A protocol for single molecule imaging and tracking of processive myosin motors

Graphical abstract


Specifications
Biochemistry, Genetics and Molecular Biology More specific subject area:

Biophysics
Protocol name: Single molecule motility assay Reagents/tools: All materials (reagents and equipment) are listed in the appropriate article section for clarity Experimental design: We describe the labelling of a recombinant myosin-5B with a quantum dot, the polymerization and labelling of actin filaments and the assembly of an in vitro single molecule motility assay to track myosin movements on actin under total internal fluorescence microscopy (TIRFM). We also describe an ensemble gliding assay in which multiple myosin-5B molecules translocate actin filaments and are visualized under fluorescence microscopy. Trial registration: NA Ethics: NA Value of the Protocol: Single molecule techniques allow tracking the movements of molecular motors with nanometer accuracy to investigate their mechanochemical and biophysical properties [1,2,3]. Here we share a protocol for QD-labelling purified processive myosin motors and observe them while they move on actin filaments immobilized on a coverslip surface. The protocol allows extracting motor trajectories using our freely available tracking software (PROOF, available as supplementary material in Gardini et al. [4]) or different tracking methods [5,6] with sufficient resolution to derive fundamental biophysical parameters such as the motor velocity, run length and step size and how they vary with nucleotides, ions, and buffer composition [7]. Such data is fundamental to dissect the molecular mechanisms at the base of motor protein function and test different chemo-mechanical models [8,9]. We also share a protocol for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties.

Description of protocol
Myosin is a family of actin-based motor proteins that hydrolyze ATP to generate force and movement and perform a broad range of functions, such as muscle contraction, hearing, vision, and cell motility [10]. Among these, several myosins transport intracellular cargoes for long distances inside living cells. For this reason, such motors are named processive, which means that they perform a large number of steps and ATP hydrolysis cycles before dissociating from actin [9]. Single molecule techniques allow direct observation of biophysical properties of motor proteins that would not be otherwise accessible. Here, we describe protocols for in vitro characterization of processive myosin motors at the single molecule level. We describe a single molecule motility assay in which single myosin molecules are labeled with single quantum dots and move along fluorescent actin filaments attached to the surface of a glass coverslip. In this configuration it is possible to measure fundamental biophysical properties of the motor such as the step size, run length, and velocity under varying nucleotides, Ca 2+ , and buffer composition. We also describe an in vitro gliding assay in which myosins attached onto a glass coverslip translocate fluorescent actin filaments in the presence of ATP in solution. By measuring the average gliding velocity of the filaments, it is possible to test protein viability and processivity of motor ensembles.
We describe protocols that were optimized to study the biophysical properties of myosin-5B [7], but can be easily generalized to the study of other myosin motors.

Myosin-5B expression and biotinylation
Recombinant myosin-5B heavy meromyosin (HMM) was expressed in Sf9 cells using the Baculovirus expression system. Description of protocols for the Baculovirus expression system is beyond the scope of the present article and protocols were performed as recommended by the manufacturer (Thermo Fisher Scientific). Protein purification was performed following standard techniques, whose description is also beyond the scope of the present article. In this section, we briefly describe the strategy that we adopted to engineer a recombinant myosin with a tag for the specific binding in the single molecule and gliding assays. The C-terminus of myosin-5B heavy chain was biotinylated using the Avi-tag-BirA system [11]. The biotin tag was used for the specific binding of myosin to either streptavidinated QDs in the single molecule motility assay (Section 5) or to a streptavidinated coverslip in the gliding assay (Section 4).To this end, a cDNA construct encoding for amino acids 1-1095 of murine myosin-5B heavy meromyosin (HMM) (Accession number NM_201600.2, MW 127 kDa) with a C-terminal Avi-tag was inserted in a modified pFastBac1 vector encoding a C-terminal Flag-tag with standard cloning techniques. After recombinant baculovirus generation, Sf9 insect cells from ThermoScientific (B82501) were infected with recombinant baculoviruses encoding Avi-tag myosin-5B HMM and calmodulin (CaM). Avi-tag myosin-5B HMM was purified via Flag capture [12] followed by ion exchange chromatography through Q-Sepharose Fast Flow resin (GE Healthcare Life Sciences).
Myosin-5B was biotinylated on the Flag-resin through incubation with BirA biotin ligase and d-biotin (Avidity). We loaded 1 ml of solution containing 20 mM ATP, 20 mM MgOAc, 100 mM d-Biotin and 150 mg BirA enzyme in a 2 ml Flag resin while the protein was trapped in the resin and incubated it for 90 min at 4 C. The protein, named herein bio-HMM-5B, was then eluted from the column following the standard Flag-resin elution protocol. Since two heavy chains dimerize to form the active myosin-5B motor, we end up with a molecule with two biotin tag at the distal end of the motor unit, as represented in Fig. 1.

F-actin polymerization and labelling
F-actin was polymerized from purified G-actin and labelled with rhodamine-phalloidin for visualization in both the single molecule motility and gliding assays.

