Kinesin-driven Microtubule Research Articles

Localized Control of the Swarming of Kinesin-Driven Microtubules Using Light.

The swarming of self-propelled cytoskeletal filaments has emerged as a new aspect in the field of molecular machines or robotics, as it has overcome one of the major challenges of controlling the mutual interaction of a large number of individuals at a time. Recently, we reported on the photoregulated swarming of kinesin-driven cytoskeletal microtubule filaments in which visible (VIS) and ultraviolet (UV) light triggered the association and dissociation of the swarm, respectively. However, systematic control of this potential system has yet to be achieved to optimize swarming for further applications in molecular machines or robotics. Here, we demonstrate the precise and localized control of a biomolecular motor-based swarm system by varying different parameters related to photoirradiation. We control the reversibility of the swarming by changing the wavelength or intensity of light and the number of azobenzenes in DNA. In addition, we regulate the swarming in local regions by introducing different-sized or shaped patterns in the UV light system. Such a detailed study of the precise control of swarming would provide new perspectives for developing a molecular swarm system for further applications in engineering systems.

Physically informed data-driven modeling of active nematics

A continuum description is essential for understanding a variety of collective phenomena in active matter. However, building quantitative continuum models of active matter from first principles can be extremely challenging due to both the gaps in our knowledge and the complicated structure of nonlinear interactions. Here, we use a physically informed data-driven approach to construct a complete mathematical model of an active nematic from experimental data describing kinesin-driven microtubule bundles confined to an oil-water interface. We find that the structure of the model is similar to the Leslie-Ericksen and Beris-Edwards models, but there are appreciable and important differences. Rather unexpectedly, elastic effects are found to play no role in the experiments considered, with the dynamics controlled entirely by the balance between active stresses and friction stresses.

Fluctuation in the Sliding Movement of Kinesin-Driven Microtubules Is Regulated Using the Deep-Sea Osmolyte Trimethylamine N-Oxide.

Nowadays, biomolecular motor-based miniaturized lab-on-a-chip devices have been attracting much attention for their wide range of nanotechnological applications. Most of the applications are dependent on the motor-driven active transportation of their associated filamentous proteins as shuttles. Fluctuation in the movement of the shuttles is a major contributor to the dispersion in motor-driven active transportation, which limits the efficiency of the miniaturized devices. In this work, by employing the biomolecular motor kinesin and its associated protein filament microtubule as a model active transport system, we demonstrate that the deep-sea osmolyte trimethylamine N-oxide (TMAO) is useful in regulating the fluctuation in the motility of microtubule shuttles. We show that the motional diffusion coefficient, a measure of the fluctuation in the movement of the kinesin-propelled microtubules, gradually decreases upon increasing the concentration of TMAO in the transportation system. We have been able to reduce the motional diffusion coefficient of microtubules more than 200 times by employing TMAO at a concentration of 2 M. We also show that upon elimination of TMAO, the motional diffusion coefficient of microtubules can be restored, which confirms that TMAO can be used as a tool to reversibly regulate the fluctuation in the sliding movement of kinesin-propelled microtubule shuttles. Such reversible regulation of the dynamic behavior of the shuttles does not require sacrificing the concentration of fuel used for transportation. Our results confirm the ability to manipulate the nanoscale motion of biomolecular motor-driven active transporters in an artificial environment. This work is expected to further enhance the tunability of biomolecular motor functions, which, in turn, will foster their nanotechnological applications based on active transportation.

Effects of confinement on the dynamics and correlation scales in kinesin-microtubule active fluids.

We study the influence of solid boundaries on dynamics and structure of kinesin-driven microtubule active fluids as the height of the container, H, increases from hundreds of micrometers to several millimeters. By three-dimensional tracking of passive tracers dispersed in the active fluid, we observe that the activity level, characterized by velocity fluctuations, increases as system size increases and retains a small-scale isotropy. Concomitantly, as the confinement level decreases, the velocity-velocity temporal correlation develops a strong positive correlation at longer times, suggesting the establishment of a "memory". We estimate the characteristic size of the flow structures from the spatial correlation function and find that, as the confinement becomes weaker, the correlation length, l_{c}, saturates at approximately 400 microns. This saturation suggests an intrinsic length scale which, along with the small-scale isotropy, demonstrates the multiscale nature of this kinesin-driven bundled microtubule active system.

