Protein expression and preparations

Rabbit skeletal muscle actin aceton powder was purchased from Pel-Freez Biological and actin was prepared as described previously [34]. The cDNA plasmid encoding human fascin was a gift of Dr. Fumio Matsumura (Rutgers University, NJ) and fascin protein was purified from E. coli with GST fusion tag [35]. The GST tag was cleaved by thrombin after purification and fascin protein without GST tag was used in this study. α-actinin purified from chicken gizzard was obtained from Sigma-Aldrich. Mouse myosin 5a HMM (m5-HMM) was prepared as described previously [36]. The cDNA encoding bovine myosin 10 HMM fragment (1-940 amino acids) was amplified by PCR followed by sub-cloning into pFastBac1 vector (Invitrogen). The GCN4 leucine-zipper sequence and the Flag epitope tag (DYKDDDDK) were engineered before the stop codon. m5-HMM or myosin 10 HMM with leucine-zipper (m10-HMM-Zip) was co-expressed with calmodulin using the baculovirus Sf9 expression system [33]. The expressed proteins were purified from Sf9 cell pellets by anti-FLAG-affinity chromatography [3]. Endogenous calmodulin of m5-HMM and m10-HMM-Zip were replaced by Alexa Fluor 488-labeled bovine calmodulin at a ratio of 5: 1 (Alexa488-CaM: myosin) as described previously [37]. In some experiments, a higher ratio of 50: 1 was used.

Single molecule motility assay

Single molecule motility assays were performed with TIRF microscopy on an IX81 microscope (Olympus America). Briefly, two diode pumped laser lines (473 nm and 556 nm, OEM Laser, UT) for TIRF illumination were introduced from a side port of a cube turret in the IX81. Both lasers were individually focused at a back-focal plane in the TIRF objective lens (Apo N, x60, N.A. 1.49 oil, Olympus America). Image magnifying lenses (2x and 4x) were used in front of an electron-multiplier CCD camera (EMCCD, Hamamatsu). Rhodamine phalloidin- and 10% biotin-labeled F-actin was bound onto the cover-glass via a biotin/avidin system. m5-HMM or m10-HMM-Zip with Alexa Fluor 488-CaM solution in the motility buffer containing 20 mM MOPS (pH 7.4), 5 mM MgCl₂, 0.1 mM EGTA, 40 mM KCl, 1 µM CaM, and 50 mM dithiothretol was then added with various concentrations of ATP as indicated. An oxygen scavenging system containing 25 µg/ml glucose oxidase, 45 µg/ml catalase, and 2.5 mg/ml glucose was also used as described previously (7). All experiments were performed at 23 °C controlled by a home-made enclosed box with a heat controller (Air-Therm, World Precision Instrument, Inc., FL). The motility of myosin molecules was captured and analyzed with Metamorph (Molecular Devices) and IDL softwares (ITT visual information).

Preparation of two dimensional (2-D) F-actin rafts

The preparation of 2-D F-actin rafts cross-linked by fascin or α-actinin was based on a previously described method [38-41]. Briefly, actin polymerization buffer (50 mM KCl, 20 mM PO₄, 1mM ATP, 2mM MgCl₂, 0.02% β-mercaptoethanol, 1 mM EGTA, 0.05% NaN₃) was first added to a Teflon well. A phospholipid solution (2 µl) composed of a 3: 7 w/w mixture of dilauryl phosphatidylcholine (DLPC) and didodecylmethyl ammonium (DDMA) in chloroform was pipetted into the well to form a lipid monolayer (Langmuir-Blodgett film) on the surface. This mixture was incubated for 60 minutes at 4 °C. Globular actin (30 µg/ml) mixed with the actin-bundling protein fascin (11 µg/ml) or α-actinin (40 µg/ml) was then added carefully underneath of the surface of the lipid monolayer and incubated for 6 hours at 4 °C or 4 hours at 23 °C. The 2-D F-actin paracrystalline bundle arrays that were formed underneath the lipid monolayer were transferred to a coverslip and labeled with rhodamine-phalloidin or phalloidin for 10 minutes at 4 °C. The coverslip was carefully attached to a slide-glass using double-sided tape, forming a flow-cell for single motility assay. To quantify the number of actin filament in the rafts, the intensity of the rhodamine-labeled F-actin rafts in the region of interest was normalized to the intensity of rhodamine-phalloidin-labeled single actin filament with the same size of the region of interest. The average number of actin filaments in fascin- and α-actinin-F-actin rafts were 3.98 ± 1.8 (n = 102, mean ± S.D.) and 4.88 ± 1.85 (n = 89, mean ± S.D.), respectively (Figure S1).

