Processive Motor Research Articles

Alternating-Site Mechanism of Kinesin-1 Characterized by Single-Molecule FRET Using Fluorescent ATP Analogues

Kinesin-1 motor proteins move along microtubules in repetitive steps of 8 nm at the expense of ATP. To determine nucleotide dwell times during these processive runs, we used a Förster resonance energy transfer method at the single-molecule level that detects nucleotide binding to kinesin motor heads. We show that the fluorescent ATP analog used produces processive motility with kinetic parameters altered <2.5-fold compared with normal ATP. Using our confocal fluorescence kinesin motility assay, we obtained fluorescence intensity time traces that we then analyzed using autocorrelation techniques, yielding a time resolution of ∼1 ms for the intensity fluctuations due to fluorescent nucleotide binding and release. To compare these experimental autocorrelation curves with kinetic models, we used Monte-Carlo simulations. We find that the experimental data can only be described satisfactorily on the basis of models assuming an alternating-site mechanism, thus supporting the view that kinesin's two motor domains hydrolyze ATP and step in a sequential way.

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.

Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells.

The Processivity of Kinesin-2 Motors Suggests Diminished Front-Head Gating

Kinesin-2 motors, which are involved in intraflagellar transport and cargo transport along cytoplasmic microtubules, differ from motors in the canonical kinesin-1 family by having a heterodimeric rather than homodimeric structure and possessing a three amino acid insertion in their neck linker domain. To determine how these structural features alter the chemomechanical coupling in kinesin-2, we used single-molecule bead experiments to measure the processivity and velocity of mouse kinesin-2 heterodimer (KIF3A/B) and the engineered homodimers KIF3A/A and KIF3B/B and compared their behavior to Drosophila kinesin-1 heavy chain (KHC). Single-motor run lengths of kinesin-2 were 4-fold shorter than those of kinesin-1. Extending the kinesin-1 neck linker by three amino acids led to a similar reduction in processivity. Furthermore, kinesin-2 processivity varied inversely with ATP concentration. Stochastic simulations of the kinesin-1 and kinesin-2 hydrolysis cycles suggest that "front-head gating," in which rearward tension prevents ATP binding to the front head when both heads are bound to the microtubule, is diminished in kinesin-2. Because the mechanical tension that underlies front-head gating must be transmitted through the neck linker domains, we propose that the diminished coordination in kinesin-2 is a result of its longer and, hence, more compliant neck linker element.

Lever Arm Length Determines The Azimuthal But Not The Axial Orientation Of Myosin V During Processive Motility

In order to assess how lever arm length affects the three-dimensional motions of myosin V during processive motility, two constructs were studied using single molecule polarized total internal reflection fluorescence (polTIRF) microscopy. MyoV6IQ and MyoV4IQ contain 6 and 4 calmodulin (CaM) binding IQ motifs, and otherwise consist of the native myosin V excluding the tail domain. Bifunctional rhodamine labeled CaM replaced a native CaM, giving probe angles βP relative to the actin axis and αP, the azimuth around actin. As with other processive myosins, αP and βP exhibited tilting of the probe with each step. With MyoV6IQ, αP often returned to its initial value after two steps, as expected for nearly straight walking. This behavior enabled us to determine the orientation of the lever arm, αL and βL, as well as θL and fL, the probe angles relative to CaM. βL was 100° and 40° in the leading and trailing heads, respectively. In MyoV4IQ, βP was similar to 6IQ, but αP seldom returned to its earlier value after two steps. This indicates considerable net azimuthal rotation, as expected for smaller step sizes. Thus, lever arm length determines the azimuthal angular path, whereas the axial orientation is likely determined by structural constraints in the motor domain. Modified gliding filament assays were performed using polTIRF to detect twirling of actin about its axis during motility. MyoV6IQ twirled almost exclusively left-handed with a pitch of 1.4 μm. MyoV4IQ twirled with both right- and left-handed pitches of 1.0 and 1.2 μm, respectively. Bidirectional twirling of MyoV4IQ contrasts with every isoform of myosin previously tested (II, native V, VI and X) all of which twirled with a single handedness. This work was supported by NIH grant AR05117.