Spontaneous circulation of active microtubules confined by optical traps

We propose an experiment to demonstrate spontaneous ordering and symmetry breaking of kinesin-driven microtubules confined to an optical trap. Calculations involving the feasibility of such an experiment are first performed which analyze the power needed to confine microtubules and address heating concerns. We then present the results of first-principles simulations of active microtubules confined in such a trap and analyze the types of motion observed by the microtubules as well as the velocity of the surrounding fluid, both near the trap and in the far-field. We find three distinct phases characterized by breaking of distinct symmetries and also analyze the power spectrum of the angular momenta of polymers to further quantify the differences between these phases. Under the correct conditions, microtubules were found to spontaneously align with one another and circle the trap in one direction.

Monopolar flocking of microtubules in collective motion

Flocking is a fascinating coordinated behavior of living organisms or self-propelled particles (SPPs). Particularly, monopolar flocking has been attractive due to its potential applications in various fields. However, the underlying mechanism behind flocking and emergence of monopolar motion in flocking of SPPs has remained obscured. Here, we demonstrate monopolar flocking of kinesin-driven microtubules, a self-propelled biomolecular motor system. Microtubules with an intrinsic structural chirality preferentially move towards counter-clockwise direction. At high density, the CCW motion of microtubules facilitates monopolar flocking and formation of a spiral pattern. The monopolar flocking of microtubules is accounted for by a torque generated when the motion of microtubules was obstructed due to collisions. Our results shed light on flocking and emergence of monopolar motion in flocking of chiral active matters. This work will help regulate the polarity in collective motion of SPPs which in turn will widen their applications in nanotechnology, materials science and engineering.

Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruption

The proper organization of the microtubule-based spindle during cell division requires the collective activity of many different proteins. These include non-motor microtubule-associated proteins (MAPs), whose functions include crosslinking microtubules to regulate filament sliding rates and assemble microtubule arrays. One such protein is PRC1, an essential MAP that has been shown to preferentially crosslink overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps remains unclear. Here, we employ a modified microtubule gliding assay to rupture PRC1-mediated microtubule pairs using surface-bound kinesins. We discovered that PRC1 crosslinks always reduce bundled filament sliding velocities relative to single-microtubule gliding rates and do so via two distinct emergent modes of mechanical resistance to motor-driven sliding. We term these behaviors braking and coasting, where braking events exhibit substantially slowed microtubule sliding compared to coasting events. Strikingly, braking behavior requires the formation of two distinct high-density clusters of PRC1 molecules near microtubule tips. Our results suggest a cooperative mechanism for PRC1 accumulation when under mechanical load that leads to a unique state of enhanced resistance to filament sliding and provides insight into collective protein ensemble behavior in regulating the mechanics of spindle assembly.

Two Modes of PRC1-Mediated Mechanical Resistance to Kinesin-Driven Microtubule Network Disruption

The proper structural organization of the microtubule-based spindle during cell division requires the activity of non-motor microtubule-associated proteins, which crosslink microtubules to regulate filament sliding rates and assemble microtubule arrays. PRC1 is an essential MAP that preferentially crosslinks overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps has been lacking.

Photo-regulated trajectories of gliding microtubules conjugated with DNA.

We regulate the persistency in motion of kinesin-driven microtubules (MTs) simply using a photoresponsive DNA (pDNA) and ultraviolet (UV)-visible light. The path persistence length of MTs, which is a measure of the persistency in their motion, increases and decreases upon illuminating the MTs with UV and visible light respectively. Moreover, pDNA is found to work as a shield for MTs against damage under UV irradiation.

Emergence of metachronal waves in active microtubule arrays

The physical mechanism behind the spontaneous formation of metachronal waves in microtubule arrays in a low Reynolds number fluid has been of interest for the past several years, yet is still not well understood. We present a model implementing the hydrodynamic coupling hypothesis from first principles, and use this model to simulate kinesin-driven microtubule arrays and observe their emergent behavior. The results of simulations are compared to known experimental observations by Sanchez et al. By varying parameters, we determine regimes in which the metachronal wave phenomenon emerges, and categorize other types of possible microtubule motion outside these regimes.

