Actin Structure-Dependent Stepping of Myosin 5a and 10 during Processive Movement

How myosin 10, an unconventional myosin, walks processively along actin is still controversial. Here, we used single molecule fluorescence techniques, TIRF and FIONA, to study the motility and the stepping mechanism of dimerized myosin 10 heavy-meromyosin-like fragment on both single actin filaments and two-dimensional F-actin rafts cross-linked by fascin or α-actinin. As a control, we also tracked and analyzed the stepping behavior of the well characterized processive motor myosin 5a. We have shown that myosin 10 moves processively along both single actin filaments and F-actin rafts with a step size of 31 nm. Moreover, myosin 10 moves more processively on fascin-F-actin rafts than on α-actinin-F-actin rafts, whereas myosin 5a shows no such selectivity. Finally, on fascin-F-actin rafts, myosin 10 has more frequent side steps to adjacent actin filaments than myosin 5a in the F-actin rafts. Together, these results reveal further single molecule features of myosin 10 on various actin structures, which may help to understand its cellular functions.

Like other members of the myosin superfamily, myosin 10 is composed of three domains: a conserved N-terminal motor domain, which binds actin filament and ATP, a neck domain which serves as a rigid lever arm to facilitate the power stroke and a C-terminal tail containing three PH domains, one MyTH4 domain and one FERM domain which serves to target the protein inside cells [5,7]. The neck of myosin 10 contains three IQ motifs each of which can bind one calmodulin (CaM) or CaM-like proteins, followed by a single α-helical (SAH) domain [15]. The SAH domain effectively extends the length of the neck to be about 27 nm which is similar to the length of the neck of the highly processive motor myosin 5a, which contains six IQ motifs [15]. The sequence comprising this SAH domain was initially thought to form a coiled-coil [5,16], but subsequent studies showed that neither truncated fragments of myosin 10 containing this sequence or full length myosin 10 in cells readily formed dimers [8,17]. Moreover subsequent studies showed that full length myosin 10 is a single-headed motor but can be independently converted into active dimer when the monomers are brought into close proximity by clustering on the surface of intracellular cargoes or actin filaments [3,9].
Processive movement is an important feature for motor proteins to transport cargo in cells. Among the 35 classes of myosins identified thus far [18], vertebrate myosin 5a was the first myosin shown to move continuously along actin filaments as a single molecule without dissociation (i.e processive movement) [19,20]. The processivity of this double-headed molecular motor has been intensively studied in vitro. Single molecule experiments using electron microscopy [21], optical trapping [19,[22][23][24][25], total internal reflection fluorescence (TIRF) microscopy [20,[26][27][28][29][30], and atomic force microscopy (AFM) [31] demonstrated that myosin 5a takes 36 nm steps along actin filaments in a hand-over-hand fashion and solution transient kinetics studies confirm that the kinetic mechanism of myosin-5 is appropriate for a processive motor [32]. Fewer or more than six IQ motifs in the neck lead to step size shorter or longer than 36 nm, respectively [25,33].
Unlike the well-characterized myosin 5a, the single molecule studies on the processivity of myosin 10 remain controversial. One study showed that myosin 10 preferred to move processively along F-actin bundled with fascin rather than on single actin filaments, and took a much shorter step size of 18 nm on the bundles [1,2]. By contrast, the work from another group showed little or no selectivity of myosin 10 for F-actin bundles [3]. Moreover, the step sizes of myosin 10 on the bundles include both 34 nm and 20 nm steps, with the shorter step size being attributed to a side step onto an adjacent actin filament within a bundle [3]. These studies used myosin 10 molecules that differed in the structure of the tail region. The former group used GCN4 leucine after the SAH domain [1,2], while the latter group used the coiled-coil of myosin 5a [3]. To clarify the processive movement of myosin 10 on different actin structures, we used a fluorescently-labeled single myosin 10 heavy meromyosin (HMM) construct that was forced into dimerization by a leucine zipper as well as a myosin 5a HMM and studied their detailed stepping mechanisms on a single actin filament and two-dimensional (2-D) F-actin rafts with different F-actin bundling proteins.
We show that myosin 10 moves processively on both single actin filaments and F-actin rafts with a step size of 31 nm. On 2-D F-actin rafts, myosin 10 prefers to move more processively on fascin-bundled F-actin rafts. Moreover, both myosins exhibit side stepping to adjacent actin filament on fascin-F-actin rafts, but not on α-actinin-F-actin rafts.