Processive Motility of Heterodimeric Kinesin That Has Defect in the Neck Linker Docking

Kinesin-1 is a motor protein that moves processively along microtubules in a hand-over-hand manner. The neck linker, a short stretch that connects two motor heads, has been shown to undergo ATP-dependent conformational changes, although its role on the processive motility is still controversial. To address this question, we employed a kinesin mutant at switch I (R203K) that can normally bind ATP but is unable to hydrolyse ATP (Klumpp et al. JBC 2003). First we observed the neck linker structural state of monomeric R203K bound to the microtubule using single molecule FRET. We found that the neck linker remained undocked conformation even in the presence of saturating ATP, suggesting that ATP-binding is not sufficient to stabilize neck linker docked state. Next we constructed heterodimeric kinesin that is composed of a wild-type head and a mutant R203K head. As recently been shown by Thoresen and Gelles (Biochemistry 2008), this heterodimer showed slow processive movement along microtubules. Then we observed the conformational changes of this heterodimer using a single molecule FRET sensor as previously developed to distinguish one-head-bound and two-head-bound states (Mori et al. Nature 2007), and found that the heterodimer showed hand-over-hand movement. Unexpectedly, they spent most of the time in the two-head-bound state where wild-type head is in the front and the mutant head is in the rear, indicating that the displacement of rear wild-type head to the forward binding site is not the rate-limiting. These results suggest that the neck linker docking in the microtubule-bound head is not essential for the tethered head to translate and bind to the forward tubulin-binding site and rather is required for promoting ATP-hydrolysis and subsequent detachment of the trailing head from microtubule.

Unique Conformation of Kinesin-1′s Neck Linker in the Nucleotide-free State

Kinesin-1 is a motor protein that moves along microtubule in a hand-over-hand manner. The neck linker-docking model has been proposed to explain processive motility but has been questioned because its free energy change is too small to account for the force production. An alternative model proposes that the tethered head freely diffuses and is captured preferentially at the forward binding site, although the structural basis is not clear. To explain this mechanism, we recently proposed that the neck linker has to adapt a backward-pointing conformation to promote ADP release (Mori et al. Nature 2007). Previous cryo-electron microscopy (cryo-EM) studies (Rice et al. Nature 1999) showed distinct densities from the gold cluster attached to the distal end of the neck linker in the nucleotide-free state, but the direction of the neck linker extension was uncertain. To identify the conformation of the neck linker in the nucleotide-free state, we attached gold cluster to the middle of the neck linker and observed the gold-labeled kinesin motor heads on the microtubule using cryo-EM at <15 A resolution. The gold-density showed ellipsoidal shape extended along the protofilament and these densities were located rearward to the beginning of the neck linker. This density distribution indicates that the mobility of the neck linker is restricted toward the minus-end of the microtubule presumably due to steric constraints, which is consistent with the recently solved nucleotide-free kinesin crystal structure (Makino et al. this meeting). This conformational preference of the neck linker after ADP release provides structural basis for the preferential binding of the tethered head: ADP release and tight microtubule-binding is prohibited at the trailing position because the neck linker is pulled forward and is permitted only at the leading position.

3P-117 アクチン変異体がミオシン5の運動性に与える影響の解析(分子モーター,第47回日本生物物理学会年会)

3P-117 アクチン変異体がミオシン5の運動性に与える影響の解析(分子モーター,第47回日本生物物理学会年会)

Antagonistic Effects of Doublecortin and MARK2/Par-1 in the Developing Cerebral Cortex

