Our results show that myosin X moves processively on individual actin filaments, with an average step size of 34 nm, and more slowly on actin bundles with an average step size of 27 nm. A processive myosin motor requires a high duty ratio and a sufficiently long lever arm to stride between appropriately oriented binding sites on the helical actin track. The myosin X construct used in this study was determined to have a high duty ratio18
although a bovine myosin X S1 construct produced a low duty ratio8
. This discrepancy has been discussed by Homma and Ikebe18
. The robust processivity reported here implies a high duty ratio in our construct.
Each myosin X lever arm has three IQ motifs (extending about 10.8 nm), so a myosin X dimer would be expected to stride maximally over 22 nm if there were no further extension to its lever arm or unraveling of its proximal tail. Knight et al.16
have suggested that the proximal tail domains of myosin X form non-dimerizing stable single α-helices (SAH domains) that act as mechanically stiff extensions to the myosin X lever arm. The SAH sequence (55 residues following the 3rd IQ motif) and the distal coiled-coil domain (the next 78 residues) are both present in the construct mainly used here, followed by the coiled-coil domain from myosin V to force dimerization.
The 34 nm step size of myosin X on actin filaments ( and ) supports the assumption that there are extensions to the lever arm of myosin X beyond the 10.8 nm length of its three CaM binding sites. The postulated 55-residue SAH domain may increase the length of lever arm by approximately 7.5 nm16
and allow the two heads of myosin X to bind 11 or 13 actin monomers apart, a sterically favorable configuration.
Myosin VI is another myosin motor that is thought to have extensions to its lever arms and thereby reach a large step size20,26,42
. However, evidence from different measurements, including angles and step sizes, suggests there are differences in the rigidity of the lever arm of myosin V, VI, and X. 2
Δα, the change of the probe azimuthal angle α after two steps, is related to the path of a moving myosin motor20
. The 2
Δα distribution of myosin VI was found to be much wider (σ = 55°)20
than that of myosin V (σ = 29°, Supplementary Fig. 2b
) or X (σ = 30°, ), suggesting that myosin VI has more flexible lever arms and walks more wiggly on actin filaments20
. The step size distributions of the three myosin motors are consistent with these angular measurements. Stepping distance of fluorescent labeled myosin X heads (twice the center of mass motion, 68 ± 8.4 nm, (s.d., n
= 1168, )) has variability closer to that of myosin V (74 ± 5.3 nm, s.d.24
, and 74 ± 7.7 nm, s.d., n
= 140, Supplementary Fig. 4a
), than to myosin VI (70 ± 23 nm ,s.d.32
). The highest variability in the step size of myosin VI is likely due to its high flexibility. Lever arm flexibility may also affect the likelihood of backward steps. Myosin VI exhibits more backward steps33,43
than myosin V (Ref.24
and Supplementary Fig. 4
). In our step size measurements on myosin X () and V (Supplementary Fig. 4
), we found that myosin X also has more backward steps than myosin V.
The differences in the lever arm rigidity of the three myosins are a consequence of their structures. Myosin V has rigid lever arms with six bound CaMs and produces robust processive stepping. In order to achieve their large step sizes, however, both myosin VI and X need extensions to their short lever arms which seem to increase the overall flexibility. Part of the extended myosin VI lever arm may contain a random coil20,26,33
or an unfolded three-helix bundle42
, which confers high flexibility. In contrast, myosin X only has one SAH to extend its three-CaM bound lever arm. The SAH is expected to be less rigid than the lever arm of myosin V, but more rigid than the extension of myosin VI. Thus the SAH domain of myosin X is likely to carry mechanical load and facilitate intra-molecular gating44
Myosin X moves more slowly on fascin-actin bundles than it does on actin filaments, consistent with its smaller average step size of 27 nm on bundled actins (). The variable step sizes and left-handed helical walking path of myosin X on actin bundles ( and ) suggest that myosin X switches onto adjacent actin filaments rather randomly. This contrasts with the more predictable “straddle mechanism” proposed by Nagy et al7
, in which the two heads of a myosin X necessarily track along two adjacent actin filaments. Our measurements show that myosin V also moves more slowly on actin bundles than on single filaments. The multiple actin binding sites available in a bundle seem to deflect myosin V and X from a straight path, producing slower velocities. Nevertheless, it may be advantageous that myosin X is able to switch onto adjacent actin filaments in a bundle or span between two actin filaments. For instance, this feature may allow myosin X to bypass “road blocks” on actin bundles during cargo transport in a crowded filopodia. Myosin X is able to pull actin filaments together at the cell periphery to initiate filopodial actin bundles15
, implying that myosin X can span across actin filaments.
Processive motility after dimerization of the native tail sequence by clustering on actin shows that processive motility of myosin X on individual actin filaments is a feature of the native molecule when two motor domains are paired. The robust processivity we observed on individual actin filaments contrasts with the findings of Nagy et al.7
, who reported that myosin X preferentially selects bundled actins for motility and shows poor processivity on single filaments. These differences are presumably due to the different truncation points in the coiled-coil domain or dimerization sequences and positions used in the two studies. The myosin X construct of Nagy et al.7
used a GCN4 leucine zipper to ensure dimerization of the heavy chains rather than the myosin V tail used here. The GCN4 was placed in almost the same position (4 residues later) as the start of our myosin V tail. The stability and flexibility of the putative SAH domain may depend on details or the position of the following coiled coil and an unstable domain structure might diminish gating within myosin X. Alternatively, it is plausible that a leucine zipper might decrease the flexibility of the neck for finding appropriate binding sites on actin filaments, thus reducing the processivity. The radii of helical paths for CaM labeled and C-terminally labeled myosin X ( and ) imply that the tail of our construct is flexible.
Myosin X has been found to walk on both individual actin filaments13,15,18,19
and actin bundles7
in cells as we found in vitro
. The postulated cellular functions of myosin X are surprisingly varied, probably requiring bivalent association with different actin filaments in formation of filopodia15
, membrane interactions in the transport of integrins4
and growth factor receptors3
to filopodial tips and in amplifying morphogenic signals9
, and possibly microtubule binding in maintenance of nuclear position and the mitotic or meiotic spindle11–13
. The flexible tail or head-tail junction in myosin X may enable interactions between its head and tail domains to regulate its activity, like myosin V45,46
and would also allow myosin X to adopt different geometries in complex, more or less dense, actin structures in the lamellipodia, filopodia, near the nucleus, and the spindle. The semi-flexible nature of the SAH domains and tail may allow myosin X to stretch, and pull multiple actin filaments into bundles at the base of filopodia15
and to squeeze through narrow cytoplasmic spaces during transport in the cell body or filopodia. It is conceivable that a myosin X carrying integrins or other cargos into a filopodium would adopt a spiral motion to ensure location of a substrate for attachment.
Conversion of non-processive monomers into processive dimers has been observed for myosin VI and postulated to be a regulatory feature of other myosins17,27–29
, Our observation that native myosin X monomers are not processive, but undergo proximity-induced dimerization and thereby become processive provides support for that postulate. The monomer-dimer conversion may regulate the local cellular function of myosin X as a structural anchor or a cargo transporter.