Processive Stepping Research Articles

Nanometer-resolution tracking of single cargo reveals dynein motor mechanisms.

Cytoplasmic dynein is essential for intracellular transport. Despite extensive in vitro characterizations, how the dynein motors transport vesicles by processive steps in live cells remains unclear. To dissect the molecular mechanisms of dynein, we develop optical probes that enable long-term single-particle tracking in live cells with high spatiotemporal resolution. We find that the number of active dynein motors transporting cargo switches stochastically between one and five dynein motors during long-range transport in neuronal axons. Our very bright optical probes allow the observation of individual molecular steps. Strikingly, these measurements reveal that the dwell times between steps are controlled by two temperature-dependent rate constants in which two ATP molecules are hydrolyzed sequentially during each dynein step. Thus, our observations uncover a previously unknown chemomechanical cycle of dynein-mediated cargo transport in living cells.

Molecular mechanism of interaction between kinesin motors affecting their residence times on microtubule lattice and end

Kinesin superfamily can be classified into 14 subfamilies. Some families of kinesin motors such as kinesin-1 are responsible for long-distance intracellular transports and thus the motors are required to reside on the microtubule (MT) lattice for a longer time than at the end. Some families such as kinesin-8 Kip3 and kinesin-5 Eg5 are responsible for the regulation of MT length by depolymerizing or polymerizing the MT from the plus end and thus the motors are required to reside at the MT end for a long time. Under the crowded condition of the motors, it was found experimentally that the residence times of the kinesin-8 Kip3 and kinesin-5 Eg5 at the MT end are reduced greatly compared to the single-motor case. However, the underlying mechanism of different families of kinesin motors having different MT-end residence times is unknown. The molecular mechanism by which the interaction between the two motors greatly reduces the residence time of the motor at the MT end is elusive. In addition, during the processive stepping on the MT lattice, when two kinesin motors meet it is unknown how the interaction between them affects their dissociation rates. To address the above unclear issues, here we make a consistent and theoretical study of the residence times of the kinesin-1, kinesin-8 Kip3 and kinesin-5 Eg5 motors on the MT lattice and at the end under both the single-motor condition and multiple-motors or crowded condition.

Effect of the Neck Linker on Processive Stepping of Kinesin Motor

Kinesin motor protein, which is composed of two catalytic domains connected together by a long coiled-coil stalk via two flexible neck linkers (NLs), can step processively on a microtubule towards the plus end by hydrolyzing adenosine triphosphate (ATP) molecules. To understand what the role is that the NL plays in the processive stepping, the dynamics of the kinesin motor are studied theoretically here by considering the mutation or deletion of an N-terminal cover strand that contributes to the docking of the NL in kinesin-1, the extension of the NL in kinesin-1, the mutation of the NL in kinesin-1, the swapping of the NL of kinesin-2 with that of kinesin-1, the joining of the stalk and neck of Ncd that moves towards the minus end of MT to the catalytic domain of kinesin-1, the replacement of catalytic domain of kinesin-1 with that of Ncd, and so on. The theoretical results give a consistent and quantitative explanation of various available experimental results about the effects of these mutations on motor dynamics and, moreover, provide predicted results. Additionally, the processive motility of kinesin-6 MKLP2 without NL docking is also explained. The available experimental data about the effect of NL mutations on the dynamics of the bi-directional kinesin-5 Cin8 are also explained. The studies are critically implicative to the mechanism of the stepping of the kinesin motor.

Effect of varying load in moving period of a step on dynamics of molecular motors.

During the processive stepping of a molecular motor on its polar track, a step consists of a long dwell period and a very short moving period. In single molecule optical trapping experiments to determine the load dependence of the motor dynamics, although the motor experiences a constant load during the dwell period, it experiences a varying load during the moving period. However, in previous theoretical studies to explain the single molecule optical trapping data, it was simply assumed that the motor experiences a constant load during both the dwell period and the following moving period. Thus, an important but unclear issue is whether the assumption is appropriate in the theoretical studies. Here, we take kinesin and myosin-V as examples to study theoretically the motor dynamics with the consideration of the varying load during the moving period and compare with that with the assumption of the constant load. The studies show that in the optical trapping experiments employed in the literature, for the kinesin with a small step size of about 8 nm it is a good approximation to make the theoretical studies by assuming that the motor experiences the constant load during the moving period. For the myosin-V with a large step size of about 36 nm, there are small but noticeable deviations of the results obtained by considering that the motor experiences the varying load during the moving period from those by assuming that the motor experiences the constant load. .

