Single Kinesin Molecules Research Articles

Reconstitution of Neuronal Motor Traffic on Septin-Associated Microtubules.

Reconstitution of intracellular transport in cell-free in vitro assays enables the understanding and dissection of the molecular mechanisms that underlie membrane traffic. Using total internal reflection fluorescence (TIRF) microscopy and microtubules, which are immobilized to a functionalized glass surface, the kinetic properties of single kinesin molecules can be imaged and analyzed in the presence or absence of microtubule-associated proteins. Here, we describe methods for the in vitro reconstitution of the motility of the neuronal kinesin motor KIF1A on microtubules associated with heteromeric septin (SEPT2/6/7) complexes. This method can be adapted for various neuronal septin complexes and kinesin motors, leading to new insights into the spatial regulation of neuronal membrane traffic by microtubule-associated septins.

Focusing properties of Azimuthally Polarized Lorentz Gauss Vortex Beam through a Dielectric Interface

Tight focusing properties of azimuthally polarized Lorentz Gaussian vortex beam through a dielectric interface are numerically studied by vector diffraction theory. The focusing properties, such as spot size, depth of focus, and maximum intensity position, are numerically calculated by properly manipulating the Lorentz parameter with/without annular obstruction values. Thus, using annular obstruction, one can generate a highly confined focal spot of long focal depth when using an azimuthally polarized Lorentz Gaussian vortex beam. Full Text: PDF References A. Ashkin et al., "Observation of a single-beam gradient force optical trap for dielectric particles", Opt. Lett. 11, 288 (1986). CrossRef A. Ambardekar, Y.Q. Li, "Optical levitation and manipulation of stuck particles with pulsed optical tweezers", Opt. Lett. 30, 1797 (2005). CrossRef P. Zemánek, C.J. Foot, "Atomic dipole trap formed by blue detuned strong Gaussian standing wave", Opt. Commun. 146, 119(1998). CrossRef S.M. Block et al., "Bead movement by single kinesin molecules studied with optical tweezers", Nature 348, 348 (1990). CrossRef D.E. Smithet al., "The bacteriophage φ29 portal motor can package DNA against a large internal force", Nature 413, 748 (2001). CrossRef L. Oroszi et al., "Direct Measurement of Torque in an Optical Trap and Its Application to Double-Strand DNA", Phys. Rev. Lett. 97, 058301 (2006). CrossRef D.P. Biss, T.G. Brown, "Cylindrical vector beam focusing through a dielectric interface", Opt. Express 9, 490 (2001). CrossRef P. Török et al., "Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: structure of the electromagnetic field. I", J. Opt. Soc. Am. A 12, 2136 (1995). CrossRef S.H. Wiersma et al., "Comparison of different theories for focusing through a plane interface", J. Opt. Soc. Am. A 14, 1482 (1997). CrossRef L.E. Helseth, "Roles of polarization, phase and amplitude in solid immersion lens systems", Opt. Commun. 191, 161 (2001). CrossRef P. Zhou et al., "Propagation properties of a Lorentz beam array", Appl. Opt. 49, 2497 (2010). CrossRef O.E. Gawhary, S. Severini, "Lorentz beams and symmetry properties in paraxial optics", J. Opt. A: Pure Appl Opt 8, 409 (2006). CrossRef J. Yang et al., "Focusing of diode laser beams: a partially coherent Lorentz model", Proc. SPIE 6824, 68240 A (2007). CrossRef O.E. Gawhary, S. Severini, "Lorentz beams as a basis for a new class of rectangularly symmetric optical fields", Opt. Commun. 269, 274 (2007). CrossRef H. Yu, L. Xiong, B. Lü, "Nonparaxial Lorentz and Lorentz–Gauss beams", Optik 121,1455(2010). CrossRef Z. Zhang, J. Pu, X. Wang, "Tight Focusing of Radially and Azimuthally Polarized Vortex Beams through a Dielectric Interface", Chin. Phys. Lett. 25, 1664 (2008). CrossRef

Fast backward steps and detachment of single kinesin molecules measured under a wide range of loads.

