This work was supported by the European Union&#8217;s Horizon 2020 research and innovation programme under Grant Agreement No. 871124 Laserlab-Europe, by the Italian Ministry of University and Research (FIRB &#8220;Futuro in Ricerca&#8221; 2013 Grant No. RBFR13V4M2), and by Ente Cassa di Risparmio di Firenze. A.V. Kashchuk was supported by Human Frontier Science Program Cross-Disciplinary Fellowship LT008/2020-C.

1. Optical setup
NOTE: The experimental setup is composed of double optical tweezers with nanometer pointing stability and &#60; 1% laser intensity fluctuations. Under these conditions, stability of the dumbbell at the nanometer level is guaranteed under typical trap stiffness (0.1 pN/nm) and tension (1 pN - few tens of pN). Figure 2 shows a detailed scheme of the optical setup.
<ol>
	<li>Optical tweezers design and construction 9,10,11.
	<ol>
		<li>Place all the components of the setup on an optical table according to the scheme in Figure 2. Note that the optical table includes active isolators to minimize mechanical vibrations. Additionally, the microscope structure is mounted on elastomeric isolators to absorb acoustic noise and mechanical resonances.</li>
		<li>Insert an optical isolator close to the laser source (&#34;OI&#34; in Figure 2), to avoid random amplitude fluctuations due to optical feedback.</li>
		<li>Seal the whole path in a closed box to reduce air current and turbulences that could impact the laser pointing stability.</li>
		<li>Create the double optical tweezers by dividing the main laser source (Nd:YAG laser, 1,064 nm wavelength in Figure 2) into two branches with orthogonal polarizations by the use of polarizing beam splitters (PBS) . Time-shared traps should be avoided because they induce oscillation of the dumbbell under tension12.</li>
		<li>Use two acousto-optic deflectors (AODs in Figure 2) driven by Direct Digital Synthesizers (DDSs) to allow fine and rapid movements of the two traps and precise regulation of the actin tension by directly driving the DDSs through the digital outputs from the field-programmable gate array FPGA board (see Figure 2). 
		&#8203;NOTE: The overall feedback response time must be &#60;10 &#181;s to rapidly correct and maintain constant force on both traps during measurements, including the unbound state, myosin interaction and movement. To this end, position detectors must have a &#62;= 100 kHz bandwidth and data must be acquired at &#62;= 200 kHz sample rate. For each data point acquired (5 &#181;s acquisition time), proportional corrections for the two traps are calculated by the FPGA and sent to the two DDS driving the AODs. The AOD response time must be below 5 &#181;s to satisfy the required feedback response time.</li>
		<li>For nm detection of the trapped beads position, put two Quadrant Photodiode Detectors (QPDs in Figure 2) in the back focal plane of the condenser. Accurate alignment of the QPDs in a plane conjugated to the back focal plane of the condenser, as well as the AODs in a plane conjugated to the back focal plane of the objective, will assure that QPDs signals will be independent of the AODs frequency.</li>
		<li>Mount the AOD on a linear translator with micrometer drive and displace it until the crystal edge gets close to the laser beam. Then, replace the objective with an iris, centered on the threaded objective housing, and regulate its aperture to fit the objective back aperture size.</li>
		<li>Move the translator towards the laser beam until the portion of the beam blocked by the piezo crystal is visible after the iris, turn the translator slightly backwards to get the beam to completely fill the iris aperture again.</li>
		<li>Repeat steps 1.1.7-1.1.8 for the second AOD. Check the response time of the feedback loop by measuring the time lag from the QPDs while rapidly moving a beam on a bead stuck on the surface of the coverslip13. 
		&#8203;NOTE: The above three steps (1.1.7-1-1-9) lead to the careful alignment of AOD crystals. These steps are important to optimize the time response of both the beam deflection and the feedback13.</li>
	</ol>
	</li>
	<li>By means of a photodiode measure the intensity fluctuations of the trapping laser at the microscope entrance which must be below 1%. Note that the photodiode bandwidth must be larger than the imaging rate.</li>