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 phalloidinand 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 MgCl 2 , 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][39][40][41]. Briefly, actin polymerization buffer (50 mM KCl, 20 mM PO 4 , 1mM ATP, 2mM MgCl 2 , 0.02% β-mercaptoethanol, 1 mM EGTA, 0.05% NaN 3 ) 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 doublesided 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.

Results
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.
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 singleand 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-headlabeled 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 headlabeled 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 rightshaded-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 stepsizes (36-nm and 30-nm, respectively, Figure S4 A and B), indicating that most molecules were double-head-labeled. 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   [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. 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 handover-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].

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 ( 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.

Discussion
In cells actin is present in a pleomorphic variety of structures including from single actin filaments, branched filament networks, loose meshworks, and bundles. There are many Factin bundling proteins that create bundles with different interfilament spacing and the actin filaments might be either parallel or antiparallel when formed in vitro depending on the bundling protein and the conditions. Our goal in this study was to determine how myosin 5a and 10 move on different actin structures. We showed that myosin 10 moved processively along both single actin filaments and F-actin rafts with 31 nm step sizes. We further demonstrated that myosin 10 moved more processively on fascin-F-actin rafts than on single filaments or on α-actinin-F-actin rafts, whereas myosin 5a showed comparable processive movement along both single filaments and F-actin rafts. Finally, myosin 5a and 10 were found to take side steps occasionally while walking along the actin filament in fascin-F-actin rafts.
The proteins used in this study were across many species. The amino acid sequences of myosin 5a and 10 are well conserved among the mammalian species including the ones used in our studies, thus we believe that our results still reflect the common properties of myosin 5a and 10 beyond the specific species. Other proteins including actin, fascin, α-actinin, and calmodulin are also well conserved among the species used in this study. Regardless of their potential specific functions and regulations in specific species, they share the general functions in building the common actin structures that  utilized in this study. In addition, the species-specific proteins that we have used allow us to directly compare our results (run lengths, velocity, etc) to the large volume of results performed in our lab and others. Similarly, due to the availability of abundant protein resources, skeletal muscle alpha-actin instead of beta-or gamma-actin was used in this study as well as most of the studies in the same field. Actins are highly conserved among the isoforms both in their amino acid sequences as well as their biological functions with respect to their polymerization to filaments. Note that De La Cruz et al [49] examined the actin isoform dependence of myosin 5a kinetics and found little difference. This study is, in general, looking at the broad question of how the myosins move on actin bundles and is less concerned with small differences in rates that might come about from the use of different actin isoforms.
Single molecule motility assays were performed to assess the processivity of the two-headed m10-HMM-Zip along F-actin filaments. We did not test the processivity of myosin 10 with single head on actin filaments, as there was no evidence that a single-headed myosin 10, which did not possess a secondary actin binding site, would be able to move processively. Compared to the highly processive motor myosin 5a [23,24,26,28,33], our results showed that myosin 10 was less processive. Only 35% of m10-HMM-Zip that initially attached to actin in the current study was shown to move processively along single actin filament for a measurable distance, while nearly 90% of attached myosin 5 molecules exhibited processive motility. Moreover, the run length of myosin 10 molecules was shorter than myosin 5, and the maximum velocity was slower than myosin 5a. The velocity of m10-HMM-Zip obtained in this study is in agreement with the data from other laboratories [1][2][3]. However, the run length of myosin 10 in our study was about 750 nm [1,3]. Nagy et al reported a much shorter run length of 170 nm [1], whereas Sun et al obtained a longer run length of 950 nm [3]. The difference in run length measurement may originate from the species of myosin 10 used in the studies and the structure of the forced myosin 10 HMM dimer, such as the length of coiled-coil region, the joint structure between neck region and the coiled-coil region and solution conditions, which are pH, ionic strength, and temperature. We used a GCN4 leucine-zipper at C-terminal fused to the myosin 10 construct after amino acid 940, while other groups used either the same GCN4 leucine-zipper at C-terminal but fused after amino acid 920 [1,2] or fused a portion of the coiled-coil tail of myosin 5a after 1042th aminoacid of myosin 10 [3]. These factors may affect the run-length and step size. The step size (31 nm) of m10-HMM-Zip in our study was shorter than that of m5-HMM (36 nm), and this is in agreement with the maximum velocity and actin activated ATPase rate of myosin 10, that is, 30.6 (nm) x 5.5 (/s) = 168 (nm/sec). Moreover, the shorter step size of myosin 10 may explain its shorter run length.