To understand force generation under a wide range of loads, the stepping of single kinesin molecules was measured at loads from -20 to 42 pN by optical tweezers with high temporal resolution. The optical trap has been improved to halve positional noise and increase bandwidth by using 200-nm beads. The step size of the forward and backward steps was 8.2 nm even over a wide range of loads. Histograms of the dwell times of backward steps and detachment fit well to two independent exponential equations with fast (~0.4 ms) and slow (>3 ms) time constants, indicating the existence of a fast step in addition to the conventional slow step. The dwell times of the fast steps were almost independent of the load and ATP concentration, while those of the slow backward steps and detachment depended on those. We constructed the kinetic model to explain the fast and slow steps under a wide range of loads.

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

Noise-Induced Acceleration of Single Molecule Kinesin-1.

The movement of single kinesin molecules was observed while applying noisy external forces that mimic intracellular active fluctuations. We found kinesin accelerates under noise, especially when a large hindering load is added. The behavior quantitatively conformed to a theoretical model that describes the kinesin movement with simple two-state reactions. The universality of the kinetic theory suggests that intracellular enzymes share a similar noise-induced acceleration mechanism, i.e., active fluctuations in cells are not just noise but are utilized to promote various physiological processes.

A bio-pen for direct writing of single molecules on user-functionalized surfaces.

Advancing ultrahigh resolution (below 10 nm) direct writing technologies could lead to impacts in areas as diverse as disease detection, genetic analysis and nanomanufacturing. Current methods based on electron-beams and photo- or dip-pen nanolithography are laborious and lack flexibility when aiming to create single molecule patterns for application specific integration. We hypothesize that a novel strategy could be developed to allow for writing of parallel and yet individually addressable patterns of single molecules on user-controlled surfaces. The strategy is based on using in vitro self-recognition of tubulin protein to assemble rigid protofilaments of microtubules, with one such microtubule to be subsequently used as a “bio-pen” capable of writing “inks” of single kinesin molecules in user-defined environments. Our results show that single kinesin inks could be written under the energy of adenosine triphosphate hydrolysis and observed by both atomic force and optical microscopy. Upon extending ink functionalities, the integration of soft and hard materials for nanostructure assembly and complex single molecule pattern formation is envisioned.

Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy

Current in vitro optical studies of microtubule dynamics tend to rely on fluorescent labeling of tubulin, with tracking accuracy thereby limited by the quantum yield of fluorophores and by photobleaching. Here, we demonstrate label-free tracking of microtubules with nanometer precision at kilohertz frame rates using interferometric scattering microscopy (iSCAT). With microtubules tethered to a glass substrate using low-density kinesin, we readily detect sequential 8 nm steps in the microtubule center of mass, characteristic of a single kinesin molecule moving a microtubule. iSCAT also permits dynamic changes in filament length to be measured with <5 nm precision. Using the arbitrarily long observation time enabled by label-free iSCAT imaging, we demonstrate continuous monitoring of microtubule disassembly over a 30 min period. The ability of iSCAT to track microtubules with nm precision together with its potential for label-free single protein detection and simultaneous single molecule fluorescence imaging represent a unique platform for novel approaches to studying microtubule dynamics.

Highly loaded behavior of kinesins increases the robustness of transport under high resisting loads.

Kinesins are nano-sized biological motors which walk by repeating a mechanochemical cycle. A single kinesin molecule is able to transport its cargo about 1 μm in the absence of external loads. However, kinesins perform much longer range transport in cells by working collectively. This long range of transport by a team of kinesins is surprising because the motion of the cargo in cells can be hindered by other particles. To reveal how the kinesins are able to accomplish their tasks of transport in harsh intracellular circumstances, stochastic studies on the kinesin motion are performed by considering the binding and unbinding of kinesins to microtubules and their dependence on the force acting on kinesin molecules. The unbinding probabilities corresponding to each mechanochemical state of kinesin are modeled. The statistical characterization of the instants and locations of binding are captured by computing the probability of unbound kinesin being at given locations. It is predicted that a group of kinesins has a more efficient transport than a single kinesin from the perspective of velocity and run length. Particularly, when large loads are applied, the leading kinesin remains bound to the microtubule for long time which increases the chances of the other kinesins to bind to the microtubule. To predict effects of this behavior of the leading kinesin under large loads on the collective transport, the motion of the cargo is studied when the cargo confronts obstacles. The result suggests that the behavior of kinesins under large loads prevents the early termination of the transport which can be caused by the interference with the static or moving obstacles.

An improved optical tweezers assay for measuring the force generation of single kinesin molecules.

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments.

