Kinesin Moves by an Asymmetric Hand-Over-Hand Mechanism
Steven Block2003, Science
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Abstract
Kinesin is a double-headed motor protein that moves along microtubules in 8-nanometer steps. Two broad classes of model have been invoked to explain kinesin movement: hand-over-hand and inchworm. In hand-over-hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, hand-over-hand mechanism.
Figures (4)
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Physical Review E, 2008
We study a discrete stochastic model of a molecular motor. This discrete model can be viewed as a minimal ratchet model. We extend our previous work on this model, by further investigating the constraints imposed by the fluctuation theorem on the operation of a molecular motor far from equilibrium. In this work, we show the connections between different formulations of the fluctuation theorem. One formulation concerns the generating function of the currents while another one concerns the corresponding large deviation function, which we have calculated exactly for this model. A third formulation concerns the ratio of the probability of observing a velocity v to the same probability of observing a velocity −v. Finally, we show that all the formulations of the fluctuation theorem can be understood from the notion of entropy production.
Nonequilibrium Fluctuations and Mechanochemical Couplings of a Molecular Motor
Physical Review Letters, 2007
We investigate theoretically the violations of Einstein and Onsager relations, and the efficiency for a single processive motor operating far from equilibrium using an extension of the two-state model introduced by Kafri et al. [Biophys. J. 86, 3373 (2004)]. With the aid of the Fluctuation Theorem, we analyze the general features of these violations and this efficiency and link them to mechanochemical couplings of motors. In particular, an analysis of the experimental data of kinesin using our framework leads to interesting predictions that may serve as a guide for future experiments. 87.16.Nn, 05.70.Ln Motor proteins are nano-machines that convert chemical energy into mechanical work and motion . Important examples include kinesin, myosin, and RNA polymerase. Despite a number of theoretical models , understanding the mechanochemical transduction mechanisms behind these motors remains a significant challenge . Recent advances in experimental techniques to probe the fluctuations of single motors provide ways to gain insight into their kinetic pathways [10]. However, a general description for fluctuations of systems driven out of equilibrium, and in particular of motors, is still lacking. Recently, the Fluctuation Theorem (FT) has emerged as a promising framework to characterize fluctuations in far-from-equilibrium regimes where Einstein and Onsager relations no longer hold . In a nutshell, FT states that the probability distribution for the entropy production rate obeys a symmetry relation, and it has been verified in a number of beautiful experiments on biopolymers and colloidal systems . In this Letter, we demonstrate that FT provides a natural framework in which thermodynamic constraints can be imposed on the operation of nanomachines far from equilibrium.
Optical trapping with superfocused high-M 2 laser diode beam
Laser Resonators, Microresonators, and Beam Control XVII, 2015
Many applications of high-power laser diodes demand tight focusing. This is often not possible due to the multimode nature of semiconductor laser radiation possessing beam propagation parameter M 2 values in double-digits. We propose a method of 'interference' superfocusing of high-M 2 diode laser beams with a technique developed for the generation of Bessel beams based on the employment of an axicon fabricated on the tip of a 100 μm diameter optical fiber with highprecision direct laser writing. Using axicons with apex angle 140 0 and rounded tip area as small as ~10 μm diameter, we demonstrate 2-4 μm diameter focused laser 'needle' beams with approximately 20 μm propagation length generated from multimode diode laser with beam propagation parameter M 2 =18 and emission wavelength of 960 nm. This is a few-fold reduction compared to the minimal focal spot size of ~11 μm that could be achieved if focused by an 'ideal' lens of unity numerical aperture. The same technique using a 160 0 axicon allowed us to demonstrate few-μm-wide laser 'needle' beams with nearly 100 μm propagation length with which to demonstrate optical trapping of 5-6 μm rat blood red cells in a water-heparin solution. Our results indicate the good potential of superfocused diode laser beams for applications relating to optical trapping and manipulation of microscopic objects including living biological objects with aspirations towards subsequent novel lab-on-chip configurations.
