Single-molecule Motility Research Articles

3P175 ミオシンVの運動性におけるトロポミオシンとトロポニンの影響(分子モーター,第48回日本生物物理学会年会)

3P175 ミオシンVの運動性におけるトロポミオシンとトロポニンの影響(分子モーター,第48回日本生物物理学会年会)

Motor Protein Function in Skeletal Muscle—A Multiple Scale Approach to Contractility

We present an approach to skeletal muscle contractility and its regulation over different scales ranging from biomechanical studies in intact muscle fibers down to the motility and interaction of single motor protein molecules. At each scale, shortening velocities as a measure for weak cross-bridge cycling rates are extracted and compared. Experimental approaches include transmitted light microscopy, second harmonic generation imaging of contracting myofibrils, and fluorescence microscopy of single molecule motility. Each method yields image sequences that are analyzed with automated image processing algorithms to extract the contraction velocity. Using this approach, we show how to isolate the contribution of the motor proteins actin and myosin and their modulation by regulatory proteins from the concerted action of electro-mechanical activation on a more complex cellular scale. The advantage of this approach is that averaged contraction velocities can be determined on the different scales ranging from isolated motor proteins to sarcomere levels in myofibrils and myofibril arrays within the cellular architecture. Our results show that maximum shortening velocities during in situ electrical activation of sarcomere contraction in intact single muscle cells can substantially deviate from sliding velocities obtained in oriented in vitro motility assays of isolated motor proteins showing that biophysical contraction kinetics not simply translate linearly between contractility scales. To adequately resolve the very fast initial mechanical activation kinetics of shortening at each scale, it was necessary to implement high-speed imaging techniques. In the case of intact fibers and single molecule motility, we achieved a major increase in temporal resolution up to frame rates of 200-1000 fps using CMOS image sensor technology. The data we obtained at this unprecedented temporal resolution and the parameters extracted can be used to validate results obtained from computational models of motor protein interaction and skeletal muscle contractility in health and muscle disease. Our approach is feasible to explain the possible underlying mechanisms that contribute to different shortening velocities at different scales and complexities.

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.

Properties of the Kinesin-3 NcKin3 motor domain and implications for neck function

Members of the Kinesin-3 family are microtubule motors involved in the transport of membranous cargo. NcKin3 from the fungus Neurospora crassa is dimeric but inactivates one of its motor heads to generate nonprocessive motility. To determine how one of the heads is inactivated, we investigated truncated monomeric constructs. None of the constructs generated processive single-molecule motility, and multimotor velocities depended linearly on the number of residues remaining in the neck. The kinetic analysis suggests futile ATP hydrolysis cycles, because a representative monomer showed a faster ATP turnover than the dimer while supporting slower motility. The K(0.5,MT) was 70-fold lower, the microtubule-bound portion of the kinetic cycle eight-fold longer and the microtubule detachment rate almost 15-fold slower than that of the dimer. Moreover, the monomer's microtubule-dependent ADP release occurred three-fold to four-fold faster than k(cat) (125 versus 34 s(-1)), whereas phosphate release was approximately equally fast (29 s(-1)). A dimeric construct containing a structure-breaking insert between motor head and neck showed a similar behaviour. These data suggest that the heads of the wild-type NcKin3 motor are strictly coupled via the neck domain, and that the dimeric structure is required for proper detachment after one ATPase cycle. This is the first direct comparison of a monomeric Kinesin-3 with its dimeric full-length counterpart, and the kinetic changes observed here may also apply to other Kinesin-3 motors.

Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin

Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast p150(Glued) homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering.

Processivity of Myosin V and X on two-dimensional (2D) paracrystalline actin array

Myosin movement in vivo takes place on a wide variety of F-actin structures, including single filaments and 2D/3D bundled networks. Using in vitro single molecule motility techniqes, we have investigated the processivity and stepping characteristics of myosin V HMM and myosin X HMM with a leucine zipper on single actin filaments and 2D actin bundles. To answer how myosin V and myosin X step on actin bundles, we observed single molecule motility of fluorescently labeled myosin V and X using total internal reflection fluorescent microscopy, and analyzed the step-size, run length, speed, and direction of the movements on actin-bundles. Actin was polymerized and cross-linked on a charged lipid monolayer in Teflon wells to create regular 2D actin arrays. Two cross-linking proteins were used: alpha-actinin, which produces non-polarized bundles with 40 nm filament spacing, and fascin which produces polarized actin bundles with 13 nm filament spacing. We applied a modified particle tracking program, which allowed us to analyze thousands of simultaneous myosin tracks and determine the run lengths and velocities typical of processive movement on the bundled networks. Myosin V moved processively on all types of in vitro actin structures. Myosin X moved well on polarized fascin cross-linked bundles, but movement was impaired or nonexistent on non-polarized alpha-actinin bundles. We hypothesize that forward runs of myosin X on alpha-actinin cross-linked bundles are inhibited because myosin X might makes “sidesteps” to a neighboring filament, which stalls the run. The presence of an SAH domain in the lever arm of myosin X could increase the working stroke or flexibility of the lever arm allowing it to more easily sidestep across the larger alpha-actinin filament spacing.