Flow chamber preparation
In both the single molecule motility and gliding assays, we assembled the in vitro system inside a small-volume flow chamber in which the coverslip was covered with nitrocellulose for protein adhesion (Fig. 2). The glass coverslip in the chamber represented in Fig. 2 is placed on the top, as during sample preparation, when solutions are fluxed in the chamber as described in the next sections. Instead, imaging was performed on an inverted microscope (see section 6) in which the coverslip faced downwards. Materials: Pure Ethanol Methods: 1 Take a glass coverslip (24 x 24 mm) and cleanse it carefully with paper soaked with pure ethanol. Then rinse it directly with pure ethanol, by handling it carefully with clean tweezers. Dry it under a gentle flow of nitrogen. No visible residues must be left on the glass surface. If perfect cleaning is not reached after a first cleaning, repeat the cleaning procedure a second time 2 Smear 2 ml of nitrocellulose solution on one surface of the coverslip by means of a second clean coverslip (24 x 60 mm), and wait for it to be completely dry (see video article in ref [13]) 3 Take a microscope slide (26 x 76 mm) and clean it carefully with paper soaked with pure ethanol.
Dry it under nitrogen flow to get rid of coarse residues. 4 Cut two narrow stripes of double sticky tape (~ 3 mm large) and stick them on one side of the microscope slide in order to create a chamber of about 20 ml final volume, as shown in Fig. 2. Final volume of the chamber can be varied by either adjusting the distance between the tape stripes or choosing tape with different thickness. 5 By handling the coverslip (prepared at step 2) with clean tweezers, close the chamber with the nitrocellulose layer facing the inside of the chamber.

Gliding assay
In the gliding assay, bio-HMM-5B is attached to the coverslip surface in the flow chamber as shown in Fig. 3 and rhodamine-labeled F-actin is fluxed inside the chamber and allowed to bind to myosin in the absence of ATP. As we flux ATP into the chamber, we observe actin filaments smoothly translocating on the coverslip surface.

Single molecule motility assay
In the single molecule motility assay, bio-HMM-5B is first attached to streptavidinated QDs at single molecule concentration. Then, a flow chamber is built in which fluorescently-labelled actin filaments are attached on a bed of inactivated N-ethylmaleimide (NEM) myosin II on the coverslip surface [14]. QD-labelled HMM-5B (QD-HMM-5B) are then fluxed into the chamber together with ATP and the movements of single QDs translocating along actin are recorded. In our configuration, HMM-5B is bound to a single streptavidinated Quantum Dot through a biotinylation domain placed at the C-terminus, in substitution of the cargo-binding site (Section 1). Therefore, position of the fluorophore reflects movements of the center of mass of the protein, differently from other labelling geometries [15,16], and interactions of the Quantum Dot with the glass surface and with the N-terminus motor domain are minimized (Fig. 4).

Labeling of quantum dots with a single myosin motor
Materials: Bio

Image acquisition
Images are acquired on a modified Nikon ECLIPSE TE300 inverted fluorescence microscope [4] equipped with a 532 nm laser for rhodamine excitation and a 488 nm laser for QD excitation. Images were acquired in total internal reflection configuration, through a Nikon Plan Apo TIRF, 1.45 NA, 60X, oil immersion objective. 91 nm pixel size images were obtained by projecting the fluorescence image onto an iXon 3 EMCCD camera, after an additional 3X magnification through an achromatic doublet telescope.
For the gliding assay: F-actin Exc/Em. 532/580 nm. Laser power on the sample was about 3 mW, 200 ms exposure time and 200 EM gain are usually fine (Fig. 5).
For the single molecule motility assay: F-actin Exc/Em. 532/580 nm, laser power on the sample was about 3 mW; QD-HMM-5B Exc/Em. 488/655 nm, laser power on the sample was about 3 mW. Exposure time is set according to ATP concentration and consequent velocity between 50 and 100 ms. EM gain was 300 (Fig. 6).

Data analysis
Gliding assay: single filaments characterized by a travelled distance >1 mm were tracked over the frames with the ImageJ Plugin "Multitracker". The filament velocity was calculated frame by frame and the average velocity with its standard error was associated to each filament. The final average velocity was calculated by weighted average with associated standard error. By this analysis we obtained an average gliding velocity of 163 AE 3 nm/s at 1 mM ATP (N = 20 filaments).
Single molecule motility assay: single QDs were localized frame by frame with nanometer precision by a custom-made software named "PROOF" that is freely available and widely described elsewhere [4]. Localization accuracy in single molecule motility experiments was around 4 nm (100 ms integration time, 300 EM gain, 3 mW laser power on the sample), calculated as standard deviation of immobile QDs on actin filaments over 20 frames, possibly limited by the compliance of the NEM-myosin attachment to the coverslip. Myosin trajectories were derived from x,y coordinates of QDs moving along actin filaments for more than ten frames. Analysis of step size, run length, and velocity based on myosin-5B trajectories under variable ATP concentration can be found in Gardini et al. [7].