Molecular Mechanism of Processive Stepping of Kinesin Motors

Kinesin-1 is a motor protein that can step processively on microtubule by hydrolyzing ATP molecules, playing an essential role in intracellular transports. To better understand the mechanochemical coupling of the motor stepping cycle, numerous structural, biochemical, single molecule, theoretical modeling and numerical simulation studies have been undertaken for the kinesin-1 motor. Recently, a novel ultraresolution optical trapping method was employed to study the mechanics of the kinesin-1 motor and new results were supplemented to its stepping dynamics. In this commentary, the new single molecule results are explained well theoretically with one of the models presented in the literature for the mechanochemical coupling of the kinesin-1 motor. With the model, various prior experimental results for dynamics of different families of N-terminal kinesin motors have also been explained quantitatively.

Processive dynamics of the usher assembly platform during uropathogenic Escherichia coli P pilus biogenesis

Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine.

KIF3A accelerates KIF3C within the kinesin-2 heterodimer to generate symmetrical phosphate release rates for each processive step

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in the central nervous system and associated with vesicles in neurons. KIF3AC is an intriguing member of the kinesin-2 family because the intrinsic kinetics of KIF3A and KIF3C when expressed as homodimers and analyzed in vitro are distinctively different from each other. For example, the single-molecule velocities of the engineered homodimers KIF3AA and KIF3CC are 293 and 7.5 nm/s, respectively, whereas KIF3AC has a velocity of 186 nm/s. These results led us to hypothesize that heterodimerization alters the intrinsic catalytic properties of the two heads, and an earlier computational analysis predicted that processive steps would alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping. To test this hypothesis directly, we measured the presteady-state kinetics of phosphate release for KIF3AC, KIF3AA, and KIF3CC followed by computational modeling of the KIF3AC phosphate release transients. The results reveal that KIF3A and KIF3C retain their intrinsic ATP-binding and hydrolysis kinetics. Yet within KIF3AC, KIF3A activates the rate of phosphate release for KIF3C such that the coupled steps of phosphate release and dissociation from the microtubule become more similar for KIF3A and KIF3C. These coupled steps are the rate-limiting transition for the ATPase cycle suggesting that within KIF3AC, the stepping kinetics are similar for each head during the processive run. Future work will be directed to define how these properties enable KIF3AC to achieve its physiological functions.

Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis.

The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop-substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.

Dynactin p150 Promotes Processive Motility of DDB Complexes by Minimizing Diffusional Behavior of Dynein

Abstract Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used Interferometric Scattering (iSCAT) microscopy to track dynein-dynactin-BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB - kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity, in quantitative agreement with the increase in diffusivity seen in isolated DDB. The data suggest a DDB activation model in which engagement of dynactin p150 with the microtubule promotes dynein processivity, serves as an allosteric activator of dynein, and enhances processive minus-end motility during intracellular bidirectional transport. TOC Highlight Dynein-dynactin-BicD2 (DDB) is highly processive, but also shows transient pausing and diffusion, which we analyzed using iSCAT microscopy. Blocking dynactin p150 results in more diffusion of isolated DDB and a plus-end shift of kinesin-1 – DDB complexes. Thus, we conclude that p150 is an allosteric activator of dynein in the DDB complex.

Resolving kinesin stepping: one head at a time

Kinesins are well known to power diverse long-range transport processes in virtually all eukaryotic cells. The ATP-dependent processive stepping as well as the regulation of kinesin' activity have, thus, been the focus of extensive studies over the past decades. It is widely accepted that kinesin motors can self-regulate their activity by suppressing the catalytic activity of the "heads." The distal random coil at the C terminus, termed "tail domain," is proposed to mediate this autoinhibition; however, a direct regulatory influence of the tail on the processive stepping of kinesin proved difficult to capture. Here, we simultaneously tracked the two distinct head domains in the kinesin-2 motor using dual-color super resolution microscopy (dcFIONA) and reveal for the first time their individual properties during processive stepping. We show that the autoinhibitory wild-type conformation selectively impacts one head in the heterodimer but not the other. Our results provide insights into the regulated kinesin stepping that had escaped experimental scrutiny so far.

