Tensile Stress on Microtubules Facilitates Dynein-Driven Cargo Transport.

Mechanical stress significantly affects the physiological functions of cells, including tissue homeostasis, cytoskeletal alterations, and intracellular transport. As a major cytoskeletal component, microtubules respond to mechanical stimulation by altering their alignment and polymerization dynamics. Previously, we reported that microtubules may modulate cargo transport by one of the microtubule-associated motor proteins, dynein, under compressive mechanical stress. Despite the critical role of tensile stress in many biological functions, how tensile stress on microtubules regulates cargo transport is yet to be unveiled. The present study demonstrates that the low-level tensile stress-induced microtubule deformation facilitates dynein-driven transport. We validate our experimental findings using all-atom molecular dynamics simulation. Our study may provide important implications for developing new therapies for diseases that involve impaired intracellular transport.

Role of tubulin C-terminal tail on mechanical properties of microtubule.

Tubulin C-terminal tail (CTT) is a disordered segment extended from each tubulin monomer of αß tubulin heterodimers, the building blocks of microtubules. The tubulin CTT contributes to the cellular function of microtubules such as intracellular transportation by regulating their interaction with other proteins and cell shape regulation by controlling microtubule polymerization dynamics. Although the mechanical integrity of microtubules is crucial for their functions, the role of tubulin CTT on microtubule mechanical properties has remained elusive. In this work, we investigate the role of tubulin CTTs in regulating the mechanical properties of microtubules by estimating the persistence lengths and investigating the buckling behavior of microtubules with and without CTT. We find that microtubules with intact CTTs exhibit twice the rigidity of microtubules lacking tubulin CTTs. Our study will widen the scope of altering microtubule mechanical properties for its application in nano bio-devices and lead to novel therapeutic approaches for neurodegenerative diseases with altered microtubule properties.

Reversible Photocontrol of Microtubule Stability by Spiropyran-Conjugated Tau-Derived Peptides.

Spatiotemporal modulation of microtubules by light has become an important aspect of the biological and nanotechnological applications of microtubules. We previously developed a Tau-derived peptide as a binding unit to the inside of microtubules. Here, we conjugated the Tau-derived peptide to spiropyran, which is reversibly converted to merocyanine by light, as a reversible photocontrol system to stabilize microtubules. Among the synthesized peptides with spiropyran/merocyanine at different positions, several peptides were bound to the inside of microtubules and stabilized the structures of microtubules. The peptide with spiropyran at the N-terminus induced polymerization and stabilization of microtubules, whereas the same peptide with the merocyanine form did not exert these effects. Reversible formation of microtubules/tubulin aggregates was achieved using the peptide with spiropyran conjugated at the N-terminus and irradiation with UV and visible light. Spiropyran-conjugated Tau-derived peptides would be useful for spatiotemporal modulation of microtubule stability through reversible photocontrol of binding.

1,2-Disubstituted 1,2-Dihydro-1,2,4,5-tetrazine-3,6-dione as a Dynamic Covalent Bonding Unit at Room Temperature.

Dynamic covalent bonds are useful tools in a wide range of applications. Although various reversible chemical reactions have been studied for this purpose, the requirement for harsh conditions, such as high temperature and low or high pH, to activate generally stable covalent bonds limits their potential applications involving biomolecules or household utilization. Here, we report the design, synthesis, characterization, and dynamic covalent bonding properties of 1,2-disubstituted 1,2-dihydro-1,2,4,5-tetrazine-3,6-dione (TETRAD). Hetero-Diels-Alder reactions of TETRAD with furan derivatives and their retro-reactions proceeded rapidly at room temperature under neutral conditions, enabling a chemically induced sol-gel transition system.

Collision-induced torque mediates the transition of chiral dynamic patterns formed by active particles.

Controlling the patterns formed by self-propelled particles through dynamic self-organization is a challenging task. Although varieties of patterns associated with chiral self-propelled particles have been reported, essential factors that determine the morphology of the patterns have remained unclear. Here, we explore theoretically how torque formed upon collision of the particles affects the dynamic self-organization of the particles and determine the patterns. Based on a particle-based model with collision-induced torque and torque associated with self-propulsion, we find that introducing collision-induced torque turns the homogeneous bi-directionally aligned particles into rotating mono-polar flocks, which helps resolve a discrepancy in the earlier observations in microfilament gliding assays.

Controlling Collective Motion of Kinesin-Driven Microtubules via Patterning of Topographic Landscapes.

