Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy
- University of Oxford, United Kingdom
- National Heart, Lung and Blood Institute, National Institutes of Health, United States
Figures
Figure 1 with 3 supplements
High-speed nanometric tracking of myosin 5 with interferometric scattering (iSCAT) microscopy.
(A) Distance traveled as a function of time for a single myosin 5 molecule biotinylated at the N-terminus and labeled with a 20 nm streptavidin-functionalized gold particle. The lateral localisation …
Figure 1—figure supplement 1
Activity of myosin 5 labeled with 20 nm gold nanoparticles at the N-terminus.
(A) Sample trajectories at [ATP] = 10 µM and at 1000 frames/s. Arrow indicates direction of motion. Scale bar: 50 nm. (B) Comparison of myosin 5 activity recorded in this work with previous single …
Figure 1—figure supplement 2
Detection of the transient state with a molecular sized fluorescent label.
(A) Representative distance time series of a myosin 5 molecule labelled with an atto-647N/streptavidin conjugate at the N-terminus and tracked using single molecule total internal fluorescence …
Figure 1—figure supplement 3
Rare transient unbinding events for the leading head of myosin 5.
(A) Representative time trace segment of an event in which the leading head detaches from the actin filament and exhibits the intermediate state behaviour reported in the main text. (B) …
Figure 2 with 1 supplement
Three-dimensional interferometric tracking of the myosin head.
(A) Distance trace (upper panel) with the simultaneously recorded iSCAT contrast (lower panel). The red subset of the traces corresponds to the unbound head state. Inset: schematic illustrating how …
Figure 2—figure supplement 1
Dual colour iSCAT imaging of myosin 5.
(A) 2D-trajectory of myosin 5 stepping along actin recorded simultaneously with 445 nm (blue trace) and 635 nm (red trace) illumination. Although the latter channel exhibits higher noise due to a …
Figure 3 with 1 supplement
Simultaneous scattering and fluorescence tracking of a single myosin 5a.
One head was labeled with a 20 nm streptavidin-functionalized gold particle (red) and the other with a fluorescent quantum dot (blue). Reported values correspond to the standard deviation σ in the …
Figure 3—figure supplement 1
Additional traces of simultaneous scattering and fluorescence tracking of a single myosin 5a.
One head was labelled with a 20 nm streptavidin-functionalized gold particle (red) and the other with a fluorescent quantum dot (blue). Reported values correspond to the standard deviation σ in the …
Figure 4 with 1 supplement
Two states of the motor domain during myosin movement.
(A) Sample 2D trajectories for a 20 nm gold particle immobilised on the surface and attached to the N-terminus of myosin 5a. The arrow indicates the direction of myosin movement. The trace shown is …
Figure 4—figure supplement 1
(A) Step size histograms for post to pre-power stroke and post to post power stroke states.
Data was collected at 100 Hz and 10 μM ATP, using 20 nm diameter gold nanoparticles. Number of steps analysed: 554. (B) The positional fluctuations of the A and B states presented as histograms of …
Figure 5
Simultaneous iSCAT and fluorescence tracking of myosin 5a.
(A) Tracking of the scattering signal from the gold nanoparticle attached to the N-terminus (black) and fluorescence signal from the GFP moiety located at the C-terminus of the same myosin 5a …
Figure 6
Conformational change within the N-terminal domain during the power stroke.
(A) Histograms for the distances between A and B states for 20, 30 and 40 nm gold nanoparticle labels located at the N-terminus. Total number of steps recorded: 124, 103 and 116, respectively. (B) …
Figure 7
Directionality and kinetics of the AB transition and the transient state.
(A and B) Simultaneous observation of the AB transition and the transient state for right and left handed walking molecules. (C and D) Position of the transient state for the same molecule before …
Figure 8 with 4 supplements
Probability density contour maps of the myosin step.
Upper panel represents the transient state of the unbound head. Contour map of a two-dimensional histogram with a 10 × 10 nm2 bin width obtained from the 1000 frames/s data (N = 486). Lower panel …
Figure 8—figure supplement 1
Spatiotemporal dynamics of the transient state as a function of ATP concentration.
(A) Contour maps of the transient state at three different ATP concentrations. Obtaining traces at high ATP concentration was challenging due to the small available field of view and rapid …
Figure 8—figure supplement 2
Dwell time distributions for pre and post-power stroke states at different ATP concentrations.
In all cases, data and fits for the pre-power stroke state are shown in red; data and fits for the post-power stroke state are shown in blue. At saturating (1 mM) ATP concentration (A) both dwell …
Figure 8—figure supplement 3
Geometrical considerations assuming myosin molecules moving parallel to the glass surface.
If the head searched the full spherical space, the average position would be simply about half way between the leading head and the next binding site. For a parallel orientation this would be 56 nm …
Figure 8—figure supplement 4
Probability density contour map of the transient state.
Contour map of a two-dimensional histogram with a 18 × 18 nm2 bin width was obtained using all, left- and right- handed walking traces, without flipping (left panel). The resulting contour plot …
Figure 9
Mechanism of the myosin step.
(A) ATP binding to the trailing head (yellow) and its detachment releases strain stored in the molecule. (B) The bound head (black with a gold nanoparticle attached) performs its power stroke which …
Author response image 1
Author response image 2
Author response image 3
Videos
Video 1
This file contains the iSCAT video from which the trajectory shown in Figure 1 of the main manuscript was obtained.
The left panel presents the raw video format after referencing with the flat field image. The flat field accounts for illumination inhomogeneities and was generated as described in the Materials and …
Video 2
This file contains a full video from which the trajectory shown in Figure 4 of the main manuscript was obtained.
The video was taken at 100 frames/s and at a magnification of 31.8 nm/pixel. The corresponding field of view shown is 2 × 2 μm2 and corresponds to a total recording time of 5 s. The video playback …