Complete, rapid and reversible regulation of the motility of a nano-biomolecular machine using an osmolyte trimethylamine-N-oxide

Nanoscale transportation in engineered environments is critical towards designing efficient and smart hybrid bio-nanodevices. Biomolecular motors, the smallest natural machines, are promising as actuators as well as sensors in hybrid nanodevices and hold enormous potentials in nanoscale transportation. Highly specific regulation of the activity of biomolecular motors is the key to control such integrated nanodevices. We present a simple method to regulate the activity of a biomolecular motor system, microtubule (MT)-kinesin by using a natural osmolyte trimethylamine-N-oxide (TMAO). Motility of kinesin-driven MTs in an in vitro gliding assay is regulated over a broad spectrum by using TMAO in a concentration dependent manner. The regulation of MT motility is rapid, reversible and repeatable over multiple cycles. Interestingly, the motility of MTs can be completely turned off using TMAO of a relatively high concentration. The halted motility of MTs is fully restored upon elimination of TMAO. Repeated cycles of TMAO addition and removal enable cyclical inhibition and restoration of the motility of MTs. These results demonstrate an ability to control nanoscale motion of a biomolecular motor in an artificial environment. This work facilitates further tunability over functions of biomolecular motors, which in turn will foster their nanotechnological applications, such as in nano-transportation.

Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides.

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 μM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.

Controlled movement of kinesin-driven microtubule along a directional track

We designed a system for the controlled directional movements of kinesin-driven microtubule by restricting the motor coating areas in a two-dimensional (2D) substrate. The t-butoxycarbonyl (Boc) was firstly introduced in an amine groups modified glass surface to construct 2D track. The polarity of the above mentioned glass can be efficiently reversed after Boc protection, resulting in the surface transferring from hydrophilicity to hydrophobicity. Through a selective treatment of Boc de-protection, the substrate surface can be distributed into hydrophilic and hydrophobic partitions. The hydrophilic areas can be observed by labeling nanoparticles or fluorescent dye, thus the different shapes were formed at the substrate. The hydrophilic parts in the substrates will allow kinesin to be selectively immobilized on this surface and result in the controlled directional movement of microtubule. With this design, the selective decoration of 2D tracks may have a potential application in field of biomolecular motors based nanodevice.

Targeted Activation of Molecular Transportation by Visible Light.

Regulated transportation of nanoscale objects with a high degree of spatiotemporal precision is a prerequisite for the development of targeted molecular delivery. In vitro integration of the kinesin-microtubule motor system with synthetic molecules offers opportunities to develop controllable molecular shuttles for lab-on-a-chip applications. We attempted a combination of the kinesin-microtubule motor system with push-pull type azobenzene tethered inhibitory peptides (azo-peptides) through which reversible, spatiotemporal control over the kinesin motor activity was achieved locally by a single, visible wavelength. The fast thermal relaxation of the cis-isomers of azo-peptides offered us quick and complete resetting of the trans-state in the dark, circumventing the requirement of two distinct wavelengths for two-way switching of kinesin-driven microtubule motility. Herein, we report the manipulation of selected, single microtubule movement while keeping other microtubules at complete rest. The photoresponsive inhibitors discussed herein would help in realizing complex bionanodevices.

Control of molecular shuttles by designing electrical and mechanical properties of microtubules.

Kinesin-driven microtubules have been focused on to serve as molecular transporters, called "molecular shuttles," to replace micro/nanoscale molecular manipulations necessitated in micro total analysis systems. Although transport, concentration, and detection of target molecules have been demonstrated, controllability of the transport directions is still a major challenge. Toward broad applications of molecular shuttles by defining multiple moving directions for selective molecular transport, we integrated a bottom-up molecular design of microtubules and a top-down design of a microfluidic device. The surface charge density and stiffness of microtubules were controlled, allowing us to create three different types of microtubules, each with different gliding directions corresponding to their electrical and mechanical properties. The measured curvature of the gliding microtubules enabled us to optimize the size and design of the device for molecular sorting in a top-down approach. The integrated bottom-up and top-down design achieved separation of stiff microtubules from negatively charged, soft microtubules under an electric field. Our method guides multiple microtubules by integrating molecular control and microfluidic device design; it is not only limited to molecular sorters but is also applicable to various molecular shuttles with the high controllability in their movement directions.

Designed Tetrapeptide Interacts with Tubulin and Microtubule.