Biomolecular motor proteins that generate forces by consuming chemical energy obtained from ATP hydrolysis play pivotal roles in organizing cytoskeletal structures in living cells. An ability to control cytoskeletal structures would benefit programmable protein patterning; however, our current knowledge is limited because of the underdevelopment of engineering approaches for controlling pattern formation. Here, we demonstrate the controlling of self-assembled patterns of microtubules (MTs) driven by kinesin motors by designing the boundary shape in fabricated microwells. By manipulating the collision angle of gliding MTs defined by the boundary shape, the self-assembly of MTs can be controlled to form protruding bundle and bridge patterns. Corroborated by the theory of self-propelled rods, we further show that the alignment of MTs determines the transition between the assembled patterns, providing a blueprint to reconstruct bridge structures in microchannels. Our findings introduce the tailoring of the self-organization of cytoskeletons and motor proteins for nanotechnological applications.

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.

Magnetic Force-Induced Alignment of Microtubules by Encapsulation of CoPt Nanoparticles Using a Tau-Derived Peptide.

Construction of magnetotactic materials is a significant challenge in nanotechnology applications such as nanodevices and nanotransportation. Artificial magnetotactic materials can be designed from magnetotactic bacteria because these bacteria use magnetic nanoparticles for aligning with and moving within magnetic fields. Microtubules are attractive scaffolds to construct magnetotactic materials because of their intrinsic motility. Nonetheless, it is challenging to magnetically control their orientation while retaining their motility by conjugating magnetic nanoparticles on their outer surface. Here we solve the issue by encapsulating magnetic cobalt-platinum nanoparticles inside microtubules using our developed Tau-derived peptide that binds to their internal pockets. The in situ growth of cobalt-platinum nanoparticles resulted in the formation of a linear-chain assembly of nanoparticles inside the microtubules. The magnetic microtubules significantly aligned with a high order parameter (0.71) along the weak magnetic field (0.37 T) and showed increased motility. This work provides a new concept for designing magnetotactic materials.

Breaking of buckled microtubules is mediated by kinesins.

Microtubule is the most rigid component of eukaryotic cytoskeleton that plays pivotal roles in many important cellular events. Microtubules are known to undergo bending or buckling in cells which often results in breaking of this cytoskeletal protein filament. Various cellular events such as cell migration, chromosome segregation, etc. are dependent on the buckling induced breaking of microtubules. However, the reason behind the breaking of buckled microtubules in cell has remained obscure yet. In this work, we have demonstrated breaking of microtubules on a 2D elastic medium by applying compressive stress. The applied compressive stress caused buckling of the microtubules which ultimately resulted in their breaking. We show that breaking of the buckled microtubules cannot be accounted for by considering the changes in curvature of the microtubules due to mechanical deformation. Our results confirm that, it is the interaction of kinesin, a microtubule-associated motor protein, with microtubules which plays the key role in breaking of the buckled microtubules on the 2D elastic medium. The breaking of buckled microtubules is ascribed to decrease in rigidity of microtubules upon interaction with kinesins. This work for the first time confirms the involvement of a microtubule-associated motor protein in breaking of microtubules under compressive stress, which will help further clarify the mechanism of breaking of buckled microtubules in cells and its significance in the cellular events.

Molecular swarm robots: recent progress and future challenges.

Recent advancements in molecular robotics have been greatly contributed by the progress in various fields of science and technology, particularly in supramolecular chemistry, bio- and nanotechnology, and informatics. Yet one of the biggest challenges in molecular robotics has been controlling a large number of robots at a time and employing the robots for any specific task as flocks in order to harness emergent functions. Swarming of molecular robots has emerged as a new paradigm with potentials to overcome this hurdle in molecular robotics. In this review article, we comprehensively discuss the latest developments in swarm molecular robotics, particularly emphasizing the effective utilization of bio- and nanotechnology in swarming of molecular robots. Importance of tuning the mutual interaction among the molecular robots in regulation of their swarming is introduced. Successful utilization of DNA, photoresponsive molecules, and natural molecular machines in swarming of molecular robots to provide them with processing, sensing, and actuating ability is highlighted. The potentials of molecular swarm robots for practical applications by means of their ability to participate in logical operations and molecular computations are also discussed. Prospects of the molecular swarm robots in utilizing the emergent functions through swarming are also emphasized together with their future perspectives.

Artificial Smooth Muscle Model Composed of Hierarchically Ordered Microtubule Asters Mediated by DNA Origami Nanostructures.