Electron Microscopy

For electron microscope imaging, 2-D F-actin paracrystalline arrays underneath Langmuir-Blodgett film were transferred to 400-mesh carbon-coated copper grids (Electron Microscopy Sciences), stained with 2% aqueous uranyl acetate for 30 sec, and the grid was washed twice with motility buffer as described above. Transmission electron microscopy (TEM) images were taken at 100 keV with 20,000 and 35,000 magnifications (TEM, JEOL-2010, JEOL). The center-to-center distance between actin filaments in the 2-D rafts was measured as described [38,41]. Briefly, images were processed using a fast Fourier transform (FFT) filter to detect spatial periodicity of actin filament (MetaMorph, Molecular Devices). Line scan intensity was measured across the filtered image of the rafts and then fit to a sum of Gaussian distributions using the maximal likelihood method [37]. The distance between the peaks of these distributions represents the distance between the centers of adjacent actin filaments.

Tracking and analysis of myosin movement

Particle localization was achieved at near-nanometer resolution using FIONA [26]. The tracking of fluorescence spots were performed using custom IDL routine works as described [42]. All tracks less than two seconds in duration were eliminated to remove non-processive as diffusive movements. A custom Origin C routine was used to calculate the total run length, which is the length between the starting point and the end point of the movement, and the total displacement, which is the sum of the individual displacements (frame by frame) during a track. The average velocity of the individual displacements was calculated from the data. Tracks with run lengths less than 250 nm were also filtered out. These criteria were strong indicators of a non-processive myosin movement. After software filtering, each track that met these criteria was manually analyzed to determine if it was in fact a processive myosin movement. To calculate the percent of attachment that were moving, a separate analysis using the IDL routine was carried out to calculate the number of processive runs and the total number of attaching/dissociating myosin molecule.

Myosin 10 exhibits less processive motility than myosin 5a along single actin filaments

A GCN4 leucine zipper, which is a dimeric coiled-coil [43] was added to the myosin 10 sequence following amino acid 940 to create a dimeric HMM-like fragment that was used in this study. To visualize the myosin molecule, we replaced endogenous calmodulin with Alexa Fluor 488-labeled calmodulin (Alexa488-CaM) [33]. Photo-bleaching experiments showed that the average numbers of Alexa488-dye was 1.9 per calmodulin and the average number of Alexa488-dye was 3.9 ± 2.4 per 10-HMM-zipper (m10-HMM-Zip), and 4.4 ± 2.9 per myosin 5-HMM (m5-HMM), meaning that about two Alexa488-CaM molecules were exchanged into one myosin molecule (Figure S2). In addition, the distribution of the number of fluorophore on myosins showed single Gaussian (Figure S2 D), and no extremely bright fluorescent spot was detected on m5-HMM and m10-HMM-Zip under TIRF microscopy, indicating that most of the myosin 5 and 10 molecules used in our study were dimers, but not oligomers or aggregated.

Both m10-HMM-Zip and m5-HMM moved processively along single actin filaments as observed by TIRF microscopy. Sequential images and kymographs of m10-HMM-Zip are shown in Figure 1A and Movie S1. The maximum velocity of m10-HMM-Zip was much less than that of m5-HMM (190 ± 40 nm/sec vs. 420 ± 38 nm/sec, mean ± S.D., Figure 1B). Moreover, the mean run-length of m10-HMM-Zip (755 ± 88 nm, n=264, mean ± S.D., Figure 1C, closed circles) was shorter than that of m5-HMM (1221 ± 255 nm, n=349, mean ± S.D., Figure 1C). In addition, at 1 mM ATP (saturating), 89% of m5-HMM and 35% of m10-HMM-Zip attachments to actin resulted in measurable processive movements, indicating that most of attached m5-HMM move processively along single actin filament while about 65% of m10-HMM-Zip bound and dissociated from single actin filament without significant movement (Table 1). Examples of such transient, nonproductive interactions are shown in Figure 1A yellow arrowheads.

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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.).

https://doi.org/10.1371/journal.pone.0074936.g001

We also examined the effects of ATP concentration (1 µM to 1mM) on the processive movement (Figure 1C inset). The mean run-lengths of both m10-HMM-zip and m5-HMM were not significantly changed with ATP concentrations, though the mean run-length of m10-HMM-zip at 10 µM ATP was slightly longer than at other ATP concentrations.

