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The physical properties of cytoskeletal microtubules have a multifaceted effect on the expression of their cellular functions. A superfamily of microtubule-associated proteins, MAP2, MAP4, and tau, promote the polymerization of microtubules, stabilize the formed microtubules, and affect the physical properties of microtubules. Here, we show differences in the effects of these three MAPs on the physical properties of microtubules. When microtubule-binding domain fragments of MAP2, tau, and three MAP4 isoforms were added to microtubules in vitro and observed by fluorescence microscopy, tau-bound microtubules showed a straighter morphology than the microtubules bound by MAP2 and the three MAP4 isoforms. Flexural rigidity was evaluated by the shape of the teardrop pattern formed when microtubules were placed in a hydrodynamic flow, revealing that tau-bound microtubules were the least flexible. When full-length MAPs fused with EGFP were expressed in human neuroblastoma (SH-SY5Y) cells, the microtubules in apical regions of protrusions expressing tau were straighter than in cells expressing MAP2 and MAP4. On the other hand, the protrusions of tau-expressing cells had the fewest branches. These results suggest that the properties of microtubules, which are regulated by MAPs, contribute to the morphogenesis of neurites.

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.

The biomolecular motor protein kinesin and its associated filamentous protein microtubule have been finding important nanotechnological applications in the recent years. Rigidity of the microtubules, which are propelled by kinesin motors in an in vitro gliding assay, is an important metric that determines the success of utilization of microtubules and kinesins in various applications, such as transportation, sensing, sorting, molecular robotics, etc. Therefore, regulating the rigidity of kinesin-propelled microtubules has been critical. In this work, we report a simple strategy to regulate the rigidity of kinesin- propelled microtubules in an in vitro gliding assay. We demonstrate that rigidity of the microtubules, propelled by kinesins in an in nitro gliding assay, can be modulated simply by using the natural osmolyte trimethylamine N-oxide (TMAO). By varying the concentration of TMAO in the gliding assay, the rigidity of microtubules can be modulated over a wide range. Based on this strategy, we are able to reduce the persistence length of microtubules, a measure of microtubule rigidity, similar to 8 fold by using TMAO at the concentration of 1.5 M. Furthermore, we found that the decreased rigidity of the kinesin-propelled microtubules can be restored upon elimination of TMAO from the in nitro gliding assay. Alteration in the rigidity of microtubules is accounted for by the non-uniformity of the force applied by kinesins along the microtubules in the presence of TMAO. This work offers a facile strategy to reversibly regulate the rigidity of kinesin-propelled microtubules in situ, which would widen the applications of the biomolecular motor kinesin and its associated protein microtubule in various fields.

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.

Microtubules play important roles in biological functions by forming superstructures, such as doublets and branched structures, in vivo. Despite the importance, it is challenging to construct these superstructures in vitro. Here, we designed a tetrameric fluorescent protein Azami-Green (AG) fused with His-tag and Tau-derived peptide (TP), TP-AG, to generate the superstructures. Main binding sites of TP-AG can be controlled to the inside and outside of microtubules by changing the polymerization conditions. The binding of TP-AG to the inside promoted microtubule formation and generated rigid and stable microtubules. The binding of TP-AG to the outside induced various microtubule superstructures, including doublets, multiplets, branched structures, and extremely long microtubules by recruiting tubulins to microtubules. Motile microtubule aster structures were also constructed by TP-AG. The generation of various microtubule superstructures by a single type of exogenous protein is a new concept for understanding the functions of microtubules and constructing microtubule-based nanomaterials.

