Figure 1.
Single molecule motility assays of myosins 10 and 5a along single actin filaments.
(A) Still images from a movie showing movement of single molecules of m10-HMM-zip along a single actin filament. A series of images on the left panel shows the actin filament (red) and the fluorescently-labeled m10-HMM-zip in green at the indicated times. The images on the right are kymographs of the data depicted on the left. Red arrow heads show the moving m10-HMM-zip molecules and the yellow arrow heads show the molecules which attached and detached from actin without significant movement. (B) Velocity of single m5-HMM (open circle) and m10-HMM-Zip (closed circle) molecules at various ATP concentrations. Maximum velocities of m5-HMM and m10-HMM-Zip are 421 ± 38 nm/sec and 218 ± 29 nm/sec, respectively (mean ± S.D.). The data were fit to the Michael-Menten equation. (C) The run length distribution of m5-HMM (open circle) and m10-HMM-Zip (closed circle) at 1 mM ATP. Run length data were fit with single exponential equation (solid line). The mean run lengths of m5-HMM and m10-HMM-Zip were 1221 ± 255 nm and 755 ± 85 nm, respectively (mean ± S.D.). Data points from runs < 250 nm were omitted from the fits. The inset is the run-length of m5-HMM (closed bar) and m10-HMM-Zip (open bar) against ATP concentrations. Error bar is standard deviation (S.D.).
Figure 2.
Analysis of processive movement of m10-HMM-Zip on single actin filament.
(A and B) Representative stepping traces of single head-labeled (A) and double-head-labeled (B) m10-HMM-Zip. Each data point represents a 0.5 s acquisition time window. (C) Histogram of the step sizes of m10-HMM-Zip. Bars show distribution of the step size for single head-labeled m10-HMM-Zip (open bar) and double-head labeled m10-HMM-Zip (shaded bar). The solid line was fit with the double-head labeled m10-HMM-Zip to a sum of two Gaussian distribution using maximal likelihood [37]. The dashed line shows the fit to the data with a single Gaussian. The multi-modal fit was justified as described previously [37]. The value for this fit between two peaks was p < 0.03. The peaks of the step size distribution for double-head labeled m10-HMM-Zip (mean ± SD) are 31.4 ± 10.2 nm and 61.5 ± 6.5 nm (n = 287). The peak of the step size distribution for single head-labeled m10-HMM-Zip (mean ± SD) is 60.8 ± 8.8 nm (n = 210). (D) Dwell time distribution of m10-HMM-Zip. The dwell time of double-head labeled m10-HMM-Zip (closed circle) was fit to a single-exponential equation, P(t) = k exp(-kt). Dwell time of single head-labeled m10-HMM-Zip (open circle) was fit to an equation on a hand-over-hand model equation; P(t) = tk2exp(-kt) [26]. The rate constants of the fits are 0.43 s-1 (single head-labeled m10-HMM-Zip) and 0.26 s-1 (double-head labeled m10-HMM-Zip).
Figure 3.
Analysis of the electron micrograph of fascin- and α-actinin-F-actin rafts.
(A) Unprocessed electron microscope images of actin rafts created with α-actinin (top) or fascin (bottom). (B) Same images transformed with a fast Fourier transform (FFT) filter, a line scan (red line) analysis of the intensities. (C) Intensity profile from the line scan in (B). The data were fit with sum of Gaussians (red lines). The peaks represent the position of actin filaments in the rafts. (D) Histogram of the center-to-center distance between actin filaments with fascin (closed circle) and α-actinin (open circle) analyzed with single Gaussian distribution (solid line). The average distances of fascin- and α-actinin-F-actin rafts were 12.5 ± 2.3 nm (n = 78, mean ± S.D.) and 39.7 ± 16.5 (n = 97, mean ± S.D.), respectively.
Figure 4.
Velocity of m5-HMM and m10-HMM-Zip on 2-D actin rafts with fascin and α-actinin.