	<li>Check the pointing stability of both traps.
	<ol>
		<li>Prepare silica beads in phosphate buffer (PB) by diluting 20 &#181;L of silica beads (1.2 &#181;m, 10% solids) in 1 mL of acetone, sonicate for 30 s, vortex briefly, and centrifuge for 2 min at 19,000 x g.</li>
		<li>Remove the supernatant, resuspend in 1 mL of acetone, and repeat the wash. Resuspend in 1 mL of 50 mM PB, wash 2 times. Finally, resuspend in 100 &#181;L of 50 mM PB.</li>
		<li>Perform optical tweezers calibration with silica beads by sticking a coverslip onto a microscope slide with a double-sided tape (about 60 &#181;m thick) to build a flow chamber. Fill the chamber with a 1 mg/mL BSA (Bovine Serum Albumin protein) and wait for 3 min.</li>
		<li>Flow a 1:1000 dilution of silica beads in PB into the chamber. Use a syringe filled with silicon grease to carefully seal the chamber. Trap a single bead in each trap and apply the power spectrum method14 to calibrate them.</li>
		<li>Prepare silica beads in pentyl acetate (at room temperature) by dissolving 20 &#181;L silica beads (1.2 &#181;m diameter, 10% solid) in 1-1.5 mL of acetone, vortex and sonicate for 30 s.</li>
		<li>Centrifuge at 18,500 x g for 2 min. Discard the supernatant and resuspend in 1 mL of acetone, then repeat the wash. Resuspend in 1 mL of pentyl acetate and repeat wash (centrifuge and resuspension) in pentyl acetate 2 times. Resuspend the pellet in 100 &#181;L of nitrocellulose 1% and 900 &#181;L pentyl acetate. Store at 4 &#176;C for 2 months.</li>
		<li>Take a 24 x 24 mm glass coverslip and clean it carefully with paper soaked with pure ethanol. Then, while holding it with clean tweezers, rinse it a second time by washing directly with pure ethanol. Let it dry under a gentle flow of nitrogen. If needed, repeat this operation to remove all visible residues on the glass surface.</li>
		<li>Take the silica beads stock, vortex it and sonicate it briefly for ~30 s.</li>
		<li>After cleaning a second coverslip (24 x 60 mm), use it to smear 2 &#181;L of silica beads solution on one surface of the coverslip, and wait for it to dry.</li>
		<li>Clean carefully a microscope slide (26 x 76 mm) that will be used to create the flow chamber.</li>
		<li>Cut two lines of double sticky tape (~3 mm large, 60-100 &#181;m thick) and attach them on one side of the microscope slide, as shown in Figure 3.</li>
		<li>By using clean tweezers, close the chamber (about 20 &#181;L final volume) by putting the coated coverslip (1.3.9) in contact with the sticky tape lines, with the nitrocellulose + beads layer facing the inside of the chamber, as shown in Figure 3a. Fill the flow chamber with 50 mM phosphate buffer and seal it with silicon grease.</li>
		<li>Image a single silica bead in brightfield microscopy at &#62;200x magnification using a charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) camera with &#62;1.4 megapixels. Use a feedback software to move the piezo stage (with nanometer accuracy or better) to compensate for thermal drifts10.</li>
		<li>Overlap the center of the left trap with the center of the bead (x-y signal levels from the QPD should match those from the calibration). Then measure the position noise and standard deviation of the position signals for this trap.</li>
		<li>Repeat the previous step for the right trap15.</li>
	</ol>
	</li>
	<li>Trap position calibration: MHz to nm
	<ol>
		<li>Prepare a flow chamber with silica beads attached on the coverslip surface (1.3.5-1.3.12) and floating polystyrene beads (use &#945;-actinin conjugated beads prepared as in the following section 2.1).</li>
		<li>Focus a silica bead on the coverslip surface slightly decentered (~5 &#181;m) from the center of the field of view (FOV) and acquire an image of the FOV. Move the bead by 10 &#181;m towards the FOV center using the piezo stage and acquire a second image. Calculate the center of the bead in the two images using a centroid algorithm or similar and calculate the distance in pixel between the two beads to obtain the nm/pixel calibration of the brightfield camera.</li>