Optical tweezers study life under tension
Nature Photonics, 2011
Optical tweezers have become one of the primary weapons in the arsenal of biophysicists, and have revolutionized the new field of single-molecule biophysics. Today's techniques allow highresolution experiments on biological macromolecules that were mere pipe dreams only a decade ago.
Dynamics of the bacterial flagellar motor with multiple stators
Proceedings of the National Academy of Sciences, 2009
The bacterial flagellar motor drives the rotation of flagellar filaments and enables many species of bacteria to swim. Torque is generated by interaction of stator units, anchored to the peptidoglycan cell wall, with the rotor. Recent experiments [Yuan J, Berg HC (2008) Proc Natl Acad Sci USA 105:1182-1185] show that at near-zero load the speed of the motor is independent of the number of stators. Here, we introduce a mathematical model of the motor dynamics that explains this behavior based on a general assumption that the stepping rate of a stator depends on the torque exerted by the stator on the rotor. We find that the motor dynamics can be characterized by two timescales: the moving-time interval for the mechanical rotation of the rotor and the waitingtime interval determined by the chemical transitions of the stators. We show that these two timescales depend differently on the load, and that their cross-over provides the microscopic explanation for the existence of two regimes in the torque-speed curves observed experimentally. We also analyze the speed fluctuation for a single motor by using our model. We show that the motion is smoothed by having more stator units. However, the mechanism for such fluctuation reduction is different depending on the load. We predict that the speed fluctuation is determined by the number of steps per revolution only at low load and is controlled by external noise for high load. Our model can be generalized to study other molecular motor systems with multiple power-generating units.
Tuning multiple motor travel via single motor velocity
Traffic (Copenhagen, Denmark), 2012
Microtubule-based molecular motors often work in small groups to transport cargos in cells. A key question in understanding transport (and its regulation in vivo) is to identify the sensitivity of multiple-motor-based motion to various single molecule properties. Whereas both single-motor travel distance and microtubule binding rate have been demonstrated to contribute to cargo travel, the role of single-motor velocity is yet to be explored. Here, we recast a previous theoretical study, and make explicit a potential contribution of velocity to cargo travel. We test this possibility experimentally, and demonstrate a strong negative correlation between single-motor velocity and cargo travel for transport driven by two motors. Our study thus discovers a previously unappreciated role of single-motor velocity in regulating multiple-motor transport.
Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy
Nature Communications, 2015
Optical and magnetic tweezers are widely employed to probe the mechanics and activity of individual biomolecular complexes. They rely on micrometer-sized particles to detect molecular conformational changes from the particle position. Real-time particle tracking with Ångström accuracy has so far been only achieved using laser detection through photodiodes. Here we demonstrate that camera-based imaging can provide a similar performance for all three dimensions. Particle imaging at kHz rates is combined with real-time data processing being accelerated by a graphics processing unit. For particles that are fixed in the sample cell we can detect 3 Å sized steps that are introduced by cell translations at rates of 10 Hz, while for DNAtethered particles 5 Å steps at 1 Hz can be resolved. Moreover, 20 particles can be tracked in parallel with comparable accuracy. Our approach provides a simple and robust way for highresolution tweezers experiments using multiple particles at a time.
3D laser nano-printing on fibre paves the way for super-focusing of multimode laser radiation
Scientific reports, 2018
Multimode high-power laser diodes suffer from inefficient beam focusing, leading to a focal spot 10-100 times greater than the diffraction limit. This inevitably restricts their wider use in 'direct-diode' applications in materials processing and biomedical photonics. We report here a 'super-focusing' characteristic for laser diodes, where the exploitation of self-interference of modes enables a significant reduction of the focal spot size. This is achieved by employing a conical microlens fabricated on the tip of a multimode optical fibre using 3D laser nano-printing (also known as multi-photon lithography). When refracted by the conical surface, the modes of the fibre-coupled laser beam self-interfere and form an elongated narrow focus, usually referred to as a 'needle' beam. The multiphoton lithography technique allows the realisation of almost any optical element on a fibre tip, thus providing the most suitable interface for free-space applications of mul...