For light-induced stabilization of microtubules (MTs) to manipulate cells, a photo-reactive diazirine group was conjugated to a Tau-derived peptide, a motif binding on the inside of MTs. Ultraviolet (UV) light irradiation induced significant stabilization of MTs

Microtubule, the most rigid filamentous protein in cytoskeleton, plays significant roles in cellular mechano-transduction and mechano-regulation of cellular functions. In cells, the mechanical stress serves as a prevalent stimulus to frequently cause deformation of the microtubules participating in various cellular events. While the experimental and simulation-based approaches have confirmed the role of mechanical stress to tune mechanical properties of microtubule. Yet, the effect of mechanical force on the structural stability and the mechanism of microtubule deformation have remained obscure. Here, we describe the mechanical stress-induced deformation of microtubules using a custom-made mechanical device. We designed the device in a way which allows the microtubules to undergo deformation as response to the applied stress while attached on a two-dimensional elastic substrate through interaction with microtubule-associated motor protein, kinesin. We provide here the method to cause controlled bucking or fragmentation of microtubules by applying compressive or tensile stress on the microtubules, respectively. Such study is crucial to understand the mechanism of deformation in microtubules in cellular environment and their consequences in physiological activities.

Filamentous microtubules, polymers of the heterodimeric protein tubulins play one of the major roles in the emergent nano-biotechnological devices. To develop the feature of those devices, it is important to understand the function of microtubule in in vitro, hence, the availability of purified αβ-tubulin is required. Additionally, fluorescently labeled tubulin has become a powerful approach for extensively studying the dynamics of these components. In this chapter, the process of purifying the heterodimeric αβ-tubulin from porcine brain will be described, as well as the process of labeling of the purified tubulin with fluorescence dye.

The filamentous cytoskeletal protein microtubule, a polymer of α and β heterodimers of tubulin, plays major roles in intracellular transport as well as in vitro molecular actuation and transportation. Functionalization of tubulin dimers through covalent linkage facilitates utilization of microtubule in the nanobioengineering. Here we present a detailed description of the methodologies used to modify tubulin dimers with DNA strand and biotin through covalent interaction.

In vitro gliding assay of the filamentous protein microtubule (MT) on a kinesin motor protein coated surface has appeared as a classic platform for studying active matters. At high densities, the gliding MTs spontaneously align and self-organize into fascinating large-scale patterns. Application of mechanical stimuli e.g., stretching stimuli to the MTs gliding on a kinesin-coated surface can modulate their self-organization and patterns according to the boundary conditions. Depending on the mode of stretching, MT at high densities change their moving direction and exhibit various kinds of patterns such as stream, zigzag and vortex pattern. In this chapter, we discuss detail procedures on how to apply mechanical stimuli to the moving MTs on a kinesin coated substrate.

Swarm robotics has been attracting much attention in recent years in the field of robotics. This chapter describes a methodology for the construction of molecular swarm robots through precise control of active self-assembly of microtubules (MTs). Detailed protocols are presented for the construction of molecular robots through conjugation of DNA to MTs and demonstration of swarming of the MTs. The swarming is mediated by DNA-based interaction and photoirradiation which act as processors and sensors respectively for the robots. Furthermore, the required protocols to utilize the swarming of MTs for molecular computation is also described.

Mechanical forces play pivotal roles in regulating various cellular functions. Biomolecular motor protein-driven intracellular transportation is one example which is affected by mechanical forces, although the mechanism at molecular level is unknown. In this chapter, we describe deformation of microtubules under compressive stress and we show that such deformation of microtubules affects the kinetics of dynein-driven cargo transportation along the microtubules. The extent of alteration in the kinetics of dynein-driven transportation is found strongly dependent on the extent of deformation of microtubules under compressive stress.

Fabrication of molecular devices using biomolecules through biomimetic approaches has witnessed a surge in interest in recent years. DNA a versatile programmable material offers an opportunity to realize complicated operations through the designing of various nanostructures such as DNA origami. Here we describe the methods to use DNA origami for the self-assembly of the biomolecular motor system, microtubule (MT)-kinesin. A rodlike DNA origami motif facilitates the self-assembly of MTs into asters. A smooth muscle like molecular contraction system could be realized following the method where DNA mediated self-assembly of MTs permits dynamic contraction in the presence of kinesins through an energy dissipative process.