		<li>Trap a single floating particle in one trap. Then, move the trap using AOD in small steps (0.2 MHz) and acquire an image of the particle and the corresponding frequency of the AOD for each step. Calculate the position of the particle in the FOV by using the centroid algorithm as before and convert it in nm by using the nm/pixel calibration obtained in the previous step.</li>
		<li>Perform a linear fit to the frequency-position data and calculate the calibration constant in nm/MHz.</li>
		<li>Repeat calibration for the second trap</li>
	</ol>
	</li>
	<li>Trap power and stiffness calibration (MHz vs W), QPD (MHz vs pN/nm)
	<ol>
		<li>Prepare a flow chamber with silica beads attached on the coverslip surface (1.3.5-1.3.12) and floating polystyrene beads (use &#945;-actinin conjugated beads prepared as in the following section 2.1) and trap a single particle in one trap. Then displace both traps through AODs in small steps (0.2 MHz) and record a Brownian motion of the particle in both traps with QPD and the corresponding frequency of the AODs.</li>
		<li>Calculate an average power on the detectors at each position and obtain trap stiffness and QPD calibration constant beta by fitting a Lorentzian function to a power spectrum of the recorded Brownian motion13.</li>
	</ol>
	</li>
</ol>
2. Sample preparation
<ol>
	<li>Prepare &#945;-actinin conjugated fluorescent beads
	<ol>
		<li>Perform conjugation16: Take amino-functionalized polystyrene beads (1 &#181;m diameter, 2.5% solids), wash them twice in 500 &#956;L distilled water and resuspended in 500 &#956;L of PBS (pH 7.0). Add 1 mM HaloTag succinimidyl ester O2 ligand and incubate at room temperature for 1 h. Wash three times with 500 &#956;L of PBS and resuspended with 100-200 &#181;M HaloTag &#945;-actinin. Incubate for 1 h at 37 &#176;C and wash three times in 500 &#956;L of PBS (use beads within 1.5 weeks or flash-frozen in liquid nitrogen and store at &#8722;80 &#176;C).</li>
		<li>Labeling: Incubate 200 &#181;L of beads solution with Rhodamine-BSA at 5 &#181;g/mL final concentration for 10 min. Wash with 50 mM PB three times and resuspend in 500 &#181;L of PB 50 mM. This can be stored in aliquots at - 80 &#176;C for months).</li>
	</ol>
	</li>
	<li>Express and purify biotinylated Myosin-5B as described previously4,17.</li>
	<li>Polymerize and label F-actin13:
	<ol>
		<li>F-actin polymerization: Mix 69 &#181;L of ultrapure water, 10 &#181;L of actin polymerization buffer 10x (100 mM Tris HCl, 20 mM MgCl2, 500 mM KCl, 10 mM ATP, 50 mM guanidine carbonate pH 7.5), 20 &#181;L of G-actin 10 mg/mL, and 1 &#181;L of DL -Dithiothreitol (DTT) 1 M. Leave it on ice for more than 1 h.</li>
		<li>F-actin labeling with rhodamine (Ex/Em: 546/575 nm): Take 25 &#181;L of polymerized F-actin and add 19.5 &#181;L of ultrapure water, 2.5 &#181;L of actin polymerization buffer 10x (100 mM Tris HCl, 20 mM MgCl2, 500 mM KCl, 10 mM ATP, 50 mM guanidine carbonate pH 7.5), 1 &#181;L of 1 M DTT, and 2 &#181;L of 250 &#181;M rhodamine phalloidin. Leave it on ice overnight. For trapping experiments rhodamine F-actin can be stored on ice and used within a week.</li>
	</ol>
	</li>
	<li>Sample assembly
	<ol>
		<li>Take the flow chamber (1.3.5-1.3.12) and incubate 1 mg/mL biotinylated BSA for 5 min. After washing with AB buffer (25 mM MOPS ,25 mM KCl, 4mM MgCl2, 1 mM EGTA, 1 mM DTT, pH 7.2) incubate 1 mg/mL streptavidin for 5 min and wash again with AB buffer. Incubate biotinylated myosin-5B heavy meromyosin at 3 nM concentration in M5B buffer (10 mM MOPS pH 7.3, 0.5 M NaCl, 0.1 mM EGTA, 3 mM NaN3) with 2 &#181;M Calmodulin (CaM) for 5 min. Wash with three volumes of 1 mg/mL biotinylated BSA supplemented with 2 &#181;M CaM in AB and incubate for 3 min.</li>
		<li>While incubating prepare the Reaction Mix (RM): 0.005% &#945;-actinin functionalized beads (section 2.1), 1 nM rhodamine F-Actin (section 2.3) in Imaging Buffer (IB: AB buffer with 1.2 &#181;M Glucose-Oxidase, 0.2 &#181;M catalase, 17 mM glucose, 20 mM DTT, 2 &#181;M CaM and ATP at the concentration needed for the experiment).</li>
		<li>Wash with RM and seal the chamber with silicone grease. The sample is now ready to be watched under the microscope.</li>
	</ol>
	</li>
</ol>
3. Measurement
<ol>
	<li>Assemble the dumbbell.
	<ol>
		<li>Look for the floating &#945;-actinin beads by moving the sample using long-range translators, switch on one trap and trap one bead.</li>
