of the experimental measurements11 are of paramount importance. Here, we describe the methods for performing successful measurements in a dual trapping/SM Fluorescence localization setup. The methodology is illustrated with the example of lactose repressor protein (LacI) fluorescently labeled (with Atto532) and detected as it binds to a DNA molecule (trapped between two optical tweezers) containing specific LacI binding sequences (i.e., operators). We demonstrate the effectiveness of the method in detecting binding of LacI to DNA and diffusion along its contour in the target search process. The method is applicable to any combination of DNA sequence and DNA-binding protein, as well as to other systems (microtubules or actin filaments and the motor proteins interacting with them).

<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>
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		<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>
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			<td><a href="http://www.ofr.com/">http://www.ofr.com</a></td>
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			<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>
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			<td><a href="http://www.newport.com/">http://www.newport.com/</a></td>
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			<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>
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			<td><a href="http://www.analog.com/">http://www.analog.com/</a></td>
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			<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>
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			<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>
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			<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>
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			<td><a href="http://www.schott.com/">http://www.schott.com</a></td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com</a></td>
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			<td height="21" style="height:21px;">electron-multiplied CCD</td>
			<td style="width:141px;">Hamamatsu&#160;</td>
			<td style="width:167px;">C9100-13</td>
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			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com/</a></td>
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		<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>
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			<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>
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			<td><a href="http://www.chroma.com/">http://www.chroma.com</a></td>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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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			<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>
		</tr>
		<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>
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			<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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			<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>
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			<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>
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			<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>
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		</tr>
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			<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 a successful experiment, one (or more) labeled proteins undergo binding/unbinding and/or monodimensional diffusion along the DNA molecule (Figure 3A). Localization of proteins along the DNA molecule allows the quantification of kinetic parameters as a function of the DNA sequence. When buffer conditions causing 1D diffusion are applied, it is possible to follow protein trajectories, and determine, for example, the diffusion coefficient D1D.
The precise position o...

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

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

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. Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

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.

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

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.

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

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

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.

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.

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.

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.

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.

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.

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

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.

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