A modified active Brownian dynamics model using asymmetric energy conversion and its application to the molecular motor system
Journal of Biological Physics, 2013
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 ATP concentration, our model describes an ATP-dependent stall force in contrast to kinesin-1.
The mechanical properties of kinesin-1: a holistic approach
Biochemical Society Transactions, 2012
During the last 25 years, a vast amount of research has gone into understanding the mechanochemical cycle of kinesin-1 and similar processive motor proteins. An experimental method that has been widely used to this effect is the in vitro study of kinesin-1 molecules moving along microtubules while pulling a bead, the position of which is monitored optically while trapped in a laser focus. Analysing results from such experiments, in which thermally excited water molecules are violently buffeting the system components, can be quite difficult. At low loads, the effect of the mechanical properties of the entire molecule must be taken into account, as stalk compliance means the bead position recorded is only weakly coupled to the movement of the motor domains, the sites of ATP hydrolysis and microtubule binding. In the present review, findings on the mechanical and functional properties of the various domains of full-length kinesin-1 molecules are summarized and a computer model is prese...
An introduction to ratchets in chemistry and biology
Mater. Horiz.
This article describes the functions and mechanisms of particle and electron ratchets, and the interplay between theory and experiment in this field of non-equilibrium transport.
The new unified theory of ATP synthesis/hydrolysis and muscle contraction, its manifold fundamental consequences and mechanistic implications and its applications in health and disease
International journal of molecular sciences, 2008
Complete details of the thermodynamics and molecular mechanisms of ATP synthesis/hydrolysis and muscle contraction are offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT) energy storage mechanism of muscle contraction. The manifold fundamental consequences and mechanistic implications of the unified theory for oxidative phosphorylation and muscle contraction are explained. The consistency of current mechanisms of ATP synthesis and muscle contraction with experiment is assessed, and the novel insights of the unified theory are shown to take us beyond the binding change mechanism, the chemiosmotic theory and the lever arm model. It is shown from first principles how previous theories of ATP synthesis and muscle contraction violate both the first and second laws of thermodynamics, necessitating their revision. It is concluded that the new paradigm, ten years after making its first appearance, is now perfe...
The inchworm episode: Reconstituting the phenomenon of kinesin motility
European journal for philosophy of science, 2021
New Mechanist philosophical models of "phenomenon reconstitution" understand the process to be driven by explanatory considerations. Here I discuss an episode of phenomenon reconstitution that occurred entirely within an experimental program dedicated to characterizing (rather than explaining) the phenomenon of kinesin motility. Rather than being driven by explanatory considerations, as standard mechanist views maintain, I argue that the phenomenon of kinesin motility was reconstituted to enhance researchers’ primary experimental tool—the single molecule motility assay.
How Two-Foot Molecular Motors May Walk
Advances in Experimental Medicine and Biology, 2005
Optical tweezers in single-molecule experiments
The European Physical Journal Plus
In the last decades, optical tweezers have progressively emerged as a unique tool to investigate the biophysical world, allowing to manipulate and control forces and movements of one molecule at a time with unprecedented resolution. In this review, we present the use of optical tweezers to perform single-molecule force spectroscopy investigations from an experimental perspective. After a comparison with other single-molecule force spectroscopy techniques, we illustrate at an introductory level the physical principles underlying optical trapping and the main experimental configurations employed nowadays in single-molecule experiments. We conclude with a brief summary of some remarkable results achieved with this approach in different biological systems, with the aim to highlight the great variety of experimental possibilities offered by optical tweezers to scientists interested in this research field.