		<li>Once the first trap is occupied, move the translator to position the trapped bead close to the coverslip surface to avoid trapping of multiple beads, and trap another bead in the second trap.</li>
		<li>Adjust the two traps to equal stiffnesses by adjusting the power of the AODs acoustic waves. Stiffnesses are usually set between 0.03 and 0.14 pN/nm. The smaller the stiffness the smaller the force noise, in particular at low forces.</li>
		<li>Then flip the motorized mirror M (Figure 2) to switch to fluorescence microscopy, and look for an actin filament floating in solution by displacing the sample through long-range translators13. Prefer long filaments (&#62;5 &#181;m) since the processive myosin will displace it and move it for a few microns before detaching.</li>
		<li>Move the sample to let one of the trapped beads approaching one end of the filament until they attach to each other. Then, adjust the beads distance to the approximate filament length and create a flow in the direction of the unbound second bead by moving the stage in its direction. The filament will be stretched by the flow and it will eventually bind to it13. The bead-actin-bead complex is called &#34;dumbbell&#34;.</li>
	</ol>
	</li>
	<li>Establish actin-myosin contact.
	<ol>
		<li>Gently separate the two traps far apart to pre-tension the filament up to about 3 pN and probe the rigidity of the dumbbell by making one trap oscillate in a triangular wave by changing the frequency of one of the two AODs and verifying the consequent transmission of the motion to the trailing bead through its position signal.</li>
		<li>Move the stage to put the dumbbell in proximity of a pedestal silica bead and allow the contact between the filament and the protein attached onto bead surface by adjusting the height of the trapped bead centers slightly below the silica bead diameter. Then position the center of the silica bead in between the trapped beads.</li>
	</ol>
	</li>
	<li>Force clamp and nm stabilization feedback:
	<ol>
		<li>Switch on the ultrafast force-clamp with 2-3 pN force and 200 nm oscillation and scan the pedestal bead in discrete steps of about 20-30 nm in the direction perpendicular to the actin filament. Wait for interactions to occur at each position (few seconds), then step ahead if no interaction is observed. As the protein-filament interaction is established, look for the position where interactions are more frequent.</li>
		<li>Ensure that when the processive myosin moves towards one end of the actin filament (usually the + end), the trapped bead attached to the + end moves towards the silica bead. Move the stage towards the -end of the filament so that the silica bead lies as close as possible to the trapped bead attached to the -end of the filament when myosin is not bound and start the nanometer-stabilization feedback. In doing so, the probability that the trapped bead attached to the +end of the filament crashes into the silica bead is minimized.</li>
	</ol>
	</li>
	<li>Record data.</li>
</ol>
4. Data analysis4
NOTE: The analysis method that is described allows for the detection and measurement of processive runs and fast stepping events based on changes in the dumbbell velocity, as caused by myosin stepping. Analysis of processive runs is performed based on a data analysis method for non-processive motors described in references3,4,13.
<ol>
	<li>Set a threshold for velocity changes to allow for stepping event detection. Since in this case both forward and backward steps are expected, the crossing of the threshold is accepted in both directions.</li>
	<li>Assign each step to the corresponding run: if the time interval between two consequent steps is shorter than 3 ms and the amplitude of the steps is &#60; 90 nm, steps are assigned to the same run, otherwise steps are assigned to different runs4.</li>
	<li>Correct run length for assistive forces4.
	<ol>
		<li>Correct run lengths under assisting forces by calculating the real run length value RL from the measured average run length value &#60;RLm&#62; from the following equation, where D is the oscillation range: 
		<img alt="Equation 1" src="/files/ftp_upload/62388/62388eq1v3.jpg" /> 
		NOTE: Details on the derivation of this equation can be found in reference4.</li>
	</ol>
	</li>
</ol>

