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 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.
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'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'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 "dumbbell"). 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.
1. Optical setup
NOTE: The experimental setup is composed of double optical tweezers with nanometer pointing stability and < 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.
2. Sample preparation
3. Measurement
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.
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 Figure 4 due to the long temporal scale). Second, once the myosin motor interacts with the filament the force carried by the moving filament is very rapidly transferred to the protein, the system velocity drops to zero (red lines in Figure 4) and stepping events occur under constant force till the end of the run. As shown in Figure 5 the force is switched from the positive to the negative direction (and vice versa) by the feedback system, which switches the force direction when the bead reaches the edge of the oscillation range set by the user. In some cases, it can happen that, when the myosin binds and displaces the filament towards the positive direction, it pushes the bead towards the (upper) edge of the oscillation range. If this happens under assistive force (i.e., directed towards positive displacement, push, in Figure 5), the run of the myosin will be interrupted by the force direction inversion at the oscillation edge (arrows in Figure 5), thus limiting the length of the run to the amplitude of the dumbbell oscillation D. This requires a correction the run length in case of assistive force (4.3.1).
Figure 1: Schematic of UFFCS applied to a processive myosin-5B motor. (a) A single myosin-5B molecule is attached to a glass bead pedestal through a streptavidin-biotin link. A single actin filament is trapped by suspending it between α-actinin coated beads (the so called "three-bead" geometry). Black arrows represent the force clamped on the right (F1) and left bead (F2), red arrow represents the net force (F) on the dumbbell. F is alternated back and forth to maintain the dumbbell within a limited oscillation range when myosin is not bound to actin. (b) Example trace showing displacement and force during the corresponding phases of dumbbell oscillation, myosin-5B attachment, and processive runs under assistive (push) and resistive (pull) loads. This figure has been modified from4. Raw data acquired at 200 kHz sample rate are plotted. Std. Dev. of force is about 0.27 pN. Please click here to view a larger version of this figure.
Figure 2: Optical scheme of the experimental setup. The optical microscope consists of: halogen lamp (H), condenser (C), sample (S), piezo translators (x-y and z), objective (O), a low-magnification camera (CCD 200X) and a high-magnification camera (CCD 2000X) used for the nm-stabilization feedback. Double optical tweezers are inserted and extracted from the optical axis of the microscope through dichroic mirrors (D2 and D3) and comprise: Nd:YAG laser (1064 nm), optical isolator (OI), λ/2 waveplates, polarizing beam splitter cubes (PBS), acousto-optic deflectors (AOD), 1064 nm interferential filters (F1 and F2), quadrant detector photodiodes (QDP). Signals from QDPs were elaborated with a FPGA, sent to two custom built direct digital synthesizers (DDS) driving the AODs (force feedback). Fluorescence excitation was provided by a duplicated Nd:YAG laser (532 nm) and the image projected on an electron multiplied camera (EMCCD). M is a movable mirror, F3 an emission filter. This figure has been modified from 3. Please click here to view a larger version of this figure.
Figure 3: Flow chamber assembly. (a) Chamber preparation. A glass coverslip, smeared with silica beads, is attached onto a microscope slide through double sticky tape stripes to form a flow-cell about 20 µL volume. b) Top view of the flow-cell. Solutions are flown from one side of the chamber with a pipette and sucked from the other side through a filter paper to create a flow along the arrow direction. Please click here to view a larger version of this figure.
Figure 4: Representative position recording. Position recording showing myosin-5B processive runs and the step and run detection algorithm. Detected beginning and end of each run are indicated by green and cyan vertical lines, respectively. Red horizontal lines indicate the detected steps. This figure has been modified from4. Please click here to view a larger version of this figure.
Figure 5: Force inversion during myosin runs. When myosin binds and moves the filament in the positive direction under assistive force (push), it can happen that it reaches the edge of the oscillation range where the force is reversed (indicated by the arrows), so that the myosin run under assistive force is interrupted. Contrary, under resistive force (pull), myosin processive stepping prevents the dumbbell from reaching the force inversion point. Therefore, in the latter case, run lengths are not limited by the oscillation range for resistive forces. This figure has been modified from4. Raw data acquired at 200 kHz sample rate are plotted. Std. Dev. of force is about 0.27 pN. Please click here to view a larger version of this figure.