Rotation-translation coupling of a double-headed Brownian motor in a traveling-wave potential
2021
1 Science Education Department, Beijing Institute of Graphic Communication, Beijing 102600, China College of Science, Hebei University of Architecture, Zhangjiakou 075000, China School of science, Tianjin University, Tianjin 300072, China 4 School of science, Hebei University of Technology, Tianjin 300401, China Institute of Systems Science and College of Information Science and Engineering, Huaqiao University, Xiamen 361021, China Corresponding author. E-mail: † [email protected]
Identification of tail binding effect of kinesin-1 using an elastic network model
Biomechanics and modeling in mechanobiology, 2015
Kinesin is a motor protein that delivers cargo inside a cell. Kinesin has many different families, but they perform basically same function and have same motions. The walking motion of kinesin enables the cargo delivery inside the cell. Autoinhibition of kinesin is important because it explains how function of kinesin inside a cell is stopped. Former researches showed that tail binding is related to autoinhibition of kinesin. In this work, we performed normal mode analysis with elastic network model using different conformation of kinesin to determine the effect of tail binding by considering four models such as functional form, autoinhibited form, autoinhibited form without tail, and autoinhibited form with carbon structure. Our calculation of the thermal fluctuation and cross-correlation shows the change of tail-binding region in structural motion. Also strain energy of kinesin showed that elimination of tail binding effect leads the structure to have energetically similar behavio...
High-Speed Single-Molecule Tracking of CXCL13 in the B-Follicle
Frontiers in immunology, 2018
Soluble factors are an essential means of communication between cells and their environment. However, many molecules readily interact with extracellular matrix components, giving rise to multiple modes of diffusion. The molecular quantification of diffusion is thus a challenging imaging frontier, requiring very high spatial and temporal resolution. Overcoming this methodological barrier is key to understanding the precise spatial patterning of the extracellular factors that regulate immune function. To address this, we have developed a high-speed light microscopy system capable of millisecond sampling in tissue samples and submillisecond sampling in controlled samples to characterize molecular diffusion in a range of complex microenvironments. We demonstrate that this method outperforms competing tools for determining molecular mobility of fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) for evaluation of diffusion. We then apply this...
Inchworm stepping of Myc-Max heterodimer protein diffusion along DNA
2020
Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes via leucine zipper with its associated partner protein Max to form a heterodimer structure, which then binds target DNA sequences to regulate gene transcription. The regulation depends on by Myc-Max binding to DNA and searching for target sequences via diffusional motions along DNA. Here, we conduct structure-based molecular dynamics (MD) simulations to investigate the diffusion dynamics of the Myc-Max heterodimer along DNA. We found that the heterodimer protein slides on the DNA in a rotation-uncoupled manner in coarse-grained simulations, as its two helical DNA binding basic regions (BRs) alternate between open and closed conformations via inchworm stepping motions. In such motions, the two BRs of the heterodimer step across the DNA strand one by one, with step sizes up about half of a DNA helical pitch length. Atomic MD simulations of the Myc-Max heterodimer in complex with DNA have ...
Reaction-path statistical mechanics of enzymatic kinetics
The Journal of chemical physics, 2022
We introduce a reaction-path statistical mechanics formalism based on the principle of large deviations to quantify the kinetics of single-molecule enzymatic reaction processes under the Michaelis-Menten mechanism, which exemplifies an out-of-equilibrium process in the living system. Our theoretical approach begins with the principle of equal a priori probabilities and defines the reaction path entropy to construct a new nonequilibrium ensemble as a collection of possible chemical reaction paths. As a result, we evaluate a variety of path-based partition functions and free energies by using the formalism of statistical mechanics. They allow us to calculate the timescales of a given enzymatic reaction, even in the absence of an explicit boundary condition that is necessary for the equilibrium ensemble. We also consider the large deviation theory under a closed-boundary condition of the fixed observation time to quantify the enzyme-substrate unbinding rates. The result demonstrates th...