How Does The Dimeric Cytoplasmic Dynein Processively Walk on a Microtubule?

Cytoplasmic dynein is a two-headed molecular motor, which can take hundreds steps along a microtubule (MT). Although the mechanism of this processive motion remains poorly understood, it is generally assumed that each of the two heads alternatively produces force on MT to move forward. To elucidate the mechanism of this processive motion, we expressed the hetero-dimeric construct of dynein motor domain, in which one domain completely lost its ATP-binding activity due to the K/T mutation in the Walker A motif in its AAA1 module (P1T mutation). Our single-molecule motility assays showed that the hetero-dimer of the wild type and the P1T mutant (Wild/P1T) moved processively on MT with its velocity approximately half of that of the wild-type homo-dimer. Because one head of the Wild/P1T hetero-dimer cannot bind ATP, its processive motion suggests that the “chemical gating” is not necessarily required for the processive stepping, but some type of “mechanical gating” may be responsible for it. We then examined if the intramolecular tension through the tail domain linking the two motor domains is responsible for this “mechanical gating”. We inserted a Gly-rich flexible linker with 20 or 40 residues between the tail domain and the hetero-dimerizer to reduce the tension. Unexpectedly, the Wild/P1T hetero-dimer with the flexible linker moved processively; their run length and velocity were similar to those of the hetero-dimer without the flexible linker. These results suggest that the tension through the tail domain does not play a critical role in the processive motion. The direct interaction of the two AAA rings in the motor domain may be responsible for the “mechanical gating” to sustain alternative steps of the two motor domains on MT.

1TP4-03 キネシンと細胞質ダイニンが混在する微小管上における各分子モーターの一分子運動解析(分子モーター,第47回日本生物物理学会年会)

1TP4-03 キネシンと細胞質ダイニンが混在する微小管上における各分子モーターの一分子運動解析(分子モーター,第47回日本生物物理学会年会)

Regulatory ATPase Sites of Cytoplasmic Dynein Affect Processivity and Force Generation

The heavy chain of cytoplasmic dynein contains four nucleotide-binding domains referred to as AAA1–AAA4, with the first domain (AAA1) being the main ATP hydrolytic site. Although previous studies have proposed regulatory roles for AAA3 and AAA4, the role of ATP hydrolysis at these sites remains elusive. Here, we have analyzed the single molecule motility properties of yeast cytoplasmic dynein mutants bearing mutations that prevent ATP hydrolysis at AAA3 or AAA4. Both mutants remain processive, but the AAA4 mutant exhibits a surprising increase in processivity due to its tighter affinity for microtubules. In addition to changes in motility characteristics, AAA3 and AAA4 mutants produce less maximal force than wild-type dynein. These results indicate that the nucleotide binding state at AAA3 and AAA4 can allosterically modulate microtubule binding affinity and affect dynein processivity and force production.

Processive Movement by a Kinesin Heterodimer with an Inactivating Mutation in One Head

A single molecule of the motor enzyme kinesin-1 keeps a tight grip on its microtubule track, making tens or hundreds of discrete, unidirectional 8 nm steps before dissociating. This high duty ratio processive movement is thought to require a mechanism in which alternating stepping of the two head domains of the kinesin dimer is driven by alternating, overlapped cycles of ATP hydrolysis by the two heads. The R210K point mutation in Drosophila kinesin heavy chain was reported to disrupt the ability of the enzyme active site to catalyze ATP P−O bond cleavage. We expressed R210K homodimers as well as isolated R210K heads and confirmed that both are essentially inactive. We then coexpressed tagged R210K subunits with untagged wild-type subunits and affinity purified R210K/wild-type heterodimers together with the inactive R210K homodimers. In contrast to the R210K head or homodimer, the heterodimer was a highly active (>50% of wild-type) microtubule-stimulated ATPase, and the heterodimer displayed high duty ratio processive movement in single-molecule motility experiments. Thus, dimerization of a subunit containing the inactivating mutation with a functional subunit can complement the mutation; this must occur either by lowering or by bypassing kinetic barriers in the ATPase or mechanical cycles of the mutant head. The observations provide support for kinesin-1 gating mechanisms in which one head stimulates the rate of essential processes in the other.

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 V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. 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 (K(d) = 0.25 +/- 0.02 microm) 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 approximately 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 approximately 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.