A Slippery Walk to the Microtubule-End

A Slippery Walk to the Microtubule-End

In vivo optical trapping indicates kinesin’s stall force is reduced by dynein during intracellular transport

Kinesin and dynein are fundamental components of intracellular transport, but their interactions when simultaneously present on cargos are unknown. We built an optical trap that can be calibrated in vivo during data acquisition for each individual cargo to measure forces in living cells. Comparing directional stall forces in vivo and in vitro, we found evidence that cytoplasmic dynein is active during minus- and plus-end directed motion, whereas kinesin is only active in the plus direction. In vivo, we found outward (∼plus-end) stall forces range from 2 to 7 pN, which is significantly less than the 5- to 7-pN stall force measured in vitro for single kinesin molecules. In vitro measurements on beads with kinesin-1 and dynein bound revealed a similar distribution, implying that an interaction between opposite polarity motors causes this difference. Finally, inward (∼minus-end) stalls in vivo were 2-3 pN, which is higher than the 1.1-pN stall force of a single dynein, implying multiple active dynein.

Measuring the force of adhesion between multiple kinesins and a microtubule using the fluid force produced by microfluidic flow

We developed a method to measure the adhesion force between the motor protein, kinesin, and a microtubule. Compared with conventional methods that use optical tweezers, our method employs the fluid force that acts on the interaction between a kinesin-coated microbead and a microtubule in a microfluidic channel. When the fluid force just exceeds the kinesin-microtubule adhesion force, the beads are released from the microtubules. Having modeled the kinesins that are bound to the microtubules and the beads as mechanical springs, adhesion forces were measured as 31.3 or 362.9 pN for fluid containing 1 mM ATP or 0 M ATP, respectively. These forces are much larger than those measured when optical tweezers were used to measure the adhesion force between a single kinesin and a microtubule. For our multi-kinesin system we elucidated the relationship between the binding force of a single kinesin molecule and that of all kinesin molecules in a contact area by varying one of two parameters: either the contact area length or the kinesin density on a bead. This study provides insight into the behavior of a bead that is supported by several kinesins in a microfluidic system, which is essential knowledge if a motor protein is to be used as a nanoactuator for in vitro molecular transport.

Two Kinesins Transport Cargo Primarily via the Action of One Motor: Implications for Intracellular Transport

The number of microtubule motors attached to vesicles, organelles, and other subcellular commodities is widely believed to influence their motile properties. There is also evidence that cells regulate intracellular transport by tuning the number and/or ratio of motor types on cargos. Yet, the number of motors responsible for cargo motion is not easily characterized, and the extent to which motor copy number affects intracellular transport remains controversial. Here, we examined the load-dependent properties of structurally defined motor assemblies composed of two kinesin-1 molecules. We found that a group of kinesins can produce forces and move with velocities beyond the abilities of single kinesin molecules. However, such capabilities are not typically harnessed by the system. Instead, two-kinesin assemblies adopt a range of microtubule-bound configurations while transporting cargos against an applied load. The binding arrangement of motors on their filament dictates how loads are distributed within the two-motor system, which in turn influences motor-microtubule affinities. Most configurations promote microtubule detachment and prevent both kinesins from contributing to force production. These results imply that cargos will tend to be carried by only a fraction of the total number of kinesins that are available for transport at any given time, and provide an alternative explanation for observations that intracellular transport depends weakly on kinesin number in vivo.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.

Myosin-1 Function Extends to Microtubule-Dependent Processes

Class 1 myosins are motor proteins found on various organelles and at defined structures at the cell periphery, where they play important roles in organelle translocation, intracellular transport, and cytoskeleton organization. Here, we investigated the cellular function of Dictyostelium myosin-1C, a long-tailed, monomeric motor with three tail homology domains called TH1, TH2, and TH3. We identified that in addition to its actin-dependent function in endocytotic processes during interphase, in Dictyostelium cells the motor co-localizes with centrosomes and spindle microtubules (MT) during mitosis. In vitro TIRF microscopy experiments and MT-cosedimentation assays using truncated myosin-1C tail constructs revealed a direct binding of myosin-1C tail to MTs. Only constructs containing TH1 and TH2 bound efficiently to MTs. Moreover, these two domains were sufficient to prevent MT depolymerization at low nanomolar concentrations of myosin-1C tail, while MT formation, i.e. nucleation and elongation, was unaffected. Additionally, we observed myosin-1C tail mediated cross-linking of MTs to F-actin. In cells, myosin-1C constructs lacking the motor domain did not associate with the spindle. This demonstrates that actin-dependent motor function is required for the cell cycle-dependent re-localization of myosin-1C from actin-rich structures at the cell periphery to MT-associated mitotic structures. Cells producing a hydrolysis deficient full-length myosin-1C mutant exhibited reduced growth rates with increased size of nuclei due to defects in spindle alignment and prolonged mitosis. Single kinesin molecule motility assays showed that MT-bound myosin-1C reduced the attachment rate of kinesin-1 to MTs without affecting its velocity and run-length. From this we propose that myosin-1C may regulate MT-dependent motility and MT dynamics. Our cell biological and functional characterization of a long-tailed class 1 myosin shows that myosin function is not limited to the F-actin network but extends to MT associated processes.