Microtubules regulate eukaryotic cell functions, which have tremendous implication in tumor progression. Thus, the design of novel approaches for controlling microtubule function is extremely important. In this manuscript, a novel tetrapeptide Ser-Leu-Arg-Pro (SLRP) has been designed and synthesized from a small peptide library consisting of 14 tetrapeptides, which perturbs microtubule function through interaction in the "anchor region". We have studied the role of peptides on microtubule function on a chemically functionalized 2D platform. Interestingly, we have found that SLRP binds with tubulin and inhibits the kinesin-driven microtubule motility on a kinesin-immobilized chemically functionalized 2D platform. Further, this peptide modulator interacts with intracellular tubulin/microtubule and depolymerizes the microtubule networks. These interesting findings of perturbation of microtubule function both on engineered platforms and inside the cell by this small peptide modulator inspired us to study the effect of this tetrapeptide on cancer cell proliferation. We found that the novel tetrapeptide modulator causes moderate cytotoxicity to the human breast cancer cell (MCF-7 cell), induces the apoptotic death of MCF-7 cell, and activates the tumor suppressor proteins p53 and cyclin-dependent kinase inhibitor 1 (p21). To the best of our knowledge, this is the shortest peptide discovered, which perturbs microtubule function both on an engineered 2D platform and inside the cell.

Role of confinement in the active self-organization of kinesin-driven microtubules

Self-organization is one of the most spectacular phenomena exhibited in the wide spectrum of biologically active systems. Many studies have attempted to investigate different parameters that regulate the self-organization of moving objects. Recent theoretical and analytical-based approaches have revealed that physical confinement has regulatory effect on the self-organization of moving objects. However, a detailed experimental study on how the varying shapes and sizes of the confinement affect the self-organization of moving objects is still lacking. Recently, biomolecular motor systems F-actin/myosin and microtubule/kinesin or microtubule/dynein have been promising to experimentally study the self-organization of moving objects. Here, we experimentally investigated the shape and size effect of confinement on the self-organization of microtubules (MTs) by employing the in vitro motility assay of MT/kinesin motor system. The MTs were confined by a lipid layer on a glass surface micro-patterned by photolithography. We demonstrated that shapes and sizes of the confinements largely influenced the self-organization of MTs. The MTs showed distinct orientations in different shapes and sizes of the confinements. This work clearly unveiled how physical confinement influences the self-organization of MTs and would help understand the effect of confinement on the self-organization of more complex biologically active systems in nature.

Microtubule sliding during Drosophila development

Development![Figure][1] Kinesin-driven microtubule sliding contributes to cytoplasmic streaming in the Drosophila oocyte . PHOTO: WEN LU, JOSHUA Z. RAPPOPORT AND VLADIMIR I. GELFAND (NORTHWESTERN UNIVERSITY) The motor protein kinesin carries cargo to locations in the cell by moving along microtubules. Kinesin-1 can also move microtubules relative to each other. Winding et al. show that it does this by using its motor domain to move along one microtubule while using a second domain to bind another microtubule. They constructed a kinesin-1 mutant that was deficient in microtubule binding but able to bind and transport other cargo and a second mutant that was able to slide microtubules but could not transport cargo. Using these mutants, they demonstrated that microtubule sliding is important in axon and dendrite outgrowth during nervous system development. In addition, Lu et al. used the mutants to show that microtubule sliding contributes to cytoplasmic streaming, which is important in distributing RNA and proteins in Drosophila oocytes. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.1520244113 and 10.1073/pnas.1522424113 (2016). [1]: pending:yes

A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation.

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell-cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule-microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes.

Dynamic formation of a microchannel array enabling kinesin-driven microtubule transport between separate compartments on a chip.

Microtubules driven by kinesin motors have been utilised as "molecular shuttles" in microfluidic environments with potential applications in autonomous nanoscale manipulations such as capturing, separating, and/or concentrating biomolecules. However, the conventional flow cell-based assay has difficulty in separating bound target molecules from free ones even with buffer flushing because molecular manipulations by molecular shuttles take place on a glass surface and molecular binding occurs stochastically; this makes it difficult to determine whether molecules are carried by molecular shuttles or by diffusion. To address this issue, we developed a microtubule-based transport system between two compartments connected by a single-micrometre-scale channel array that forms dynamically via pneumatic actuation of a polydimethylsiloxane membrane. The device comprises three layers-a control channel layer (top), a microfluidic channel layer (middle), and a channel array layer (bottom)-that enable selective injection of assay solutions into a target compartment and dynamic formation of the microchannel array. The pneumatic channel also serves as a nitrogen supply path to the assay area, which reduces photobleaching of fluorescently labelled microtubules and deactivation of kinesin by oxygen radicals. The channel array suppresses cross-contamination of molecules caused by diffusion or pressure-driven flow between compartments, facilitating unidirectional transport of molecular shuttles from one compartment to another. The method demonstrates, for the first time, efficient and unidirectional microtubule transport by eliminating diffusion of target molecules on a chip and thus may constitute one of the key aspects of motor-driven nanosystems.