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.

Autonomous assembly and disassembly of gliding molecular robots regulated by a DNA-based molecular controller.

In recent years, there has been a growing interest in engineering dynamic and autonomous systems with robotic functionalities using biomolecules. Specifically, the ability of molecular motors to convert chemical energy to mechanical forces and the programmability of DNA are regarded as promising components for these systems. However, current systems rely on the manual addition of external stimuli, limiting the potential for autonomous molecular systems. Here, we show that DNA-based cascade reactions can act as a molecular controller that drives the autonomous assembly and disassembly of DNA-functionalized microtubules propelled by kinesins. The DNA controller is designed to produce two different DNA strands that program the interaction between the microtubules. The gliding microtubules integrated with the controller autonomously assemble to bundle-like structures and disassemble into discrete filaments without external stimuli, which is observable by fluorescence microscopy. We believe this approach to be a starting point toward more autonomous behavior of motor protein-based multicomponent systems with robotic functionalities.

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.

Effects of three microtubule-associated proteins (MAP2, MAP4, and Tau) on microtubules' physical properties and neurite morphology.

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.

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.

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.

Generation of stable microtubule superstructures by binding of peptide-fused tetrameric proteins to inside and outside.

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.

Light-induced stabilization of microtubules by photo-crosslinking of a Tau-derived peptide.

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 via the formation of a covalent bond of the peptide and showed toxicity.

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.

Functionalization of Tubulin: Approaches to Modify Tubulin with Biotin and DNA.

The filamentous cytoskeletal protein microtubule, a polymer of α and ß 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.

Dynamic Pattern Formation of Active Matters Triggered by Mechanical Stimuli.

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.

Fabrication of Artificial Muscle from Microtubules, Kinesins, and DNA Origami Nanostructures.

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.

Construction of Molecular Robots from Microtubules for Programmable Swarming.

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.

Cargo Transport by Microtubule-Associated Motor Protein Along Mechanically Deformed Microtubules.

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.

Mechanical Deformation of Microtubules on a Two-Dimensional Elastic Medium.

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.

Controlling the Rigidity of Kinesin-Propelled Microtubules in an In Vitro Gliding Assay Using the Deep-Sea Osmolyte Trimethylamine N-Oxide.

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 vitro 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, ∼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 vitro 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.

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.

Deformation of microtubules regulates translocation dynamics of kinesin.

Microtubules, the most rigid components of the cytoskeleton, can be key transduction elements between external forces and the cellular environment. Mechanical forces induce microtubule deformation, which is presumed to be critical for the mechanoregulation of cellular events. However, concrete evidence is lacking. In this work, with high-speed atomic force microscopy, we unravel how microtubule deformation regulates the translocation of the microtubule-associated motor protein kinesin-1, responsible for intracellular transport. Our results show that the microtubule deformation by bending impedes the translocation dynamics of kinesins along them. Molecular dynamics simulation shows that the hindered translocation of kinesins can be attributed to an enhanced affinity of kinesins to the microtubule structural units in microtubules deformed by bending. This study advances our understanding of the role of cytoskeletal components in mechanotransduction.

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.

Controlling the length of self-assembled microtubes through mechanical stress-induced scission.

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.