Cooperation is a strategy that has been adopted by groups of organisms to execute complex tasks more efficiently than single entities. Cooperation increases the robustness and flexibility of the working groups and permits sharing of the workload among individuals. However, the utilization of this strategy in artificial systems at the molecular level, which could enable substantial advances in microrobotics and nanotechnology, remains highly challenging. Here, we demonstrate molecular transportation through the cooperative action of a large number of artificial molecular machines, photoresponsive DNA-conjugated microtubules driven by kinesin motor proteins. Mechanical communication via conjugated photoresponsive DNA enables these microtubules to organize into groups upon photoirradiation. The groups of transporters load and transport cargo, and cargo unloading is achieved by dissociating the groups into single microtubules. The group formation permits the loading and transport of cargoes with larger sizes and in larger numbers over long distances compared with single transporters. We also demonstrate that cargo can be collected at user-determined locations defined by ultraviolet light exposure. This work demonstrates cooperative task performance by molecular machines, which will help to construct molecular robots with advanced functionalities in the future.

The cyclization of peptides is a valuable strategy for the development of binding motifs to target proteins with improved affinity. Microtubules (MTs) are important targets for therapeutics, and a variety of MT-targeted drugs and peptides have recently been developed. We have previously designed a Tau-derived peptide (TP) that binds to the interior of MTs. In the present study, the development of a cyclic TP (TCP) for enhanced binding to tubulin and the stabilization of MTs are described. The fluorescently labeled cyclic peptide containing three glycine linkers (TCP3-TMR) exhibited a remarkably enhanced binding affinity to tubulin. The cyclic peptide was also demonstrated to stabilize MTs by enhancing polymerization and reducing depolymerization. Moreover, MTs were effectively formed by the treatment of cyclic peptides in the presence of guanosine triphosphate (GTP), while the linear peptide showed no such effect. These findings indicate that TCP is a useful binding motif that can stabilize MTs and is valuable for various therapeutic and material applications. Microtubules (MTs) consisting of tubulins are important targets of drugs for MT-related diseases. We have previously designed a linear Tau-derived peptide (TP) that binds to the interior of MTs. In this article, a cyclic TP (TCP) was developed for enhanced binding to tubulin and stabilization of MTs. The fluorescently labeled TCP exhibited a remarkably enhanced binding affinity to tubulin compared to the linear TP. The stabilization of MTs by binding of TCP was demonstrated, such as formation of typically unstable MTs in the presence of guanosine triphosphate.

Microtubules, one of the major components of the cytoskeleton, play important roles as pathways for neuronal transport of cellular traffic. The loss of structural stability of microtubules causes detrimental effects on neurons and may contribute to several neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, etc. The triazolopyrimidine class compound cevipabulin is a synthetic microtubule-stabilizing agent that has recently emerged as a drug for the treatment of Alzheimer's disease. However, the mechanism of microtubule stabilization by cevipabulin has not yet been revealed. Here, we explored the effect of cevipabulin on stabilizing microtubules polymerized from purified tubulins in vitro. We observed the effects of the concentration of microtubule-stabilizing drugs, incubation time, and modification of the cevipabulin structure on the stabilization of microtubules in comparison to those of the most commonly used anticancer drug, paclitaxel. This study will provide insight into the action of cevipabulin in the treatment of neurodegenerative diseases. Stabilization of microtubules occurs by paclitaxel bound to the taxoid site and cevipabulin (and its derivative) bound to the taxoid site and/or vinca alkaloid domain of the beta-tubulin in tubulin heterodimer.

Mechanical stress on cells has profound influences on biological processes, such as cell shape regulation, the formation of tissue patterns, and development. Recently, mechanosensing properties of the microtubule, an important cytoskeletal component, have drawn attention. In this work, we studied cargo transport by dynein, a microtubule-associated motor protein, along microtubules deformed under mechanical stress. We reveal that the microtubule deformation took place as a response to the applied stress and that the deformation of microtubules facilitated the transport of dynein-driven quantum dots. This finding will provide opportunities to explore the role of microtubules as molecular mechanotransducers in cellular processes.