A torque component in the kinesin-1 power stroke
Nature Chemical Biology, 2005
Kinesin-1 is a twin-headed molecular motor that moves along microtubules in 8-nm steps, using a walking action in which the two heads interact alternately with the microtubule 1-4. Constructs with only one head can also produce impulses of force and motion 5-7 , indicating that the walking action is an amplification strategy that leverages an underlying forcegenerating event. Recent work suggests that directional force is produced either by directionally biased selection of microtubule binding sites 8,9 or by a conformational change subsequent to the binding event 10-12. We report here that surface-attached rat kinesin-1 monomers drive counterclockwise rotation of sliding microtubules around their axes, and that by manipulating the assay geometry, we could reduce or block the torsional motion with negligible effects on the axial motion. We can account for this behavior on the simple assumption that kinesin heads tend to bind to the closest available tubulin heterodimer in the lattice, but only in the case where an additional biasing process is present that shifts the start position for diffusion-to-capture toward the microtubule plus end by B1 nm.
Kinesin's cover-neck bundle folds forward to generate force
Proceedings of the National Academy of Sciences, 2008
Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a β-sheet with the neck linker to yield the cover-neck bundle . We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the N-terminal “dangling end,” unlike the neck linker and nucleotide-binding pocket, is not involved with any ...
Alternating Site ATPase Pathway of Rat Conventional Kinesin
Journal of Biological Chemistry, 2005
The pathway of ATP hydrolysis by rat kinesin was established by pre-steady-state kinetic methods. A 406-residue long N-terminal fragment was shown by sedimentation equilibrium analysis to form a dimer with a K d of 46 nM. The pathway of ATP hydrolysis follows the Gilbert-Johnson pathway determined previously for a similarsized N-terminal fragment of Drosophila conventional kinesin. However, the rates of ADP release were at least 3-fold faster, and ATP hydrolysis was ϳ5-fold faster. Paralleling our previous mechanistic data, these results support an alternating site ATPase pathway, including a captive head state as an intermediate in the kinesin ATPase cycle. The kinetic data presented in this report once again point to the importance of the captive head state and argue against a pathway that short-circuits this key intermediate. In addition, several unique aspects of the rat kinesin kinetics reveal new aspects of the ATPase-coupling mechanism. These studies provide a baseline set of kinetic parameters against which future studies of rat kinesin mutants may be evaluated and directly correlated with the structure of the dimeric kinesin.
Biophysical Tools to Study Cellular Mechanotransduction
Bioengineering, 2017
The cell membrane is the interface that volumetrically isolates cellular components from the cell's environment. Proteins embedded within and on the membrane have varied biological functions: reception of external biochemical signals, as membrane channels, amplification and regulation of chemical signals through secondary messenger molecules, controlled exocytosis, endocytosis, phagocytosis, organized recruitment and sequestration of cytosolic complex proteins, cell division processes, organization of the cytoskeleton and more. The membrane's bioelectrical role is enabled by the physiologically controlled release and accumulation of electrochemical potential modulating molecules across the membrane through specialized ion channels (e.g., Na + , Ca 2+ , K + channels). The membrane's biomechanical functions include sensing external forces and/or the rigidity of the external environment through force transmission, specific conformational changes and/or signaling through mechanoreceptors (e.g., platelet endothelial cell adhesion molecule (PECAM), vascular endothelial (VE)-cadherin, epithelial (E)-cadherin, integrin) embedded in the membrane. Certain mechanical stimulations through specific receptor complexes induce electrical and/or chemical impulses in cells and propagate across cells and tissues. These biomechanical sensory and biochemical responses have profound implications in normal physiology and disease. Here, we discuss the tools that facilitate the understanding of mechanosensitive adhesion receptors. This article is structured to provide a broad biochemical and mechanobiology background to introduce a freshman mechano-biologist to the field of mechanotransduction, with deeper study enabled by many of the references cited herein.
An automatedin vitromotility assay for high-throughput studies of molecular motors
Lab on a Chip, 2018
Molecular motors, essential to force-generation and cargo transport within cells, are invaluable tools for powering nanobiotechnological lab-on-a-chip devices.