Dynamical mechanism of stepping of the molecular motor myosin V along actin filament and simulation in an actual system

On the base of the random motion of myosin, the structural asymmetry of the actin filament, and the interaction between the motor and the track, the present study proposes a physical mechanism for the processive stepping of the two-headed molecular motor myosin V along the actin filament. The detachment from actin filament and forward swing of the trailing head, which are caused by increased entropy and the release of the bending energy in the neck region, are the initial steps of the stepping process. The detached head then rotates and randomly moves around the still attached head. A step is completed after the detached head detects the next binding site. In the mechanism, the direction of myosin V movement was determined on the basis of the configuration of the actomyosin system. The role of ATP is the energy provider for the local random motion. The applicability of the dynamic mechanism was evaluated on a simulation system that preserved most of the characteristics of the actomyosin system.

Mg2+ -free ATP regulates the processivity of native cytoplasmic dynein.

Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 μm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.

Kinesin-2 heterodimerization alters entry into a processive run along the microtubule but not stepping within the run

Heterodimeric KIF3AC and KIF3AB, two members of the mammalian kinesin-2 family, generate force for microtubule plus end-directed cargo transport. However, the advantage of heterodimeric kinesins over homodimeric ones is not well-understood. We showed previously that microtubule association for entry into a processive run was similar in rate for KIF3AC and KIF3AB at ∼7.0 μm-1 s-1 Yet, for engineered homodimers of KIF3AA and KIF3BB, this constant is significantly faster at 11 μm-1 s-1 and much slower for KIF3CC at 2.1 μm-1 s-1 These results led us to hypothesize that heterodimerization of KIF3A with KIF3C and KIF3A with KIF3B altered the intrinsic catalytic properties of each motor domain. Here, we tested this hypothesis by using presteady-state stopped-flow kinetics and mathematical modeling. Surprisingly, the modeling revealed that the catalytic properties of KIF3A and KIF3B/C were altered upon microtubule binding, yet each motor domain retained its relative intrinsic kinetics for ADP release and subsequent ATP binding and the nucleotide-promoted transitions thereafter. These results are consistent with the interpretation that for KIF3AB, each motor head is catalytically similar and therefore each step is approximately equivalent. In contrast, for KIF3AC the results predict that the processive steps will alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping during a processive run. This study reveals the impact of heterodimerization of the motor polypeptides for microtubule association to start the processive run and the fundamental differences in the motile properties of KIF3C compared with KIF3A and KIF3B.

Combined Poltirf and Sub-Pixel Particle Tracking of Cytoplasmic Dynein Supports a Winch-Like Stepping Mechanism

Cytoplasmic dynein is a molecular motor that carries cargos toward the minus end of microtubules. Dynein walks processively, but less regularly along microtubules than kinesin and is capable of taking both backwards and sideways steps. Structural dynamics of dynein's AAA+ ring domain during processive stepping are not well understood. We use a combination of polarized TIRF and sub-pixel particle tracking to measure the position and orientation of fluorescent nanorods rigidly attached to AAA5 and AAA6 of the individual dynein ring domains via biotin-NeutrAvidin linkage. We observe rotational changes of the ring and how they correlate with translocation steps. The dynein ring undergoes frequent, small rotations, typically less than 20 degrees, about twice as often as steps. Stepping and tilting both depend on ATP, although some ATP-independent rotations are observed. Rotations which accompany translocation steps have larger magnitudes than ones that are not correlated with steps. Not all steps are correlated with angle changes, either. A heterodimeric mutant construct, with one biotinylated ring carrying a quantum rod and one non-biotinylated dead-head ring walks slowly, indicating that the rod does not destroy motility. Our results are inconsistent with a purely powerstroke stepping mechanism, analogous to that of myosin which would predict larger angle changes tightly coupled to stepping, but instead they support a winch-like mechanism that involves bending of the coiled-coil stalk under intermolecular torque between the heads in the double-headed bound state. Supported by NIH Grant P015GM087253.

Local heat activation of single myosins based on optical trapping of gold nanoparticles.

Myosin is a mechano-enzyme that hydrolyzes ATP in order to move unidirectionally along actin filaments. Here we show by single molecule imaging that myosin V motion can be activated by local heat. We constructed a dark-field microscopy that included optical tweezers to monitor 80 nm gold nanoparticles (GNP) bound to single myosin V molecules with nanometer and submillisecond accuracy. We observed 34 nm processive steps along actin filaments like those seen when using 200 nm polystyrene beads (PB) but dwell times (ATPase activity) that were 4.5 times faster. Further, by using DNA nanotechnology (DNA origami) and myosin V as a nanometric thermometer, the temperature gradient surrounding optically trapped GNP could be estimated with nanometer accuracy. We propose our single molecule measurement system should advance quantitative analysis of the thermal control of biological and artificial systems like nanoscale thermal ratchet motors.