Abnormal neuronal migration is manifested in brain malformations such as lissencephaly. The impairment in coordinated cell motility likely reflects a faulty mechanism of cell polarization or coupling between polarization and movement. Here we report on the relationship between the polarity kinase MARK2/Par-1 and its substrate, the well-known lissencephaly-associated gene doublecortin (DCX), during cortical radial migration. We have previously shown using in utero electroporation that reduced MARK2 levels resulted in multipolar neurons stalled at the intermediate zone border, similar to the phenotype observed in the case of DCX silencing. However, whereas reduced MARK2 stabilized microtubules, we show here that knock-down of DCX increased microtubule dynamics. This led to the hypothesis that simultaneous reduction may alleviate the phenotype. Coreduction of MARK2 and DCX resulted in a partial restoration of the normal neuronal migration phenotype in vivo. The kinetic behavior of the centrosomes reflected the different molecular mechanisms activated when either protein was reduced. In the case of reducing MARK2 processive motility of the centrosome was hindered, whereas when DCX was reduced, centrosomes moved quickly but bidirectionally. Our results stress the necessity for successful coupling between the polarity pathway and cytoplasmic dynein-dependent activities for proper neuronal migration.

1P-153 ネックリンカーが正常にドッキングできないヘテロニ量体キネシンの連続歩行運動(分子モーター(1),第46回日本生物物理学会年会)

1P-153 ネックリンカーが正常にドッキングできないヘテロニ量体キネシンの連続歩行運動(分子モーター(1),第46回日本生物物理学会年会)

Myosin VI Walks “Wiggly” on Actin with Large and Variable Tilting

Myosin VI is an unconventional motor protein with unusual motility properties such as its direction of motion and path on actin and a large stride relative to its short lever arms. To understand these features, the rotational dynamics of the lever arm were studied by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy during processive motility of myosin VI along actin. The axial angle is distributed in two peaks, consistent with the hand-over-hand model. The changes in lever arm angles during discrete steps suggest that it exhibits large and variable tilting in the plane of actin and to the sides. These motions imply that, in addition to the previously suggested flexible tail domain, there is a compliant region between the motor domain and lever arm that allows myosin VI to accommodate the helical position of binding sites while taking variable step sizes along the actin filament.

How kinesin waits between steps

Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules by hydrolysing ATP and moving processively in 8-nm steps. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains ('heads'), a process driven by a conformational change in the neck-linker domain of kinesin. However, the 'waiting conformation' of kinesin between steps remains controversial-some models propose that kinesin adopts a one-head-bound intermediate, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.

Force-Induced Bidirectional Stepping of Cytoplasmic Dynein

Cytoplasmic dynein is a minus-end-directed microtubule motor whose mechanism of movement remains poorly understood. Here, we use optical tweezers to examine the force-dependent stepping behavior of yeast cytoplasmic dynein. We find that dynein primarily advances in 8 nm increments but takes other sized steps (4-24 nm) as well. An opposing force induces more frequent backward stepping by dynein, and the motor walks backward toward the microtubule plus end at loads above its stall force of 7 pN. Remarkably, in the absence of ATP, dynein steps processively along microtubules under an external load, with less force required for minus-end- than for plus-end-directed movement. This nucleotide-independent walking reveals that force alone can drive repetitive microtubule detachment-attachment cycles of dynein's motor domains. These results suggest a model for how dynein's two motor domains coordinate their activities during normal processive motility and provide new clues for understanding dynein-based motility in living cells.

Myo4p is a monomeric myosin with motility uniquely adapted to transport mRNA

The yeast Saccharomyces cerevisiae uses two class V myosins to transport cellular material into the bud: Myo2p moves secretory vesicles and organelles, whereas Myo4p transports mRNA. To understand how Myo2p and Myo4p are adapted to transport physically distinct cargos, we characterize Myo2p and Myo4p in yeast extracts, purify active Myo2p and Myo4p from yeast lysates, and analyze their motility. We find several striking differences between Myo2p and Myo4p. First, Myo2p forms a dimer, whereas Myo4p is a monomer. Second, Myo4p generates higher actin filament velocity at lower motor density. Third, single molecules of Myo2p are weakly processive, whereas individual Myo4p motors are nonprocessive. Finally, Myo4p self-assembles into multi-motor complexes capable of processive motility. We show that the unique motility of Myo4p is not due to its motor domain and that the motor domain of Myo2p can transport ASH1 mRNA in vivo. Our results suggest that the oligomeric state of Myo4p is important for its motility and ability to transport mRNA.