DNA has been well-known for its applications in programmable self-assembly of materials. Nonetheless, utility of DNA origami, which offers more opportunity to realize complicated operations, has been very limited. Here we report self-assembly of a biomolecular motor system, microtubule-kinesin mediated by DNA origami nanostructures. We demonstrate that a rodlike DNA origami motif facilitates self-assembly of microtubules into asters. A smooth-muscle like molecular contraction system has also been realized using the DNA origami in which self-assembled microtubules exhibited fast and dynamic contraction in the presence of kinesins through an energy dissipative process. This work provides potential nanotechnological applications of DNA and biomolecular motor proteins.

Stabilization of microtubules by cevipabulin.

We report the utility of cevipabulin as a stabilizing agent for microtubules. Cevipabulin-stabilized microtubules were more flexible compared to the microtubules stabilized by paclitaxel, the most commonly used microtubule stabilizing agent. Similar to the paclitaxel-stabilized microtubules, cevipabulin-stabilized microtubules were driven by kinesins in an in vitro gliding assay. The velocity of cevipabulin-stabilized microtubules was significantly higher than that of paclitaxel-stabilized microtubules. These findings will enrich the variety of microtubules with difference in mechanical and dynamic properties and widen their applications in nanotechnology.

Molecular Encapsulation Inside Microtubules Based on Tau-Derived Peptides.

Microtubules are cytoskeletal filaments that serve as attractive scaffolds for developing nanomaterials and nanodevices because of their unique structural properties. The functionalization of the outer surface of microtubules has been established for this purpose. However, no attempts have been made to encapsulate molecules inside microtubules with 15 nm inner diameter. The encapsulation of various molecular cargos inside microtubules constitutes a new concept for nanodevice and nanocarrier applications of microtubules. Here, we developed peptide motifs for binding to the inner surface of microtubules, based on a repeat domain of the microtubule-associated protein Tau. One of the four Tau-derived peptides, 2N , binds to a taxol binding pocket of ß-tubulin located inside microtubules by preincubation with tubulin dimer and subsequent polymerization of the peptide-tubulin complex. By conjugation of 2N to gold nanoparticles, encapsulation of gold nanoparticles inside microtubules was achieved. The methodology for molecular encapsulation inside microtubules by the Tau-derived peptide is expected to advance the development of microtubule-based nanomaterials and nanodevices.

Substrate selectivity and its mechanistic insight of the photo-responsive non-nucleoside triphosphate for myosin and kinesin.

Linear motor proteins including kinesin and myosin are promising biomaterials for developing nano-devices. Photoswitchable substrates of these biomotors can be used to optically regulate the motility of their associated cytoskeletal filaments in in vitro systems. Here, we describe the discovery of the myosin selective azobenzene-tethered triphosphate. It enables the specific photocontrol over myosin in a reversible mode with the composite motility assay composed of both kinesin and myosin. The mechanistic insight into this myosin selectivity is also explained with the docking simulation study.

Motility of Microtubules on the Inner Surface of Water-in-Oil Emulsion Droplets.

Water-in-oil emulsion systems have recently attracted much attention in various fields. However, functionalization of water-in-oil emulsion systems, which is required for expanding their applications in industries and research, has been challenging. We now demonstrate the functionalization of a water-in-oil emulsion system by anchoring a target protein molecule. A microtubule (MT)-associated motor protein kinesin-1 was successfully anchored to the inner surface of water-in-oil emulsion droplets by employing the specific interaction of nickel-nitrilotriacetic acid-histidine tag. The MTs exhibited a gliding motion on the kinesin-functionalized inner surface of the emulsion droplets, which confirmed the success of the functionalization of the water-in-oil emulsion system. This result would be beneficial in exploring the roles of biomolecular motor systems in the cellular events that take place at the cell membrane and might also contribute to expanding the nanotechnological applications of biomolecular motors and water-in-oil emulsion systems in the future.

Buckling of microtubules on elastic media via breakable bonds.

Buckling of microtubules observed in cells has been reconstructed on a two-dimensional elastic medium consisting of kinesins grafted over compressible substrates, enabling precise control of experimental conditions and quantitative analysis. However, interpretations of the observations have ambiguities due to inevitable experimental difficulties. In this study, with computer simulations, we investigated importance of the mode of interaction of microtubule with elastic medium in the buckling behavior of microtubule. By taking into consideration of forced-induced detachments of kinesins from microtubules, our simulations reproduced the previous experimental results, and showed deviations from predictions of the elastic foundation model. On the other hand, with hypothetical linkers permanently bound to microtubules, our simulation reproduced the predictions of the elastic foundation model. By analyzing the results of the simulations, we investigated as to why the difference arose. These findings indicate the importance of the mode of interaction of microtubule with the medium in the buckling behavior of microtubule. Our findings would bring new insights on buckling of microtubules in living cells.