Large deviation theory for the kinetics and energetics of turnover of enzyme catalysis in a chemiostatic flow
The Journal of chemical physics, 2018
In the framework of large deviation theory, we have characterized nonequilibrium turnover statistics of enzyme catalysis in a chemiostatic flow with externally controllable parameters, like substrate injection rate and mechanical force. In the kinetics of the process, we have shown the fluctuation theorems in terms of the symmetry of the scaled cumulant generating function (SCGF) in the transient and steady state regime and a similar symmetry rule is reflected in a large deviation rate function (LDRF) as a property of the dissipation rate through boundaries. Large deviation theory also gives the thermodynamic force of a nonequilibrium steady state, as is usually recorded experimentally by a single molecule technique, which plays a key role responsible for the dynamical symmetry of the SCGF and LDRF. Using some special properties of the Legendre transformation, here, we have provided a relation between the fluctuations of fluxes and dissipation rates, and among them, the fluctuation ...
Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner
Proceedings of the National Academy of Sciences, 2010
Cilia are microtubule-based protrusions of the plasma membrane found on most eukaryotic cells. Their assembly is mediated through the conserved intraflagellar transport mechanism. One class of motor proteins involved in intraflagellar transport, kinesin-2, is unique among kinesin motors in that some of its members are composed of two distinct polypeptides. However, the biological reason for heterodimerization has remained elusive. Here we provide several interdependent reasons for the heterodimerization of the kinesin-2 motor KLP11/KLP20 of Caenorhabditis elegans cilia. One motor domain is unprocessive as a homodimer, but heterodimerization with a processive partner generates processivity. The “unprocessive” subunit is kept in this partnership as it mediates an asymmetric autoregulation of the motor activity. Finally, heterodimerization is necessary to bind KAP1, the in vivo link between motor and cargo.
Functional asymmetry in kinesin and dynein dimers
Biology of the Cell, 2012
Active transport along the microtubule lattice is a complex process that involves both the Kinesin and Dynein superfamily of motors. Transportation requires sophisticated regulation much of which occurs through the motor's tail domain. However, a significant portion of this regulation also occurs through structural changes that arise in the motor and the microtubule upon binding. The most obvious structural change being the manifestation of asymmetry. To a first approximation in solution, kinesin dimers exhibit twofold symmetry, and microtubules, helical symmetry. The higher symmetries of both the kinesin dimers and microtubule lattice are lost on formation of the kinesin-microtubule complex. Loss of symmetry has functional consequences such as an asymmetric handover hand mechanism in plus-end directed kinesins, asymmetric microtubule binding in the Kinesin-14 family, spatially biased stepping in dynein, and cooperative binding of additional motors to the microtubule. This review focuses on how the consequences of asymmetry affect regulation of motor heads within a dimer, dimers within an ensemble of motors, and suggests how these asymmetries may affect regulation of active transport within the cell.