Myosin 10 walks hand-over-hand manner along actin filaments with a step size of 30.6 nm

The step size of myosin 5a and myosin 10 along single actin filament was analyzed by FIONA [26] at 0.3 µM ATP for m5-HMM and 1 µM ATP for m10-HMM-Zip. As the ATPase rate [17,44] and velocity of myosin 10 were slower than those of myosin 5a (Figure 1A, Figure S2A), a lower concentration of ATP (0.3 µM) resulted in sufficiently slow movement of myosin 5a so that sufficient number of photons could be collected for identification of individual steps, while a higher concentration of ATP (1 µM) allowed fluorescently-labeled myosins 10 to take sufficient steps before photobleaching for analysis of step-size and dwell time. Both myosins exhibited two classes of step sizes, long ones and ones where the stepping distance were approximately one half that of the long ones (Figure 2 and Figure S3). Consistent with our previous studies [45], we assigned the long stepping traces to be from molecules in which only one of the two heads of the HMM was labeled with Alexa Fluor 488. In these traces the distance that the labeled rear head traveled as it dissociated and moved to find a new forward binding site was being measured. This would be followed by a “silent” step where the unlabeled trail head detaches and reattaches in the forward position while the labeled (originally) forward head remains attached at a fixed position. The short stepping traces represent molecules where both heads were labeled with Alexa Fluor 488 and therefore the movement of the center-of-mass is measured each time either of the heads takes a step. The stepping traces of single- and double-head-labeled m10-HMM-Zip and m5-HMM are shown in Figure 2 A and B, and Figure S3 A and B. Consistent with previously published studies [26,37,45], our experiment also showed that myosin 5a moves in a hand-over-hand manner with a step size of 36 nm (Figure S3). However, myosin 10 took shorter steps at low ATP concentration (Figure 2). When the step size of single- and double-head-labeled m10-HMM-zip (Figure 2 C) were fit with a single Gaussian and the sum of two Gaussians, respectively, the double-head-labeled m10-HMM-Zip was shown to take a step size (the center of mass) of 30.6 ± 11.2 nm (n = 287, mean ± S.D.). In cases where only a single head of m10-HMM-Zip was labeled, the step size was 63.3 ± 10.7 nm (n = 210, mean ± S.D.), approximately twice as large for double head-labeled m10-HMM (Figure 2 C). Additionally, about 6% of the double head-labeled m10-HMM-zip took a step of 61 nm, which probably represents either the sum of two fast steps where an obvious intermediate dwell was not found or where the dye on one of the two heads photo-bleached at the end (Figure 2 C right-shaded-bars). When the exchange ratio was increased to 50: 1 of Alexa488-CaM to myosin, the step-size histograms of m5-HMM and m10-HMM-Zip showed predominantly short step-sizes (36-nm and 30-nm, respectively, Figure S4 A and B), indicating that most molecules were double-head-labeled.

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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).

https://doi.org/10.1371/journal.pone.0074936.g002

A plot of the dwell-time distributions supports this interpretation of the short and long steps. The distribution of dwell times for the long stepping events was best fit by the sum of two exponentials as expected for the case where every other step would be “silent” [26,37]. The detachment rate constant determined from the fit (0.26 s^-1, Figure 2 D open circles) is approximately half that of the double-head-labeled m10-HMM-Zip (0.43 s^-1, Figure 2 D close circles), confirming only half of the steps were observed with single-head labeled HMM since movements of the unlabeled head would be silent. The above results suggest that the two heads of myosin 10 move alternatively along single actin filaments, supporting the hand-over-hand model of myosin 10 movement [3]. Of note, the distribution of the step size of m10-HMM-Zip (S.D. = 16 nm) (Figure 2 C) was slightly wider than that of m5-HMM (S.D.: 9 nm, Figure S3 C), which may result from the flexibility of the SAH domain located between the third calmodulin-binding site and the GCN4 leucine zipper.

Myosin 10 moves more processively on fascin-F-actin rafts than on α-actinin-F-actin rafts

We next examined the processive motility of m10-HMM-zip and m5-HMM on 2-D F-actin rafts on a lipid monolayer that were cross-linked by either α-actinin or fascin [38,40,41], both of which were reported to form parallel F-actin bundles on the 2-D F-actin rafts [40,46]. The alignment of actin filaments in 2-D rafts were visualized by electron microscopy (Figure 3) and the center-to-center distance between actin filaments was analyzed using a fast Fourier transform (FFT) filter to detect spatial periodicity. In an α-actinin-F-actin raft, the inter-filament distance is 35 nm while this is 12 nm in a fascin-F-actin raft (Figure 3 D), which is consistent with previous studies [38,41,47,48].