(A and B) The velocity distributions of m5-HMM (A) and m10-HMM-Zip (B) on 2D F-actin (FA) rafts with α-actinin (open circle) and fascin (closed circle). Solid lines represent a single Gaussian fit to the data. The average velocities of m5-HMM on α-actinin- and fascin-F-actin rafts were 398 ± 88 nm/sec (n = 2355) and 430 ± 144 nm/sec (n = 2200), respectively. The average velocities of m10-HMM-Zip on α-actinin- and fascin-F-actin rafts were 124 ± 36 nm/sec (n = 157) and 165 ± 43 nm/sec (n = 1633), respectively. (C and D) The run-length of m5-HMM (C) and m10-HMM-Zip (D) on 2D-actin rafts with α-actinin (open circle) and fascin (closed circle). The run-lengths of m5-HMM on F-actin rafts with α-actinin (open circle) and fascin (closed circle) were 998 ± 87 nm (n = 2335, means ± S.D.) and 1255 ± 135 nm (n = 2200, means ± S.D.), respectively. The run-lengths of m10-HMM-Zip on F-actin rafts with α-actinin and fascin were 564 ± 23 nm (inset: open circle, n = 157, means ± S.D.) and 1465 ± 125 nm (closed circle, n = 1633, means ± S.D.), respectively.
Figure 5.
Stepping traces of double-head labeled m5-HMM and double-head labeled m10-HMM-Zip on F-actin rafts with fascin.
Stepping traces of m5-HMM (A) and m10-HMM-Zip (B) over time. (C) XY-plots of the stepping positions of m5-HMM data shown above in panel (A). (D) XY plots of the stepping positions of m10-HMM-Zip data shown above in panel (B). Small step sizes (red numbers in A and B) represent the side-steps from one actin filament to the other on the F-actin rafts. Red solid lines in C and D indicate 45 degree angles with respect to the X-axis along each actin filament in the rafts.
Figure 6.
Distribution of step sizes for double-head labeled m5-HMM and double-head labeled m10-HMM-Zip on 2D-F-actin rafts bundled with fascin and α-actinin.
Distribution of step sizes for the movement of m5-HMM on fascin-F-actin rafts (A) and on α-actinin rafts (B). Distribution of step sizes for the movement of m10-HMM-Zip on fascin-F-actin rafts (C) and on α-actinin rafts (D). The solid lines in panels A and C represent the fit of the data with a sum of two Gaussian distribution using maximal likelihood as described above. Red line in (C) represents the fit to the shorter step size. The solid lines in panels B and D represent the fit with single Gaussian curve. The peaks of step size of m5-HMM were (A) 36.2 ± 6.3 nm and 21.6 ± 3.5 nm (n = 158, means ± S.D.) on fascin-F-actin rafts and (B) 35.1 ± 8.3 nm (n = 154, means ± S.D.) on α-actinin-F-actin rafts. The peaks of step size of m10-HMM-Zip were (C) 30.8 ± 6.8 nm and 21.3 ± 3.8 nm (n = 209, means ± S.D.) and (D) 31.5 ± 4.8 nm (n = 153, means ± S.D.).
Figure 7.
Stepping model of myosin on actin rafts.
Myosin stepping model on F-actin rafts. Two heads of myosin (light brown color) bind on an actin monomer (green) in actin filaments. The center of the mass of the myosin is depicted by a red circle. When myosin steps forward (black arrow), the detached head binds a new actin monomer (green). (A) The model for stepping on fascin-F-actin rafts. Left-hand side shows the myosin molecule stepping forward along an actin filament in the rafts. The length of the red arrow represents the magnitude of the step-size. Black arrows represent the direction of the stepping. Right-hand side model shows a myosin molecule taking a side step to an adjacent actin filament in the rafts. Note that when a myosin takes a side step onto an adjacent actin filament the center of mass movement takes two short steps. Yellow arrow represents the direction of actin filament in the rafts. (B) The model of stepping on α-actinin-F-actin rafts. A myosin molecule cannot reach an adjacent actin filament and thus cannot have a side step.