Yasuharu Takagi

The SAH domain extends the functional length of the myosin lever

Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5–2IQ). Electron microscopy of this chimera (Myo5–2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5–6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5–6IQ but much greater than for Myo5–2IQ. Myo5–2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5–6IQ in in-vitro single molecule assays. In comparison, Myo5–2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5–6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.

Label-free, all-optical detection, imaging, and tracking of a single protein

Optical detection of individual proteins requires fluorescent labeling. Cavity and plasmonic methodologies enhance single molecule signatures in the absence of any labels but have struggled to demonstrate routine and quantitative single protein detection. Here, we used interferometric scattering microscopy not only to detect but also to image and nanometrically track the motion of single myosin 5a heavy meromyosin molecules without the use of labels or any nanoscopic amplification. Together with the simple experimental arrangement, an intrinsic independence from strong electronic transition dipoles and a detection limit of <60 kDa, our approach paves the way toward nonresonant, label-free sensing and imaging of nanoscopic objects down to the single protein level.

Mammalian Myosin-18A: A Highly Divergent Myosin

Background: The myosin superfamily has many classes that have evolved to carry out different functions. Results: Mouse myosin-18A binds actin weakly in an ATP-independent manner and has very low enzymatic activity. Conclusion: Not all myosins exhibit motor activity. Significance: This work demonstrates that myosins may have functions unrelated to their ability to hydrolyze ATP. The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s−1) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Background: Nonmuscle myosin IIB (NMIIB) is a key player in cell motility. Results: Although the individual NMIIB molecules are not processive, NMIIB thick filaments show robust processive motion. Conclusion: NMIIB forms processive thick filament in vitro, which is likely the functional unit in cells. Significance: We demonstrate how processive systems can be formed from nonprocessive individual molecules. Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ∼14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20–25%) motor. The ADP release step (∼0.35 s−1) of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s−1). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (∼0.4 s−1). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ∼6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.

Human Myosin Vc Is a Low Duty Ratio, Nonprocessive Molecular Motor

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a Vmax of 1.8 ± 0.3 s-1 and a KATPase of 43 ± 11 μm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (∼1.5 s-1), rather than the ADP release step (∼12.0–16.0 s-1). Nevertheless, the ADP affinity of actomyosin Vc (Kd = 0.25 ± 0.02 μm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was ∼10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of ∼24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

Significance Mutations in unconventional myosin 15 cause nonsyndromic autosomal recessive deafness, a common form of hereditary hearing loss in humans. Myosin 15 is required for the development of hair cell mechanosensory stereocilia that detect sounds within the inner ear. To our knowledge, our work offers the first insight into the biophysical properties of purified myosin 15. Using ensemble and single molecule techniques, we show that myosin 15 is a high-duty ratio motor, which is a characteristic of myosins that can move processively along actin filaments. We also introduce a new strategy for producing myosins by chaperone coexpression in Spodoptera frugiperda insect cells. This approach may help optimize expression of skeletal and cardiac muscle myosins, which are emerging as translational drug targets but are presently refractory to larger-scale purification. Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s−1 at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (kcat ∼6 s−1) was similar to the actin-detachment rate (kdet = 6.2 s−1) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.

Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments

Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy

Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head–head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps. DOI: http://dx.doi.org/10.7554/eLife.05413.001

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

Significance Filopodia act as organelles for sensing and exploring the environment, as well as producing traction forces during cellular locomotion. Myosin-10 is a molecular motor crucial for intrafilopodial trafficking and filopodia formation. To decipher how myosin-10 generates force and movement, we used electron microscopy and a combination of ensemble biochemical and single molecule mechanical techniques to help elucidate its structure and mechano-chemical coupling. Our results clarify current controversies about myosin-10 structure and function by revealing that it generates an unexpectedly large biphasic power stroke and moves processively along actin, but detaches rapidly at relatively low force. These adaptations may be advantageous features for a myosin motor that carries bulky cargo within the narrow confines of the filopodium. Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein’s mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12–14 s−1) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.

Temperature dependent measurements reveal similarities between muscle and non-muscle myosin motility.

We examined the temperature dependence of muscle and non-muscle myosin (heavy meromyosin, HMM) with in vitro motility and actin-activated ATPase assays. Our results indicate that myosin V (MV) has a temperature dependence that is similar in both ATPase and motility assays. We demonstrate that skeletal muscle myosin (SK), smooth muscle myosin (SM), and non-muscle myosin IIA (NM) have different temperature dependence in ATPase compared to in vitro motility assays. In the class II myosins we examined (SK, SM, and NM) the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while for in vitro motility assays it is controversial. In MV the rate-limiting step for both in vitro motility and ATPase assays is known to be ADP release. Consequently, in MV the temperature dependence of the ADP release rate constant is similar to the temperature dependence of in vitro motility. Interestingly, the temperature dependence of the ADP release rate constant of SM and NM was shifted toward the in vitro motility temperature dependence. Our results suggest that the rate-limiting step in SK, SM, and NM may shift from attachment-limited in solution to detachment limited in the in vitro motility assay. Internal strain within the myosin molecule or by neighboring myosin motors may slow ADP release which becomes rate-limiting in the in vitro motility assay. Within this small subset of myosins examined, the in vitro sliding velocity correlates reasonably well with actin-activated ATPase activity, which was suggested by the original study by Barany (J Gen Physiol 50:197–218, 1967).