KINESIN BYPASSING BLOCKAGES ON MICROTUBULE RAILS

Kinesins are motor proteins which convert the chemical energy of ATP into mechanical energy to move along proteinaceous microtubule rails and to transport different cargoes to defined intracellular destinations. It is well documented that following the track of a single protofilament is the thermodynamically most effective mechanism of kinesin movement along microtubules. However, the question arises what happens when a kinesin molecule encounters a hindrance along the protofilament. The present study describes a simple, cell-free approach which enables to study the effects of structural blockages on kinesin-based transport. This experimental approach uses dimeric conventional kinesin moving nanometre-sized gold beads along immobilized microtubules whose surface has been irreversibly decorated by blocking proteins. We demonstrated that the continuous bead transport temporarily stopped at sites of blockages, but usually continued after a certain resting time. Our results suggest that single dimeric kinesin molecules are able to change to another protofilament if the next tubulin dimer where the second head should bind is blocked. A bypassing mechanism is discussed which is considered to be one fundamental prerequisite to realize a kinesin-mediated cargo-transport along microtubules over long distances, required for e.g., the fast axonal transport in motor neurons.

Pressure-Induced Changes in the Structure and Function of the Kinesin-Microtubule Complex

Kinesin-1 is an ATP-driven molecular motor that “walks” along a microtubule by working two heads in a “hand-over-hand” fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ends at the same speed. The sliding velocity of kinesin motors was reversibly changed by pressure, and reached half-maximal value at ∼100 MPa. The pressure-velocity relationship was very close to the force-velocity relationship of single kinesin molecules, suggesting a similar inhibitory mechanism on kinesin motility. Further analysis showed that the pressure mainly affects the stepping motion, but not the ATP binding reaction. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of the kinesin head and microtubule. These pressure-induced effects could prevent kinesin motors from completing the stepping motion.

Cooperativity of multiple kinesin-1 motors mechanically coupled through a shared load

Recent experiments using single-molecule techniques have characterized the mechanical properties of single kinesin molecules in vitro at a range of loads and ATP concentrations. These experiments have shown that kinesin moves processively along microtubules by alternately advancing each of its motor domains in a hand-over-hand fashion, using Brownian motion and the energy from ATP hydrolysis. We have extended the theoretical analysis of kinesin through a mechanistic model that is capable of describing transient and steady-state behavior. Transient dynamics are needed to describe the effect of external perturbations (e.g. interactions with other kinesin molecules). Quantitative metrics are tailored to characterize the synchronization of nonlinear, nonsmooth systems such as kinesin. These metrics are employed to analyze the simulation results and to quantify the effect of the cargo linker stiffness, the load, and the difference in intrinsic velocity on the synchronization of two coupled motor proteins. Herein, the mechanistic model and the new analysis techniques are demonstrated for the case of two coupled kinesin motors.

Kinesin and Dynein-Dynactin at Intersecting Microtubules: Motor Density Affects Dynein Function

Kinesin and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery, and the behavior of molecular motors at these intersections has implications for long-range transport efficiency and accuracy. To test motor function at microtubule intersections, crossovers were arranged in vitro using flow to orient successive layers of filaments. Single kinesin and cytoplasmic dynein-dynactin molecules fused with green-fluorescent protein, and artificial bead cargos decorated with multiple motors, were observed while they encountered intersections. Single kinesins tend to cross intersecting microtubules, whereas single dynein-dynactins have a more varied response. For bead cargos, kinesin motion is independent of motor number. Dynein beads with high motor numbers pause, but their actions become more varied as the motor number decreases. These results suggest that regulating the number of active dynein molecules could change a motile cargo into one that is anchored at an intersection, consistent with dynein's proposed transport and tethering functions in the cell.