Name: dissecting mechanoenzymatic properties of processive myosins with ultrafast force-clamp spectroscopy
Uploaded: 2021-07-01T13:30:00
Description: Watch this Scientific Journal Video about Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy at JoVE.com

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.
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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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Labview FPGA module</td>
			<td>National Instruments</td>
			<td>version 8.1</td>
			<td>Fast Force-Clamp</td>
		</tr>
		<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 ...

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