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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.

https://doi.org/10.1371/journal.pone.0074936.g003

Single molecule motility assay showed different degrees of processivity of m10-HMM-Zip and m5-HMM moving on α-actinin- and fascin-F-actin rafts, demonstrating that the type of track could influence the movement (Figure 4, Figure S5, Movies S2, S3, S4, and S5). This could be manifest in both the tendency to move processively and in the run length. First, when the same concentration of m5-HMM and m10-HMM-Zip was used, only 23.5% of m10-HMM-Zip molecules that attached to the α-actinin-F-actin rafts moved processively while 58.8% of the attached molecules moved on fascin-F-actin rafts (Table 1). In contrast, m5-HMM showed no such selective movement on α-actinin-F-actin rafts and fascin-F-actin rafts (65.8% vs. 72.3%, Table 1). The average velocity of m10-HMM-Zip with 1 mM ATP on α-actinin-F-actin rafts (124 ± 36 nm/s, mean ± S.D, n = 157, Figure 4 B inset) and on fascin-F-actin rafts (165 ± 43 nm/s, mean ± S.D., n = 1633, Figure 4 B) were similar to that obtained using single actin filaments as the track. Under the same condition, the average velocities of m5-HMM with 1mM ATP on α-actinin-F-actin rafts and fascin-F-actin rafts were 398 ± 88 nm/s (mean ± S.D, n=2335, Figure 4 A open circles) and 430 ± 144 nm/s (mean ± S.D, n = 2200, Figure 4 A close circles). Both were similar to that the velocity on single actin filaments (420 ± 38 nm/s, Figure 1 B). Second, the run-length of m10-HMM-Zip on α-actinin-F-actin rafts was significantly shorter than on fascin-F-actin rafts (695 ± 89 nm vs. 1465 ± 125 nm, mean ± S.D.), whereas the run-lengths of m5-HMM on fascin- and α-actinin-F-actin rafts were similar, 1255 ± 135 nm and 998 ± 87 nm, respectively (Figure 4 C and D, Table 1). These results suggest that fascin-F-actin rafts favor the processivity of m10-HMM-Zip.

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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.

https://doi.org/10.1371/journal.pone.0074936.g004

Distinct stepping patterns of myosin 10 on fascin- and α-actinin-F-actin rafts

The stepping behavior and step size of m10-HHM-zip and m5-HMM on the above 2D-F-actin rafts were analyzed by FIONA. When a myosin 5 molecule moved along fascin-F-actin rafts, most of its steps were 36.2 nm, while 14% of its steps were shorter with a value of 21.6 nm (Figures 5 A and 6 A). The shorter step could be correlated in space to a side step to an adjacent parallel actin filament. Each side step should be marked by two back-to-back shorter steps since the center of mass of these double-head labeled molecules are being followed (Figure 7). This means that m5-HMM takes one side step out of every seven total steps. On α-actinin-F-actin rafts, the step size of myosin 5 exhibited a single peak at 35.1 nm, consistent with the wider spacings of the actin filaments (Figure 6 B). By contrast, the major step size of m10-HMM-Zip on fascin-F-actin rafts was 30.8 nm, while 28% of the step size was 21.3 nm (Figures 5 B and 6 C). Similar to myosin 5-HMM, m10-HMM-Zip showed a major step size of 31.5 nm without detectable side steps on α-actinin-F-actin rafts (Figure 6 D). These results suggest that m10-HMM-Zip took more frequent side-steps on fascin-F-actin rafts than did m5-HMM. We also determined the direction of the myosin step on fascin-F-actin rafts according to the fluorescence intensity in both vertical and lateral directions (XY coordinates, Figure 5 C and D), confirming that most m5-HMM and m10-HMM-Zip moved straight on actin filaments (red-straight line), but occasionally took shorter side steps with right-hand direction against the direction of the F-actin rafts.

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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.

https://doi.org/10.1371/journal.pone.0074936.g005

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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.).

https://doi.org/10.1371/journal.pone.0074936.g006

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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.

https://doi.org/10.1371/journal.pone.0074936.g007