Analysis of the weak interactions of ADP‐Unc104 and ADP‐kinesin with microtubules and their inhibition by MAP2c

Microtubule based motors like conventional kinesin (Kinesin-1) and Unc104 (Kinesin-3), and classical microtubule associated proteins (MAPs), including MAP2, are intimately involved in neurite formation and organelle transport. The processive motility of both these kinesins involves weak microtubule interactions in the ADP-bound states. Using cosedimentation assays, we have investigated these weak interactions and characterized their inhibition by MAP2c. We show that Unc104 binds microtubules with five-fold weaker affinity and two-fold higher stoichiometry compared with conventional kinesin. Unc104 and conventional kinesin binding affinities are primarily dependent on positively charged residues in the Unc104 K-loop and conventional kinesin neck coiled-coil and removal of these residues affects Unc104 and conventional kinesin differently. We observed that MAP2c acts primarily as a competitive inhibitor of Unc104 but a mixed inhibitor of conventional kinesin. Our data suggest a specific model in which MAP2c differentially interferes with each kinesin motor by inhibiting its weak attachment to the tubulin C-termini. This is reminiscent of the defects we have observed in Unc104 and kinesin mutants in which the positively charged residues in K-loop and neck coiled-coil domains were removed.

Allosteric inhibition of kinesin-5 modulates its processive directional motility

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division, represent alternatives to antimitotic agents that target tubulin. While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol, act through poorly understood allosteric mechanisms, not competing with ATP binding. Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays. We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

Processive bidirectional motion of dynein–dynactin complexes in vitro

Cytoplasmic dynein is the primary molecular motor responsible for transport of vesicles, organelles, proteins and RNA cargoes from the periphery of the cell towards the nucleus along the microtubule cytoskeleton of eukaryotic cells. Dynactin, a large multi-subunit activator of dynein, docks cargo to the motor and may enhance dynein processivity. Here, we show that individual fluorescently labelled dynein-dynactin complexes exhibit bidirectional and processive motility towards both the plus and minus ends of microtubules. The dependence of this activity on substrate ATP concentration, nucleotide analogues and inhibitors suggests that bidirectional motility is an active energy-transduction property of dynein-dynactin motor mechano-chemistry. The unique motility characteristics observed may reflect the flexibility of the dynein structure that leads to an enhanced ability to navigate around obstacles in the cell.

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.

RETRACTED: The Two Motor Domains of KIF3A/B Coordinate for Processive Motility and Move at Different Speeds

KIF3A/B, a kinesin involved in intraflagellar transport and Golgi trafficking, is distinctive because it contains two nonidentical motor domains. Our hypothesis is that the two heads have distinct functional properties, which are tuned to maximize the performance of the wild-type heterodimer. To test this, we investigated the motility of wild-type KIF3A/B heterodimer and chimaeric KIF3A/A and KIF3B/B homodimers made by splicing the head of one subunit to the rod and tail of the other. The first result is that KIF3A/B is processive, consistent with its transport function in cells. Secondly, the KIF3B/B homodimer moves at twice the speed of the wild-type motor but has reduced processivity, suggesting a trade-off between speed and processivity. Third, the KIF3A/A homodimer moves fivefold slower than wild-type, demonstrating distinct functional differences between the two heads. The heterodimer speed cannot be accounted for by a sequential head model in which the two heads alternate along the microtubule with identical speeds as in the homodimers. Instead, the data are consistent with a coordinated head model in which detachment of the slow KIF3A head from the microtubule is accelerated roughly threefold by the KIF3B head.

Microtubule−Kinesin Interface Mutants Reveal a Site Critical for Communication

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.