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 all through the run and probing the motor dependence with full control on force directionality. Regarding the last point, the three-bead geometry as we use here is very efficient in applying and measuring forces along the filament direction, minimizing contributions from transverse or vertical components. However, when the motor protein is expected to actively produce transverse or vertical forces, or even torques, other configurations such as the single bead geometry are more appropriate2,7,18. Moreover, thanks to its spatial and temporal resolution, UFFCS represents a unique tool for the understanding of basics of molecular interactions that would otherwise be hindered with conventional single-molecule techniques. In fact, UFFCS made it possible to investigate how assistive and resistive forces regulates the mechanical response of myosin-5B, thus giving new insight into its collective behavior within the actin mesh in the cell4.
However, the success of these experiments relies on the fulfillment of some important requirements that must be addressed very carefully by following all the instructions found in this protocol: the precise alignment and isolation of the optical setup is fundamental to reach an optimal spatial resolution; careful calibration of the optical system is necessary to determine the values of the applied forces with high precision; the setting of a fast feedback system is necessary to reach the high temporal resolution; finally all the components that are assembled in the sample chamber must be prepared in a controlled environment, keeping them as sterile as possible, since any impurity in the sample chamber could compromise the experiment, and all indications about their optimal storage and handling should be strictly respected for the success of the experimental protocol. Importantly, data analysis should be carefully adapted to the different kinds of motor-filament interactions to properly interpret results and avoid artifacts.
In this protocol are included all the steps to perform ultrafast force-clamp experiments on processive myosin-5 motors, from the setup of experimental apparatus to sample preparation, measurement and data analysis, that could be conveniently adapted to study a variety of unconventional myosins and other classes of processive motors such as kinesins and dyneins.
The authors have nothing to disclose.
This work was supported by the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 871124 Laserlab-Europe, by the Italian Ministry of University and Research (FIRB “Futuro in Ricerca” 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.
Aliphatic Amine Latex Beads | ThermoFisher | A37362 | 1.0-μm diameter, 2% (w/v) |
Acetone | Sigma | 32201 | |
Actin polymerization buffer | Cytoskeleton | BSA02 | 10X |
AODs (acousto-optic deflectors) | AA Opto Electronic | DTS-XY 250 | Laser beam deflectors |
ATP | Sigma | A7699 | |
Biotinylated-BSA | ThermoFisher | 29130 | |
BSA | Sigma | B4287 | |
Calmodulin from porcine brain (CaM) | Merck Millipore | 208783 | |
Catalase from bovine liver | Sigma | C40 | |
Condenser | Olympus | OlympusU-AAC, Aplanat, Achromat | NA 1.4, oil immersion |
Creatine phosphate disodium salt tetrahydrate | Sigma | 27920 | |
Creatine Phosphokinase from rabbit muscle | Sigma | C3755 | |
DDs | AA Opto Electronic | AA.DDS.XX | Two-channel digital synthesizer |
DL-Dithiothreitol (DTT)/td> | Sigma | 43819 | |
EGTA | Sigma | E4378 | |
G-actin protein | Cytoskeleton | AKL99 | |
Glucose | Sigma | G7528 | |
Glucose Oxidase from Aspergillus niger | Sigma | G7141 | |
HaloTag succinimidyl ester O2 ligand | Promega | P1691 | |
High vacuum silicone grease heavy | Merck Millipore | 107921 | |
KCl | Sigma | P9541 | |
KH2PO4/K2HPO4 | Sigma | P5379/ P8281 | |
Labview | National Instruments | version 8.1 | Data acquisition |
Labview FPGA module | National Instruments | version 8.1 | Fast Force-Clamp |
Matlab | MathWorks | 2016 | Data analysis |
MgCl2 | Fluka | 63020 | |
Microscope Objective | Nikon | Plan-Apo 60X | NA 1.2, WD 0.2 mm, water imm. |
MOPS | Sigma | M1254 | |
Nitrocellulose | Sigma | N8267 | 0.45 pore size |
Pentyl acetate solution | Sigma | 46022 | |
Pure Ethanol | Sigma | 2860 | |
QPDs | UDT | DLS-20 | D Position Detecto |
Rhodamine BSA | Molecular Probes | A23016 | |
Rhodamine Phalloidin | Sigma | P1951 | |
Silica beads | Bangslabs | SS04N | 1.21 mm, 10% solids |
Sodium azide | Sigma | S2002 | |
Streptavidin protein | Sigma | 189730 |