The glutaraldehyde fixation method for fixing tissues is attractive for its ease of use and straightforward surface chemistry. We investigated the effect of glutaraldehyde-induced microtubule immobilization on kinesin-driven cargo transport along microtubules and found that at low glutaraldehyde concentrations, the microtubule–kinesin interaction remains unperturbed. Such findings may facilitate the application of the glutaraldehyde fixation method for many in vitro studies aiming to build nanodevices powered by the microtubule–motor protein interaction. The graphical abstract shows that the kinesin-driven quantum dot transport along microtubule immobilized on a substrate using glutaraldehyde concentration ≤0.10% (v/v) remain unaltered, whereas, at higher glutaraldehyde concentration, >0.10% (v/v), the quantum dot transportation is slowed down.

Trimethylamine N-oxide is found to be effective in regulating the interaction between microtubules and kinesins over a wide temperature range. The lifetime of the motility of microtubules on kinesins at high temperatures is prolonged using trimethylamine N-oxide. The activation energy of microtubule motility is increased by trimethylamine N-oxide. Prolonged operation at high temperatures decreased the activation energy of MT motility despite the increase in concentration of 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.

Microtubules (MTs) play important roles in biological functions by forming superstructures, such as doublets, triplets, and branched structures, in vivo. Formation of these superstructures by exogenous molecules in vitro will be useful not only for understanding the functions of MTs but also as components of MT-based nanomaterials. Here, we developed a tetrameric fluorescent protein Azami-Green (AG) fused with a His-tag and Tau-derived peptide (TP), TP–AG, which can bind to the inside or outside of MTs depending on the polymerization conditions. The binding of TP–AG to the inside of MTs induced the formation, stabilized, and increased the rigidity of the MTs. The binding of TP–AG to the outside of MTs induced various types of MT superstructures, including doublets, multiplets, and branched structures, by recruiting tubulins to MTs. The formation of motile MT aster structures by TP–AG was also observed. The generation of MT superstructures by these exogenous proteins provides guidelines for the design of MT-based nanomaterials.

We demonstrate that the rigidity of the microtubules (MTs), propelled by kinesins in an in vitro gliding assay, can be modulated using the deep-sea osmolyte trimethylamine N-oxide (TMAO). By varying the concentration of TMAO in the gliding assay, the rigidity of the MTs is modulated over a wide range. By employing this approach, we are able to reduce the persistence length of MTs, a measure of MT rigidity, ∼8 fold using TMAO of the concentration of 1.5 M. The rigidity of gliding MTs can be restored by eliminating the TMAO from the gliding assay. This work offers a simple strategy to regulate the rigidity of kinesin-propelled MTs in situ and would widen the applications of biomolecular motors in nanotechnology, materials science, and bioengineering.

Biomolecular motor proteins that generate forces by consuming chemical energy obtained from ATP hydrolysis are pivotal for organizing broad cytoskeletal structures in living cells. The control of such cytoskeletal structures benefits programmable protein patterning; however, our current knowledge is limited owing to the underdevelopment of an engineering approach for controlling pattern formation. Here, we demonstrate the tailoring of assembled patterns of microtubules (MTs) driven by kinesin motors by designing the boundary shape in fabricated microwells. We found an MT bundle structure along the microwell wall and a bridging structure perpendicular to the wall. Corroborated by the theory of self-propelled rods, we further showed that the alignment of MTs defined by the boundary shape determined the transition of the assembled patterns, providing a blueprint to reconstruct bridge structures in microchannels. Our findings provide a geometric rule to tailor the self-organization of cytoskeletons and motor proteins for nanotechnological applications., 19 pages, 4 figures, and supporting information 13 pages