Controlling the bias of rotational motion of ring-shaped microtubule assembly.

Biomolecular motor system microtubule (MT)-kinesin is considered a building block for developing artificial microdevices. Recently, an active self-organization method has been established to integrate MT filaments into ring-shaped assembly that can produce rotational motion both in the clockwise and in the counterclockwise directions. In this work, we have investigated the effect of parameters such as MT and kinesin concentration, length, and rigidity of MT and type of kinesin (structure of tail region) on the preferential rotation of the ring-shaped MT assembly produced in an active self-organization. We elucidated that these factors can significantly affect the bias of rotation of the ring-shaped MT assembly, which seems to be related to the fluctuation of leading tip of moving MT filaments. This new finding might be important for designing handedness regulated artificial biomachine using the ring-shaped MT assembly in future.

Effect of length and rigidity of microtubules on the size of ring-shaped assemblies obtained through active self-organization.

The microtubule (MT)-kinesin biomolecular motor system has attracted considerable attention due to its possible applications in artificial biomachines. Recently, an active self-organization (AcSO) method has been established to integrate MT filaments into highly organized assembled structures. The ring-shaped MT assembly, one of the structures derived from the AcSO of MTs, can convert the translational motion of MTs into rotational motion. Due to this attractive feature, the ring-shaped MT assembly appears to be a promising candidate for developing artificial devices and for future nanotechnological applications. In this work, we have investigated the effect of length and rigidity of the MT filaments on the size of the ring-shaped MT assembly in the AcSO process. We show that the size of the ring-shaped MT assembly can be controlled by tuning the length and rigidity of MT filaments employed in the AcSO. Longer and stiffer MT filaments led to larger ring-shaped assemblies through AcSO, whereas AcSO of shorter and less stiff MT filaments produced smaller ring-shaped assemblies. This work might be important for the development of biomolecular motor based artificial biomachines, especially where size control of ring-shaped MT assembly will play an important role.

Biomolecular motor modulates mechanical property of microtubule.

The microtubule (MT) is the stiffest cytoskeletal filamentous protein that takes part in a wide range of cellular activities where its mechanical property plays a crucially significant role. How a single biological entity plays multiple roles in cell has been a mystery for long time. Over the recent years, it has been known that modulation of the mechanical property of MT by different cellular agents is the key to performing manifold in vivo activities by MT. Studying the mechanical property of MT thus has been a prerequisite in understanding how MT plays such diversified in vivo roles. However, the anisotropic structure of MT has been an impediment in obtaining a precise description of the mechanical property of MT along its longitudinal and lateral directions that requires employment of distinct experimental approach and has not been demonstrated yet. In this work, we have developed an experimental system that enabled us to investigate the effect of tensile stress on MT. By using our newly developed system, (1) we have determined the Young's modulus of MT considering its deformation under applied tensile stress and (2) a new role of MT associated motor protein kinesin in modulating the mechanical property of MT was revealed for the first time. Decrease in Young's modulus of MT with the increase in interaction with kinesin suggests that kinesin has a softening effect on MT and thereby can modulate the rigidity of MT. This work will be an aid in understanding the modulation of mechanical property of MTs by MT associated proteins and might also help obtain a clear insight of the endurance and mechanical instability of MTs under applied stress.

Prolonged morphometric study of barnacles grown on soft substrata of hydrogels and elastomers.

A long-term investigation of the shell shape and the basal morphology of barnacles grown on tough, double-network (DN) hydrogels and polydimethylsiloxane (PDMS) elastomer was conducted in a laboratory environment. The elastic modulus of these soft substrata varied between 0.01 and 0.47 MPa. Polystyrene (PS) (elastic modulus, 3 GPa) was used as a hard substratum control. It was found that the shell shape and the basal plate morphology of barnacles were different on the rigid PS substratum compared to the soft substrata of PDMS and DN hydrogels. Barnacles on the PS substratum had a truncated cone shape with a flat basal plate while on soft PDMS and DN gels, barnacles had a pseudo-cylindrical shape and their basal plates showed curvature. In addition, a large adhesive layer was observed under barnacles on PDMS, but not on DN gels. The effect of substratum stiffness is discussed in terms of barnacle muscle contraction, whereby the relative stiffness of the substratum compared to that of the muscle is considered as the key parameter.