The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1 Powerstroke
Journal of Biological Chemistry, 2012
Background: Kar3Vik1 binds side-by-side microtubule protofilaments and utilizes a minus-end-directed powerstroke. Results: Microtubule collision occurs through Vik1 followed by Kar3 binding and ADP release, which destabilize Vik1 and generate the intermediate poised for ATP binding. Conclusion: The transient Kar3Vik1 two-head-bound state intermediate was identified. Significance: This study provides new insights into force generation by kinesin-14 motors. Kar3, a Saccharomyces cerevisiae microtubule minus-end-directed kinesin-14, dimerizes with either Vik1 or Cik1. The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain and binds microtubules independently of Kar3 but lacks a nucleotide binding site. The only known function of Kar3Vik1 is to cross-link parallel microtubules at the spindle poles during mitosis. In contrast, Kar3Cik1 depolymerizes microtubules during mating but cross-links antiparallel microtubules in the spindle overlap zone during mitosis. A recent study showed that Kar3Vik1 binds across adjacent microtubule protofilaments and uses a minus-end-directed powerstroke to drive ATP-dependent motility. The presteady-state experiments presented here extend this study and establish an ATPase model for the powerstroke mechanism. The results incorporated into the model indicate that Kar3Vik1 collides with the microtubule at 2.4 M ؊1 s ؊1 through Vik1, promoting microtubule binding by Kar3 followed by ADP release at 14 s ؊1. The tight binding of Kar3 to the microtubule destabilizes the Vik1 interaction with the microtubule, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the postpowerstroke ATP hydrolysis at 26 s ؊1 is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke. Detachment of Kar3Vik1 from the microtubule at 6 s ؊1 completes the cycle and allows the motor to return to its initial conformation. The results also reveal key differences in the ATPase cycles of Kar3Vik1 and Kar3Cik1, supporting the fact that these two motors have distinctive biological functions. Kinesin-14 represents a subfamily of kinesins that are nonprocessive, promote microtubule (MT) 2 minus-end-directed force generation, and contain C-terminal motor domains that are dimerized through an N-terminal coiled coil (1-7). Unlike the well known N-terminal motor domain kinesins that use an asymmetric handover hand mechanism for MT plus-end-directed processive stepping (8-11), kinesin-14s use an MT minus-end-directed rotation or bending of the coiled-coil stalk to generate force (12-14). This rotation coupled to ATP turnover is designated the powerstroke and is used to slide one MT relative to another (15-19). During mitosis, kinesin-14s are known to cross-link parallel MTs at the spindle poles but crosslink antiparallel interpolar MTs in the spindle overlap zone (20-24). Furthermore, at the spindle poles and the spindle overlap zone, MT plus-end-directed kinesin-5 is present and provides an outward force to counterbalance the inward force of kinesin-14 to achieve spindle integrity yet allow MT dynamics and other motor and protein interactions required for chromosome segregation (25-28). Saccharomyces cerevisiae kinesin-14 Kar3 is an intriguing model to study kinesin-14 mechanochemistry and head-head communication because Kar3 forms heterodimers with either Cik1 or Vik1, both of which lack a nucleotide binding site, and Kar3Cik1 and Kar3Vik1 have different physiological roles during the yeast life cycle (21, 22, 29-32). Moreover, the C-terminal domain of Vik1 exhibits the structural fold of a kinesin motor domain, and it can bind MTs with high affinity. However, the absence of a nucleotide binding site indicates that nucleotide cannot modulate the interactions of Vik1 with the MT lattice (33). Recent studies using equilibrium binding, cryo-EM, x-ray crystallography, and site-directed cross-links at the base of the coiled coil with analysis by in vitro motility assays clearly demonstrated that both Kar3 and Vik1 interact with the MT for motility (14). Furthermore, an unprecedented mode of MT binding was revealed for Kar3Vik1 by high resolution unidirectional metal shadowing, which strongly emphasizes the surface features when viewed by EM. The images of the shadowed MT⅐Kar3Vik1 complexes in the presence of ADP, used to mimic the beginning of the cycle when Kar3Vik1 collides with the MT, revealed that Kar3 and Vik1 bound side-by-side MT protofilaments rather than binding along a single protofilament in a head-to-tail fashion (14). Because the complexes were formed with ADP, the results suggested that the initial event of
Kar3Vik1, a member of the Kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern
Journal of Cell Biology, 2012
Kinesin-14 motors generate microtubule minus-end–directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1–microtubule interaction and positioning the motor for the start of the powerstroke. The result...