Boundary conditions are important for pattern formation in active matter. However, it is still not well-understood how alterations in the boundary conditions (dynamic boundary conditions) impact pattern formation. To elucidate the effect of dynamic boundary conditions on the pattern formation by active matter, we investigate an in vitro gliding assay of microtubules on a deformable soft substrate. The dynamic boundary conditions were realized by applying mechanical stress through stretching and compression of the substrate during the gliding assay. A single cycle of stretch-and-compression (relaxation) of the substrate induces perpendicular alignment of microtubules relative to the stretch axis, whereas repeated cycles resulted in zigzag patterns of microtubules. Our model shows that the orientation angles of microtubules correspond to the direction to attain smooth movement without buckling, which is further amplified by the collective migration of the microtubules. Our results provide an insight into understanding the rich dynamics in self-organization arising in active matter subjected to time-dependent boundary conditions.

We constructed GFP-encapsulated microtubules (MTs) using Tauderived peptide which binds to their interior. The encapsulation of GFP dramatically increased the rigidity of MTs, resulting in their enhanced velocity on a kinesin-coated substrate. Moreover, the GFP-encapsulated MTs were significantly more stable compared to normal MTs.

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.

Microtubules (MTs) are key cytoskeletal components that modulate various cellular activities with their dynamic structural changes, including polymerization and depolymerization. To monitor the dynamics of MTs in living cells, many drug-based fluorescent probes have been developed; however, these also potentially disturb the polymerization/depolymerization of MTs. Here, we report nondrug, peptide-based fluorescent probes to monitor MTs in living cells. We employed a Tau-derived peptide (TP) that has been shown to bind MTs without inhibiting polymerization/depolymerization in vitro. We show that a tetramethylrhodamine (TMR)-labeled TP (TP–TMR) is internalized into HepG2 cells and binds to intracellular MTs, enabling visualization of MTs as clear, fibrous structures. The binding of TP–TMR shows no apparent effects on polymerization/depolymerization of MTs induced by MT-targeted drugs and temperature change. The main uptake mechanism of TP–TMR was elucidated as endocytosis, and partial endosomal escape resulted in the binding of TP–TMR to MTs. TP–TMR exhibited no cytotoxicity compared with MT-targeted drug scaffolds. These results indicate that TP scaffolds can be exploited as useful MT-targeted tools in living cells, such as in long-term imaging of MTs.

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.

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.

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.

It is still challenging to control dynamic self-organization patterns of self-propelled particles. Although varieties of patterns associated with chirality have been observed, essential control factors determining patterns remain unclear. Here, we explore numerically how torque upon particle collision affects dynamic self-organization. Based on the particle-based model with both collision-induced torque and torque in self-propulsion, we find that introducing collision-induced torque turns homogeneous bi-polar orientation templated by bi-directional alignment into rotating mono-polar flocks., 24 pages, 7 figures

We demonstrate that mechanical stress-induced scission is an effective strategy to control the length of self-assembled microtubes. By applying mechanical stress with variable magnitude and mode, the length of microtubes can be tightly regulated. We have succeeded in reducing the average length of microtubes ∼twenty-fold through stretching and compression. The mechanical stress-induced scission of self-assembled, long microtubes into smaller fragments has no adverse effect on the functionality of the microtubes. This work will foster the applications of length-controlled, self-assembled microtubes in various fields.

This study introduces a modeling method for a supermolecular structure of microtubules for the development of a force generation material using motor proteins. 3D imaging by confocal laser scanning microscopy (CLSM) was used to obtain 3D volume density data. The density data were then interpreted by a set of cylinders with the general-purpose 3D modeling software Blender, and a 3D network structure of microtubules was constructed. Although motor proteins were not visualized experimentally, they were introduced into the model to simulate pulling of the microtubules toward each other to yield shrinking of the network, resulting in contraction of the artificial muscle. From the successful force generation simulation of the obtained model structure of artificial muscle, the modeling method introduced here could be useful in various studies for potential improvements of this contractile molecular system.

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.