Detection of fractional steps in cargo movement by the collective operation of kinesin-1 motors

The stepping behavior of single kinesin-1 motor proteins has been studied in great detail. However, in cells, these motors often do not work alone but rather function in small groups when they transport cellular cargo. Until now, the cooperative interactions between motors in such groups were poorly understood. A fundamental question is whether two or more motors that move the same cargo step in synchrony, producing the same step size as a single motor, or whether the step size of the cargo movement varies. To answer this question, we performed in vitro gliding motility assays, where microtubules coated with quantum dots were driven over a glass surface by a known number of kinesin-1 motors. The motion of individual microtubules was then tracked with nanometer precision. In the case of transport by two kinesin-1 motors, we found successive 4-nm steps, corresponding to half the step size of a single motor. Dwell-time analysis did not reveal any coordination, in the sense of alternate stepping, between the motors. When three motors interacted in collective transport, we identified distinct forward and backward jumps on the order of 10 nm. The existence of the fractional steps as well as the distinct jumps illustrate a lack of synchronization and has implications for the analysis of motor-driven organelle movement investigated in vivo.     

Myosin V stepping mechanism

We observe the myosin V stepping mechanism by traveling wave tracking. This technique, associated with optical tweezers, allows one to follow a scattering particle in a two-dimensional plane, with nanometer accuracy and a temporal resolution in the microsecond range. We have observed that, at the millisecond time scale, the myosin V combines longitudinal and vertical motions during the step. Because at this time scale the steps appear heterogeneous, we deduce their general features by aligning and averaging a large number of them. Our data show that the 36-nm step occurs in three main stages. First, the myosin center of mass moves forward 5 nm; the duration of this short prestep depends on the ATP concentration. Second, the motor performs a fast motion over 23 nm; this motion is associated to a vertical movement of the myosin center of mass, whose distance from the actin filament increases by 6 nm. Third, the myosin head freely diffuses toward the next binding site and the vertical position is recovered. We propose a simple model to describe the step mechanism of the dimeric myosin V.     

A one-headed class V myosin molecule develops multiple large (approximately 32-nm) steps successively

Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large ( approximately 36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP- and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large (approximately 32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.     

Role of the lever arm in the processive stepping of myosin V

Myosin V is a two-headed molecular motor that binds six light chains per heavy chain, which creates unusually long lever arms. This motor moves processively along its actin track in discrete 36-nm steps. Our model is that one head of the two-headed myosin V tightly binds to actin and swings its long lever arm through a large angle, providing a stroke. We created single-headed constructs with different-size lever arms and show that stroke size is proportional to lever arm length. In a two-headed molecule, the stroke provides the directional bias, after which the unbound head diffuses to find its binding site, 36 nm forward. Our two-headed construct with all six light chains per head reconstitutes the 36-nm processive step seen in tissue-purified myosin V. Two-headed myosin V molecules with only four light chains per head are still processive, but their step size is reduced to 24 nm. A further reduction in the length of the lever arms to one light chain per head results in a motor that is unable to walk processively. This motor produces single small approximately 6-nm strokes, and ATPase and pyrene actin quench measurements show that only one of the heads of this dimer rapidly binds to actin for a given binding event. These data show that for myosin V with its normal proximal tail domain, both heads and a long lever arm are required for large, processive steps.     

Load-dependent ADP binding to myosins V and VI: implications for subunit coordination and function

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a "hand-over-hand" fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is approximately 2 pN for both myosin V and myosin VI. The distinct load dependence of ADP binding allows these motors to perform different cellular functions.     

Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors

Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus- and minus-end–directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ∼20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance.     

Unconventional myosin traffic in cells reveals a selective actin cytoskeleton

Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments.     

Myosin V and Kinesin act as tethers to enhance each others' processivity

Organelle transport to the periphery of the cell involves coordinated transport between the processive motors kinesin and myosin V. Long-range transport takes place on microtubule tracks, whereas final delivery involves shorter actin-based movements. The concept that motors only function on their appropriate track required further investigation with the recent observation that myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of myosin V's diffusion on microtubules is a significant enhancement of the processive run length of kinesin when both motors are present on the same cargo. The degree of run length enhancement correlated with the net positive charge in loop 2 of myosin V. On actin, myosin V also undergoes longer processive runs when kinesin is present on the same cargo. The process that causes run length enhancement on both cytoskeletal tracks is electrostatic. We propose that one motor acts as a tether for the other and prevents its diffusion away from the track, thus allowing more steps to be taken before dissociation. The resulting run length enhancement likely contributes to the successful delivery of cargo in the cell.     