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The authors declare no competing interests.

Although single molecule techniques, such as the three-bead assay, are technically challenging and low throughput, UFFCS improves the detection of molecular interactions thanks to the high signal-to-noise ratio of the data. UFFCS allows the study of the load-dependence of motor proteins, with the main advantages of applying the force very rapidly upon binding of the motor to the filament to probe early and very rapid events in force production and weak binding states under controlled force; maintaining the force constant...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62388">Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy</a>. The title was updated.
The title was updated from:
Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy
to:
Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62388">Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy</a>. The title was updated.

Erratum: Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy

In the last decades optical tweezers have been a valuable tool to elucidate the mechanochemistry of protein interactions at the single molecule level, due to the striking possibility of concurrent manipulation and measurement of conformational changes and enzymatic kinetics 1,2. In particular, the capability to apply and measure forces in the range of those exerted by molecular motors in the cell, together with the capacity to measure sub-nanometer conformational changes, made optical tweezers a unique single-molecule tool for unraveling the chemomechanical properties of motor proteins and their mechanical regulation.
Ultrafast force-clamp spectroscopy (UFFCS) is a single-molecule force-spectroscopy technique based on optical tweezers, developed to study the fast kinetics of molecular motors under load in a three-bead geometry (Figure 1a)3,4. UFFCS reduces the time lag for force application to the motor protein to the physical limit of optical tweezers, i.e., the mechanical relaxation time of the system, thus allowing the application of the force rapidly after the beginning of a myosin run (few tens of microseconds)3. This capability has been exploited to investigate the early mechanical events in fast skeletal 3 and cardiac5 muscle myosin to reveal the load dependence of the powerstroke, the weak- and strong-binding states, as well as the order of biochemical (Pi) and mechanical (powerstroke) events.
The three-bead geometry is usually employed to study non-processive motors, a single bead geometry with a force-clamp has been commonly used to investigate processive non-conventional myosins such as myosin Va6. However, there are several reasons to prefer a three-bead UFFCS assay also for processive myosins. First, the rapid application of load right after actin-myosin binding allows the measurement of the early events in force development as in non-processive motors. In addition, in the case of processive motors it also allows an accurate measurement of the motor&#39;s run lengths and run durations under constant force all through their progression (Figure 1b). Moreover, because of the high rate of the force feedback, the system can maintain the force constant during fast changes in position, such as the myosin working stroke, thereby guaranteeing a constant load during motor stepping. The high-temporal resolution of the system allows the detection of sub-ms interactions, opening the possibility of investigating weak binding of myosin to actin. Finally, the assay geometry guarantees that the force is applied along the actin filament, with negligible transverse and vertical components of the force. This point is of particular relevance since the vertical force component has been shown to influence significantly the load-dependence of motor&#39;s kinetics7,8. By using this technique, we could apply a range of assistive and resistive loads to processive myosin-5B and directly measure the load dependence of its processivity for a wide range of forces4.
As shown in Figure 1a, in this system a single actin filament is suspended between two polystyrene beads trapped in the focus of double optical tweezers (the &#34;dumbbell&#34;). An imbalanced net force F= F1-F2 is imposed on the filament, through a fast feedback system, which makes the filament move at constant velocity in one direction until it reaches a user-defined inversion point where the net force is reversed in the opposite direction. When the motor protein is not interacting with the filament, the dumbbell is free to move back and forth in a triangular wave shape (Figure 1b, bottom panel) spanning the pedestal bead on which a single motor protein is attached. Once the interaction is established the force carried by the dumbbell is very rapidly transferred to the motor protein and the motor starts displacing the filament by stepping under the force intensity and direction that was applied by the feedback system at the time of the interaction, until myosin detaches from actin. Being the displacement produced by the stepping of the motor dependent on the polarity of the trapped actin filament, according to the direction of the applied force the load can be either assistive, i.e., pushing in the same direction of the motor displacement (push in Figure 1b upper panel), or resistive, i.e., pulling in the opposite direction with respect to the motor displacement (pull in Figure 1b upper panel) making it possible to study the chemomechanical regulation of the motor processivity by both the intensity and the directionality of the applied load.