Family-specific kinesin structures reveal neck-linker length based on initiation of the coiled-coil
The Journal of biological chemistry, 2016
Kinesin-1, 2, 5, and 7 generate processive hand-over-hand 8-nm steps to transport intracellular cargoes toward the microtubule plus end. This processive motility requires gating mechanisms to coordinate the mechanochemical cycles of the two motor heads to sustain the processive run. A key structural element believed to regulate the degree of processivity is the neck-linker, a short peptide of 12-18 residues, which connects the motor domain to its coiled-coil stalk. While a shorter neck-linker has been correlated with longer run lengths, the structural data to support this hypothesis have been lacking. To test this hypothesis, seven kinesin structures were determined by X-ray crystallography. Each included the neck-linker motif, followed by helix α7 which constitutes the start of the coiled-coil stalk. In the majority of the structures, the neck-linker length differed from predictions because helix α7, which initiates the coiled-coil, started earlier in the sequence than predicted. A...
Kinesin-2 motors: Kinetics and biophysics
Journal of Biological Chemistry, 2018
Optical manipulation of a dielectric particle along polygonal closed-loop geometries within a single water droplet
Scientific Reports, 2021
We report a new method to optically manipulate a single dielectric particle along closed-loop polygonal trajectories by crossing a suite of all-fiber Bessel-like beams within a single water droplet. Exploiting optical radiation pressure, this method demonstrates the circulation of a single polystyrene bead in both a triangular and a rectangle geometry enabling the trapped particle to undergo multiple circulations successfully. The crossing of the Bessel-like beams creates polygonal corners where the trapped particles successfully make abrupt turns with acute angles, which is a novel capability in microfluidics. This offers an optofluidic paradigm for particle transport overcoming turbulences in conventional microfluidic chips.
Direct measurement of the strength of microtubule attachment to yeast centrosomes
Molecular Biology of the Cell, 2017
Direct measurement of the strength of microtubule attachment to yeast centrosomes ABSTRACT Centrosomes, or spindle pole bodies (SPBs) in yeast, are vital mechanical hubs that maintain load-bearing attachments to microtubules during mitotic spindle assembly, spindle positioning, and chromosome segregation. However, the strength of microtubulecentrosome attachments is unknown, and the possibility that mechanical force might regulate centrosome function has scarcely been explored. To uncover how centrosomes sustain and regulate force, we purified SPBs from budding yeast and used laser trapping to manipulate single attached microtubules in vitro. Our experiments reveal that SPB-microtubule attachments are extraordinarily strong, rupturing at forces approximately fourfold higher than kinetochore attachments under identical loading conditions. Furthermore, removal of the calmodulin-binding site from the SPB component Spc110 weakens SPB-microtubule attachment in vitro and sensitizes cells to increased SPB stress in vivo. These observations show that calmodulin binding contributes to SPB mechanical integrity and suggest that its removal may cause pole delamination and mitotic failure when spindle forces are elevated. We propose that the very high strength of SPB-microtubule attachments may be important for spindle integrity in mitotic cells so that tensile forces generated at kinetochores do not cause microtubule detachment and delamination at SPBs. INTRODUCTION The centrosome is the microtubule-organizing center of the cell, responsible for nucleation of microtubules and organization of the bipolar mitotic spindle. Centrosomes serve as mechanical hubs, subjected to force from interpolar microtubules (Dumont and
Additive manufacturing of laminar flow cells for single-molecule experiments
Scientific Reports, 2019
A microfluidic laminar flow cell (LFC) forms an indispensable component in single-molecule experiments, enabling different substances to be delivered directly to the point under observation and thereby tightly controlling the biochemical environment immediately surrounding single molecules. Despite substantial progress in the production of such components, the process remains relatively inefficient, inaccurate and time-consuming. Here we address challenges and limitations in the routines, materials and the designs that have been commonly employed in the field, and introduce a new generation of LFCs designed for single-molecule experiments and assembled using additive manufacturing. We present single- and multi-channel, as well as reservoir-based LFCs produced by 3D printing to perform single-molecule experiments. Using these flow cells along with optical tweezers, we show compatibility with single-molecule experiments including the isolation and manipulation of single DNA molecules ...
Processive movement of single kinesins on crowded microtubules visualized using quantum dots
The EMBO Journal, 2006
Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions.
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