Evidence for S2 flexibility by direct visualization of quantum dot–labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro

Figures

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  Figure 1.. Summary of actomyosin kinetics and experimental system. (A) Kinetic scheme for myosin (M) attachment to actin (A). D, ADP; T, ATP; Pi, phosphate. Kw, equilibrium constant for weak binding of myosin to actin; kws, forward rate constant for the weak to strong transition. We assume that k-ws is insignificant. See text for other rate constants. (B) Top: Mf/A assay showing SMM-Spy Snoop-LMM (RLC-LMM) cofilament (orange) labeled with QD-SpyC (blue dot) attached to the RLC in the lever arm domain (pink) and QD-SnoopC (red dot) moving over biotinylated actin (green) attached to PEG brush-coated coverslip (not shown). Filaments are shown widely separated for clarity with S2 (black) detached completely from the filament backbone, which may not reflect the actual process (see text). Pre-working step, green box 1; post-working step, gold box 2; and after full extension of S2, gray box 3, by other working heads (not shown) giving L = 40–50 nm. The myosin head then must return to its starting position relative to the filament backbone, purple box 4. Bottom: Graph of mixed-kinetic model predictions of single head behavior in a filament. Black arrows indicate approximate predicted dwell time and…

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  Figure S1.. Snoop-LMM reacts spontaneously with SnoopC. Reaction time course of 0.5 µM Snoop-LMM mixed with 1 µM SnoopC in filament buffer. Percent reactants covalently linked was determined by SDS-PAGE and gel densitometry. Error bars are SD; n = 3.

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  Figure S2.. Snoop-LMM SMM cofilaments are stable and can be labeled with SnoopC-QDs. TIRF microscopy images of various rhodamine labeled SMM filament preparations in filament buffer. Scale bar, 5 µm. (A) Control 20 µg ml−1 SMM filaments on a glass coverslip. Arrows indicate aggregates of filaments; carrots indicate single filaments. (B) Overlaid image of 20 µg ml−1 95% SMM 5% Snoop-LMM cofilaments (red) reacted at 1 µM with 0.12 µM 585 nm QD-SnoopC overnight at 4°C. (C) Same as B, except with 0.12 µM 655 nm QD-SnoopC. For B and C, arrows point to colocalized filaments and QDs.

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  Figure 2.. Representative displacement trajectory of SMM filament moving on stationary actin filament. Fluorescence of 605 nm QD on RLC (black trace) and 705 nm QD on LMM (red trace) incorporated into RLC-LMM cofilament was simultaneously acquired, tracked, and reoriented along the axis of motion. [ATP] = 10 µM. (A and B) Insets reveal dwells (horizontal lines) followed by jumps (vertical lines) in the RLC trace. The LMM traces were translated from the RLC traces by −80 nm in the insets for better comparison.

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  Figure 3.. Representative events showing dwell times followed by jumps at varying [ATP]. For all panels, the black squares and line are the displacement of the SpyC-605QD (RLC), and the red circles and line are the displacement of the SnoopC-705QD (LMM). The horizontal black line is the estimated time that the RLC trace pauses (dwell time) while the LMM traces rises, and the vertical black line is the jump distance (J). (A) 5 µM ATP. (B) 7.5 µM ATP. (C) 10 µM ATP. (D) 12.5 µM ATP. (E) 15 µM ATP. (F) 20 µM ATP.

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  Figure 4.. Dwell times (ton) and jump lengths (J) at varying [ATP]. (A) Dwell times identified before the jump at varying [ATP]. Green diamonds, RLC-LMM cofilaments, 5 µM ATP, n = 9; 7.5 µM ATP, n = 50; 10 µM ATP, n = 45; 12.5 µM ATP, n = 46; 15 µM ATP, n = 12; 20 µM ATP, n = 11. Red diamonds, LMM-LMM cofilaments, 10 µM ATP, n = 16. The asterisks for the 10 µM ATP condition indicate the two means are statistically significantly different using a two-sample t test with P = 0.000645. The black line is a fit of Eq. 1 to the means. (B) Jump lengths (J) following the dwell times identified in A. The asterisks for the 10 µM ATP data indicate the means are statistically significantly different with P = 0.01. For A and B, the black vertical whiskers indicate SEM; the longer black horizontal line is the mean.

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  Figure S3.. Representative false-positive events from LMM-LMM trajectories. The horizontal black line is the estimated time the black trace pauses (dwell time) while the reference trace rises, and the vertical black line is the estimated jump distance (J). All data were collected at 10 µM [ATP] (see Fig. 4, A and B).

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  Figure 5.. Distribution of jump durations. Histogram showing the distribution of the time each jump took from the end of the dwell to where it returned to the LMM trace. The average = 52.9 ± 2.3 ms (SE), n = 171.

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  Figure 6.. Frequency of events per track and distribution of time between events. (A) Histogram of the frequency of detected events as a probability density given the total number of trajectories. All data refer to RLC-LMM except that indicated as LMM (orange) for the LMM-LMM data. Half of the tracks that contained zero events were removed from the probability calculation (see text). (B) Plot of the mean probability density (black squares) for each [ATP] from data shown in A, excluding the LMM-LMM data. The LMM-LMM data are shown in orange circles. The color-coded lines show a fit to Eq. 2. (C) Histogram showing the distribution of the time between events for all tracks that contained multiple events. The black line is an exponential distribution with the mean time between events equal to that of the data and the amplitude equal to the maximum of the data. The red line is a fit to Eq. 3.