In the next sections all the steps to measure actin-myosin-5B interactions under different loads with an ultrafast force-clamp spectroscopy setup are fully described, including 1) the setting up of the optical setup, optical traps alignment and calibration procedures, 2) the preparations of all the components and their assembly in the sample chamber, 3) the measurement procedure, 4) representative data and data analysis to extract important physical parameters, such as the run length, the step size and the velocity of the motor protein.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Aliphatic Amine Latex Beads</td>
			<td>ThermoFisher</td>
			<td>A37362</td>
			<td>1.0-&#956;m diameter, 2% (w/v)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>32201</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin polymerization buffer</td>
			<td>Cytoskeleton</td>
			<td>BSA02</td>
			<td>10X</td>
		</tr>
		<tr>
			<td>AODs (acousto-optic deflectors)</td>
			<td>AA Opto Electronic</td>
			<td>DTS-XY 250</td>
			<td>Laser beam deflectors</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-BSA</td>
			<td>ThermoFisher</td>
			<td>29130</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>B4287</td>
			<td></td>
		</tr>
		<tr>
			<td>Calmodulin from porcine brain (CaM)</td>
			<td>Merck Millipore</td>
			<td>208783</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase from bovine liver</td>
			<td>Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr>
			<td>Condenser</td>
			<td>Olympus</td>
			<td>OlympusU-AAC, Aplanat, Achromat</td>
			<td>NA 1.4, oil immersion</td>
		</tr>
		<tr>
			<td>Creatine phosphate disodium salt tetrahydrate</td>
			<td>Sigma</td>
			<td>27920</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine Phosphokinase from rabbit muscle</td>
			<td>Sigma</td>
			<td>C3755</td>
			<td></td>
		</tr>
		<tr>
			<td>DDs</td>
			<td>AA Opto Electronic</td>
			<td>AA.DDS.XX</td>
			<td>Two-channel digital synthesizer</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)/td&#62;</td>
			<td>Sigma</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>G-actin protein</td>
			<td>Cytoskeleton</td>
			<td>AKL99</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase from Aspergillus niger</td>
			<td>Sigma</td>
			<td>&#160;G7141</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag succinimidyl ester O2 ligand</td>
			<td>Promega</td>
			<td>P1691</td>
			<td></td>
		</tr>
		<tr>
			<td>High vacuum silicone grease heavy</td>
			<td>Merck Millipore</td>
			<td>107921</td>
			<td></td>
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		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4/K2HPO4</td>
			<td>Sigma</td>
			<td>P5379/ P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td>version 8.1</td>
			<td>Data acquisition</td>
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		<tr>
			<td>Labview FPGA module</td>
			<td>National Instruments</td>
			<td>version 8.1</td>
			<td>Fast Force-Clamp</td>
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		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>2016</td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fluka</td>
			<td>63020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Nikon</td>
			<td>Plan-Apo 60X</td>
			<td>NA 1.2, WD 0.2 mm, water imm.</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Sigma</td>
			<td>N8267</td>
			<td>0.45 pore size</td>
		</tr>
		<tr>
			<td>Pentyl acetate solution</td>
			<td>Sigma</td>
			<td>46022</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure Ethanol</td>
			<td>&#160;Sigma</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>QPDs</td>
			<td>UDT</td>
			<td>DLS-20</td>
			<td>D Position Detecto</td>
		</tr>
		<tr>
			<td>Rhodamine BSA</td>
			<td>Molecular Probes</td>
			<td>A23016</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>&#160;Sigma</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica beads</td>
			<td>Bangslabs</td>
			<td>SS04N</td>
			<td>1.21 mm, 10% solids</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>&#160;Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin protein</td>
			<td>&#160;Sigma</td>
			<td>189730</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissecting mechanoenzymatic properties of processive myosins with ultrafast force-clamp spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Representative data consist in position records over time as shown in Figure 4. In the position record two kinds of displacement are visible. Firstly, when the myosin motor is not interacting with the actin filament the trapped beads are moving at constant velocity against the viscous drag force of the solution showing a linear displacement oscillating within the oscillation range set by the operator in a triangular wave3 (not visible in <strong class...

Watch this Scientific Journal Video about Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy at JoVE.com

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

dissecting-mechanoenzymatic-properties-processive-myosins-with

Research

JoVE Journal

Biology

1.3K Views.  National Institute of Optics, National Research Council. Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Video: Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

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.

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 ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...