The Winch Model Can Explain both Coordinated and Uncoordinated Stepping of Cytoplasmic Dynein

Cytoplasmic dynein moves processively along microtubules, but the mechanism of how its heads use the energy from ATP hydrolysis, coupled to a linker swing, to achieve directed motion, is still unclear. In this article, we present a theoretical model based on the winch mechanism in which the principal direction of the linker stroke is toward the microtubule-binding domain. When mechanically coupling two identical heads (each with postulated elastic properties and a minimal ATPase cycle), the model reproduces stepping with 8-nm steps (even though the motor itself is much larger), interhead coordination, and processivity, as reported for mammalian dyneins. Furthermore, when we loosen the elastic connection between the heads, the model still shows processive directional stepping, but it becomes uncoordinated and the stepping pattern shows a greater variability, which reproduces the properties of yeast dyneins. Their slower chemical kinetics allows processive motility and a high stall force without the need for coordination.

Coupling of two non-processive myosin 5c dimers enables processive stepping along actin filaments.

Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30–36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells.

Mechanistic Insights of Dynein Motor Action from Electron Microscopy Studies

Dynein motors are the largest of the cytoskeletal motor proteins. They perform critical functions in eukaryotic cells, carrying cargoes along microtubule tracks and exerting pulling forces on microtubules in a variety of cellular processes. Dynein motors use ATP hydrolysis to perform these functions by moving towards the minus ends of microtubules. A major goal of our research is to understand the structure and conformational changes that allow dynein, a member of the AAA+ superfamily of ring-shaped ATPases, to perform these functions.We have used cryo-electron microscopy and single-particle image processing to determine (ab initio) three-dimensional structures of a native (full-length) flagellar dynein (dynein-c from the single-celled alga Chlamydomonas) and of an engineered cytoplasmic dynein motor domain (from the slime mold Dictyostelium) lacking the cargo-binding tail domain, in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the “linker” domain, involving a hinge near its middle.Analysis of a mutant in which the linker “undocks” from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. We find flexing of the tail domain of dynein-c and the stalks of both dynein isoforms, relative to the AAA+ ring in the frozen-hydrated state. Fitting the dynein-c structures into three-dimensional cryo-tomograms of Chlamydomonas flagella suggests how the mechanism of linker remodeling could drive sliding between microtubules. This also has implications for the processive stepping of cytoplasmic dynein dimers undergoing cargo transport and force exertion.This work was funded by the B.B.S.R.C. (UK), the Human Frontiers Science Programme Organization, The Wellcome Trust (UK) and MEXT (Japan).

Spontaneous Detachment of the Leading Head Contributes to Myosin VI Backward Steps

Myosin VI is an ATP driven molecular motor that normally takes forward and processive steps on actin filaments, but also on occasion stochastic backward steps. While a number of models have attempted to explain the backwards steps, none offer an acceptable mechanism for their existence. We therefore performed single molecule imaging of myosin VI and calculated the stepping rates of forward and backward steps at the single molecule level. The forward stepping rate was proportional to the ATP concentration, whereas the backward stepping rate was independent. Using these data, we proposed that spontaneous detachment of the leading head is uncoupled from ATP binding and is responsible for the backward steps of myosin VI.

A modified active Brownian dynamics model using asymmetric energy conversion and its application to the molecular motor system

We consider a modified energy depot model in the overdamped limit using an asymmetric energy conversion rate, which consists of linear and quadratic terms in an active particle's velocity. In order to analyze our model, we adopt a system of molecular motors on a microtubule and employ a flashing ratchet potential synchronized to a stochastic energy supply. By performing an active Brownian dynamics simulation, we investigate effects of the active force, thermal noise, external load, and energy-supply rate. Our model yields the stepping and stalling behaviors of the conventional molecular motor. The active force is found to facilitate the forwardly processive stepping motion, while the thermal noise reduces the stall force by enhancing relatively the backward stepping motion under external loads. The stall force in our model decreases as the energy-supply rate is decreased. Hence, assuming the Michaelis-Menten relation between the energy-supply rate and the an ATP concentration, our model describes ATP-dependent stall force in contrast to kinesin-1.