Myosin X is an unconventional myosin that has been implicated in filopodial development in mammals. We have recently characterized its steady-state and transient state MgATPase activity. Myosin X contains a region of predicted coiled-coil 120 residues long. However, the highly charged nature, and pattern of charges in the proximal 36-residues, appears incompatible with coiled-coil formation. We have shown that this domain forms a stable single alpha-helical domain (SAH domain) and functions to extend the neck region of the myosin which forms part of the lever arm of myosin. Thus, the powerstroke is lengthened. We have carried out optical trapping experiments with a forced dimer of myosin X where a leucine zipper was added at the end of the predicted coiled-coil region. This molecule is shown to be dimeric by electron microscopy. We have measured its mechanical properties using optical trapping nanometry and find that it has a power stroke of about 17 nm. Increasing the calcium concentration increased the power stroke size to 23 nm consistent with three IQ motifs and a SAH domain in the lever arm. We believe the increase power stroke length is due to binding of an additional calmodulin to the third IQ motif in the presence of calcium. The attachment lifetimes are consistent with the ADP release rate measured in vitro. At low trap stiffness, the myosin X shows processive movement (forward and backward steps) occurring with steps of about 35 nm. This is consistent with electron micrographs showing the molecule attached by two head to actin monomer that are separated by 36 nm in the actin filament. In collaboration with a former postdoc, Takeshi Sakamoto, we show that single molecule TIRF assays show that the molecule moves processively along actin in the absence of load with 36 nm steps. We examined the movement of myosin X on parallel bundles. The myosin walks predominantly along a single actin filament, but takes frequent side steps onto adjacent filaments Full length myosin 18A does not form filaments, but rather exists in an equilibrium between a monomer and an antiparallel dimer. When myosin 18A is mixed with nonmuscle myosin IIA, the two molecules co-polymerize to form heteropolymeric filaments which become shorter as the ratio of myosin 18A:myosin IIA increases. At high ratios, no filaments are seen, but rather dimeric molecules in solution. In collaboration with Philipp Kukura of Oxford University, we have used a light microscopy based interferometric scattering technique to examine the processive movement of myosin 5 HMM on actin. Using this technique we were able to image single, unlabeled molecules of myosin 5 HMM move along actin with a precision of a few nanometers. The molecule took 36 nm steps and moved at the same speed as previously reported for fluorescently-labeled myosin 5. By attaching a 20 nm gold particle to the amino-terminus we are able to measure the movement at sampling rates up to 1000 Hz and follow the movement of the unattached labeled myosin head. Interesting, even with a 20 nm gold particle attached the myosin moves at the same velocity as the unlabeled molecule. Myosin 3B is a monomeric myosin which we have expressed in Sf9 cells along with calmodulin and regulatory light chain. It binds regulatory light chain and calmodulin as purified in the absence of calcium. Calmodulin displaces the regulatory light chain in the presence of calcium and this is accompanied by a significant increase in the actin-activated MgATPase activity.

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Gardini, Lucia; Heissler, Sarah M; Arbore, Claudia et al. (2018) Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level. Nat Commun 9:2844
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