Second, we characterized the processive movement of myosin 5 and myosin 10 on highly ordered F-actin structures by using the 2-D F-actin rafts with α-actinin and fascin to control the center-to-center distance between the actin filaments in the rafts. We found that the run length and the number of movements of m10-HMM-Zip on the fascin-F-actin rafts was more than that on α-actinin-F-actin rafts, suggesting that myosin 10 prefers to move on fascin-F-actin rafts, which is consistent with the observation of a previous study [1] and the subcellular localization of myosin 10 at the tips of filopodia, where F-actin is mainly cross-linked by fascin [50,51]. By contrast, myosin 5a shows no preference for either single filaments, or F-actin rafts cross-linked by fascin or α-actinin [1,2].
A substantial portion of the myosin 10 did not start walking on actin filaments or actin rafts when observe under TIRF microscopy at saturating ATP concentrations. There are several possible reasons for the higher number of abortive binding events with myosin 10 than with myosin 5. First, it is possible that a myosin must bind with a proper stereochemical position on actin in order to commence a processive run and it is possible that myosin 5 is more successful in doing this than myosin 10. Second, since these measurements are not done under FIONA conditions, a very short processive run of only two or three steps would likely not be detected as a processive run. Given that myosin 10 is inherently less processive than myosin 5 (as shown by the shorter run length) such short runs might be more frequently "observed" with it and scored as a nonprocessive event.
On fascin-F-actin rafts, myosin 10 took mostly 31 nm steps along with a more occasional 21 nm side step, and myosin 5a took mostly 36 nm steps with less frequent 22 nm side steps. This stepping pattern is consistent with a previous report, except that the step size of myosin 10 on fascin-F-actin bundle (27 nm) was shorter than that on single actin filament (34 nm) [3]. However, our results contrast with that of Nagy et al who reported a uniform 18 nm step size on fascin-F-actin bundle [1]. Our results support a stepping model where both myosins take multiple processive steps along an actin filament within the bundle and occasionally take a side step to an adjacent actin filament (Figure 7). In this model, the two heads of myosin bind on single actin filament in the F-actin rafts and occasionally straddle two adjacent actin filaments when taking a side step. This differs from the model proposed by Nagy et al where myosin-10 was suggest to move along an actin bundle with its two heads "waddling" between two adjacent actin filaments.
We did not observed side-steps of m5-HMM and m10-HMM-Zip on α-actinin-F-actin rafts, suggesting that the heads of both myosin molecules could not reach adjacent actin filaments in these rafts due to the wider distances (40 nm) between the center-to-center of actin filaments. However, a recent study showed that myosin 5a could take a side step or switch to another actin filament on a three-dimensional α-actinin actin bundle [52]. In this case, the actin filaments within the bundle were of mixed polarity and this resulted in bi-directional movements of individual myosin 5a molecules within the bundle [52].
There are many actin binding proteins that might create road blocks or obstacles to movement along a single actin filament. Two mechanisms might allow for myosins to dodge these obstacles in order to continue moving processively. Electron microscopic images of myosin 5a bound to actin in the presence of ATP shows that myosin 5a prefers to bind with its two motor domains 13 actin monomers (~36 nm) apart when moving along a single actin filament which effectively allows it to walk "straight" and avoid spirally around the filament [21,53]. However, the lead motor can also bind to the 11 th or 15 th actin monomer which might allow the molecule to take a shorter or longer step if the preferred actin monomer is blocked. There is no comparable electron microscopic information on the interaction of myosin 10 with actin in the presence of ATP. The ability of myosins 5a and 10 to take side steps on fascin-actin rafts provides a second method to avoid obstacles that might prevent the unbound myosin head from attaching to its preferred actin monomer when searching for a new actin binding site. Of note, 2-D actin rafts was used to facilitate the detailed analysis of the stepping manners of myosin 5 and 10 along individual actin filaments by eliminating the complicated structures in 3-D bundles, as seen in cells. As such, the 2-D rafts may not provide the exact road blocks similar to those mentioned above that referred to cellular conditions. However, it provides a simplified, alternative approach to study the 3-D bundles that are difficult structures to study the stepping manner along single filaments.