Minus-End-Directed Motor Ncd Exhibits Processive Movement that Is Enhanced by Microtubule Bundling In Vitro

Drosophila Ncd, a kinesin-14A family member, is essential for meiosis and mitosis. Ncd is a minus-end-directed motor protein that has an ATP-independent microtubule binding site in the tail region, which enables it to act as a dynamic crosslinker of microtubules to assemble and maintain the spindle. Although a tailless Ncd has been shown to be nonprocessive, the role of the Ncd tail in single-molecule motility is unknown. Here, we show that individual Ncd dimers containing the tail region can move processively along microtubules at very low ionic strength, which provides the first evidence of processivity for minus-end-directed kinesins. The movement of GFP-Ncd consists of both a unidirectional and a diffusive element, and it was sensitive to ionic strength. Motility of a truncation series of Ncd and removal of the tubulin tail suggested that the Ncd tail serves as an electrostatic tether to microtubules. Under higher ionic conditions, Ncd showed only a small bias in diffusion along "single" microtubules, whereas it exhibited processive movement along "bundled" microtubules. This property may allow Ncd to accumulate preferentially in the vicinity of focused microtubules and then to crosslink and slide microtubules, possibly contributing to dynamic spindle self-organization.

How myosin VI coordinates its heads during processive movement

A processive molecular motor must coordinate the enzymatic state of its two catalytic domains in order to prevent premature detachment from its track. For myosin V, internal strain produced when both heads of are attached to an actin track prevents completion of the lever arm swing of the lead head and blocks ADP release. However, this mechanism cannot work for myosin VI, since its lever arm positions are reversed. Here, we demonstrate that myosin VI gating is achieved instead by blocking ATP binding to the lead head once it has released its ADP. The structural basis for this unique gating mechanism involves an insert near the nucleotide binding pocket that is found only in class VI myosin. Reverse strain greatly favors binding of ADP to the lead head, which makes it possible for myosin VI to function as a processive transporter as well as an actin-based anchor. While this mechanism is unlike that of any other myosin superfamily member, it bears remarkable similarities to that of another processive motor from a different superfamily--kinesin I.

Long-term outcome of bilateral pallidal deep brain stimulation for primary cervical dystonia

Ten patients with severe cervical dystonia (CD) unresponsive to medical treatment underwent bilateral globus pallidus internus (GPi) deep brain stimulation (DBS) and were followed for 31.9 +/- 20.9 months. At last follow-up, the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) severity score improved by 54.8%, the TWSTRS disability score improved by 59.1%, and the TWSTRS pain score improved by 50.4%. Bilateral GPi DBS is an effective long-term therapy in patients with CD.

Dynactin Enhances the Processivity of Kinesin‐2

Kinesin-2 is a major microtubule-based motor in most cell types. Its in vitro motile properties have been analyzed extensively and been found to differ considerably from kinesin-1. Although recombinant kinesin-2 heterodimers exhibit processive movement, the processivity of the native kinesin-2 holoenzyme has never been evaluated. Kinesin-2 can interact with dynactin, a 'processivity factor' for cytoplasmic dynein, which may alter its motile properties. In this study, we analyze the in vitro motility of single native kinesin-2 molecules and determine the effects of dynactin on motor processivity. We find that individual native kinesin-2 molecules travel processively. Dynactin has no effect on velocity but significantly increases the run length of kinesin-2 movements. These results show that the interaction with dynactin has important functional consequences on the activity of the kinesin-2 motor.

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.

Processivity of Chimeric Class V Myosins

Unconventional myosin V takes many 36-nm steps along an actin filament before it dissociates, thus ensuring its ability to move cargo intracellularly over long distances. In the present study we assessed the structural features that affect processive run length by analyzing the properties of chimeras of mouse myosin V and a non-processive class V myosin from yeast (Myo4p) (Reck-Peterson, S. L., Tyska, M. J., Novick, P. J., and Mooseker, M. S. (2001) J. Cell Biol. 153, 1121-1126). Surprisingly a chimera containing the yeast motor domain on the neck and rod of mouse myosin V (Y-MD) showed longer run lengths than mouse wild type at low salt. Run lengths of mouse myosin V showed little salt dependence, whereas those of Y-MD decreased steeply with ionic strength, similar to a chimera containing yeast loop 2 in the mouse myosin V backbone. Loop 2 binds to acidic patches on actin in the weak binding states of the cycle (Volkmann, N., Liu, H., Hazelwood, L., Krementsova, E. B., Lowey, S., Trybus, K. M., and Hanein, D. (2005) Mol. Cell 19, 595-605). Constructs containing yeast loop 2, which has no net charge compared with +6 for wild type, showed a higher K(m) for actin in steady-state ATPase assays. The results imply that a positively charged loop 2 and a high affinity for actin are important to maintain processivity near physiologic ionic strength.

A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.

1H1330 ミオシンVの1分子機能観察(31.ビデオセッション,一般演題,日本生物物理学会第40回年会)

1H1330 ミオシンVの1分子機能観察(31.ビデオセッション,一般演題,日本生物物理学会第40回年会)

Myosin VI is a processive motor with a large step size.

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.