The mitotic kinesin CENP-E is a processive transport motor

In vivo studies suggest that centromeric protein E (CENP-E), a kinesin-7 family member, plays a key role in the movement of chromosomes toward the metaphase plate during mitosis. How CENP-E accomplishes this crucial task, however, is not clear. Here we present single-molecule measurements of CENP-E that demonstrate that this motor moves processively toward the plus end of microtubules, with an average run length of 2.6 +/- 0.2 mum, in a hand-over-hand fashion, taking 8-nm steps with a stall force of 6 +/- 0.1 pN. The ATP dependence of motor velocity obeys Michaelis-Menten kinetics with K(M,ATP) = 35 +/- 5 muM. All of these features are remarkably similar to those for kinesin-1-a highly processive transport motor. We, therefore, propose that CENP-E transports chromosomes in a manner analogous to how kinesin-1 transports cytoplasmic vesicles.     

Dimerized Drosophila myosin VIIa: a processive motor

The molecular mechanism of processive movement of single myosin molecules from classes V and VI along their actin tracks has recently attracted extraordinary attention. Another member of the myosin superfamily, myosin VII, plays vital roles in the sensory function of Drosophila and mammals. We studied the molecular mechanism of Drosophila myosin VIIa, using transient kinetics and single-molecule motility assays. Myosin VIIa moves along actin filaments as a processive, double-headed single molecule when dimerized by the inclusion of a leucine zipper at the C terminus of the coiled-coil domain. Its motility is approximately 8-10 times slower than that of myosin V, and its step size is 30 nm, which is consistent with the presence of five IQ motifs in its neck region. The kinetic basis for the processive motility of myosin VIIa is the relative magnitude of the release rate constants of phosphate (fast) and ADP (slow) as in myosins V and VI. The ATPase pathway is rate-limited by a reversible interconversion between two distinct ADP-bound actomyosin states, which results in high steady-state occupancy of a strongly actin-bound myosin species. The distinctive features of myosin VIIa (long run lengths, slow motility) will be very useful in video-based single-molecule applications. In cells, this kinetic behavior would allow myosin VIIa to exert and hold tension on actin filaments and, if dimerized, to function as a processive cargo transporter.     

A point mutation in the regulatory light chain reduces the step size of skeletal muscle myosin

Current evidence favors the theory that, when the globular motor domain of myosin attaches to actin, the light chain binding domain or "lever arm" rotates, and thereby generates movement of actin filaments. Myosin is uniquely designed for such a role in that a long alpha-helix (approximately 9 nm) extending from the C terminus of the catalytic core is stabilized by two calmodulin-like molecules, the regulatory light chain (RLC) and the essential light chain (ELC). Here, we introduce a single-point mutation into the skeletal myosin RLC, which results in a large (approximately 50%) reduction in actin filament velocity (V(actin)) without any loss in actin-activated MgATPase activity. Single-molecule analysis of myosin by optical trapping showed a comparable 2-fold reduction in unitary displacement or step size (d), without a significant change in the duration of the strongly attached state (tau(on)) after the power stroke. Assuming that V(actin) approximately d/tau(on), we can account for the change in velocity primarily by a change in the step size of the lever arm without incurring any change in the kinetic properties of the mutant myosin. These results suggest that a principal role for the many light chain isoforms in the myosin II class may be to modulate the flexural rigidity of the light chain binding domain to maximize tension development and movement during muscle contraction.     

Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ≈20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (MADPP_i) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP → MADPP_i. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.     

Transient binding of dynein controls bidirectional long-range motility of early endosomes

In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3-based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the long-range motility behavior of the organelles.     

Evidence for myosin motors on organelles in squid axoplasm.

Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.     

Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin

During skeletal muscle contraction, regular arrays of actin and myosin filaments slide past each other driven by the cyclic ATP-dependent interaction of the motor protein myosin II (the cross-bridge) with actin. The rate of the cross-bridge cycle and its load-dependence, defining shortening velocity and energy consumption at the molecular level, vary widely among different isoforms of myosin II. However, the underlying mechanisms remain poorly understood. We have addressed this question by applying a single-molecule approach to rapidly ( approximately 300 mus) and precisely ( approximately 0.1 nm) detect acto-myosin interactions of two myosin isoforms having large differences in shortening velocity. We show that skeletal myosin propels actin filaments, performing its conformational change (working stroke) in two steps. The first step ( approximately 3.4-5.2 nm) occurs immediately after myosin binding and is followed by a smaller step ( approximately 1.0-1.3 nm), which occurs much faster in the fast myosin isoform than in the slow one, independently of ATP concentration. On the other hand, the rate of the second phase of the working stroke, from development of the latter step to dissociation of the acto-myosin complex, is very similar in the two isoforms and depends linearly on ATP concentration. The finding of a second mechanical event in the working stroke of skeletal muscle myosin provides the molecular basis for a simple model of actomyosin interaction. This model can account for the variation, in different fiber types, of the rate of the cross-bridge cycle and provides a common scheme for the chemo-mechanical transduction within the myosin family.     

Movements of truncated kinesin fragments with a short or an artificial flexible neck

To investigate the role of the neck domain of kinesin, we used optical trapping nanometry to perform high-resolution measurements of the movements and forces produced by recombinant kinesin fragments in which the neck domains were shortened or replaced by an artificial random coil. Truncated kinesin fragments (K351) that contain a motor domain consisting of ≈340 aa and a short neck domain consisting of ≈11 aa showed fast movement (800 nm/s) and 8-nm steps. Such behavior was similar to that of recombinant fragments containing the full-length neck domain (K411) and to that of native kinesin. Kinesin fragments lacking the short neck domain (K340), however, showed very slow movement (<50 nm/s), as previously reported. Joining an artificial 11-aa sequence that was expected to form a flexible random chain to the motor domain (K340–chain) produced normal fast (≈700 nm/s) and stepwise movement. The results suggest that the neck domain does not act as a rigid lever arm to magnify the structural change at the catalytic domain as has been believed for myosin, but it does act as a flexible joint to guarantee the mobility of the motor domain.     

The unique insert at the end of the myosin VI motor is the sole determinant of directionality

Myosin VI moves toward the pointed (minus) end of actin filaments, the reverse direction of other myosin classes. The myosin VI structure demonstrates that a unique insert at the end of the motor repositions its lever arm and is at least in part responsible for the reversal of directionality. However, it has been proposed that there must be additional modifications within the motor that contribute to its large step size and to the reversal of directionality. To ascertain the inherent directionality of the motor core, we attached the myosin V lever arm to myosin VI, with and without the unique insert. If the insert was maintained, the motor moved toward the minus end of actin filaments, but if removed, movement was redirected toward the plus end. Single-molecule studies revealed that further adaptations within the motor increase the magnitude and variability of the plus-end directed converter movements, and unexpectedly provide the source of the highly variable myosin VI step size. Thus, the unique insert is necessary and sufficient to reverse an inherently plus-end directed myosin.     

High-resolution tracking of microtubule motility driven by a single kinesin motor.

Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions.     

Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro

Although many types of membrane-bound organelles rely upon microtubule-based transport for their proper placement within the cytoplasm, the molecular mechanisms that regulate intracellular motility remain largely unknown. To address this problem, we have studied the microtubule-dependent dispersion and aggregation of pigment granules from an immortalized Xenopus melanophore cell line. We have reconstituted pigment granule motility along bovine brain microtubules in vitro using a microscope-based motility assay. Pigment granules, or melanosomes, move along single microtubules bidirectionally; however, analysis of the polarities of this movement shows that melanosomes that have been purified from dispersed cells exhibit mostly plus end-directed motility, while movement of organelles from aggregating cells is biased toward the minus end. Removal of all soluble proteins from the melanosome fractions by density gradient centrifugation does not diminish organelle motility, demonstrating that all the components required for transport have a stable association with the melanosome membranes. Western blotting shows the presence of the plus end-directed motor, kinesin-II, and the minus end-directed motor, cytoplasmic dynein in highly purified melanosomes. Therefore, purified melanosomes retain their ability to move along microtubules as well as their regulated state. Direct biochemical comparison of melanosomes from aggregated and dispersed cells may elucidate the molecular mechanisms that regulate organelle transport in melanophores.     

The kinetic mechanism of myosin V

Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590-593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin.