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3D Structure of Ring-shaped Microtubule Swarms Revealed by High-speed Atomic Force Microscopy
Abstract
Biomolecular motor (microtubule (MT)-kinesin) based molecular swarm robots are important due to their applications in cooperative task achievements, while the structural details are still unknown. In this work, high-speed atomic force microscopy was used to observe the MT swarm ring structure at nanometer-level resolution. MTs were observed to pile up in multiple layers to form swarm rings. The number of MTs involved in force generation was estimated which will specifically impact the understanding of the force-generation capability of swarms.
... The morphology of the swarm (circular ring-shaped and bundle shaped) could also be controlled by changing the flexibility and length of the MTs. MTs in a swarm system could be oriented by piling up in multiple layers instead of being organized in a single layer, which was recently investigated by high speed atomic force microscopy ( Fig. 2(c)) [18]. ...
... (c) High resolution AFM image of the orientation of MTs in a circular swarm robot. The figures are used with permission from[10,18]. ...
Biomolecular motor-based micro-sized robots have recently created an innovation in the field of science and technology as molecular transporters. Groups of these tiny robots can work substantially better than individual ones in terms of the transported distance and number or size of cargo. Site-specific molecular delivery, the main feature of these robots, has helped to improve the workability of robots in a more controllable manner.
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.
We report the swarming of microtubules driven by the biomolecular motor kinesin and dissociation of microtubule swarms under UV and visible light irradiation, respectively. We introduced para tert-butyl-substituted azobenzene, a photoresponsive molecule, to the backbone of single strand DNA, which functions as a photoswitch. Due to the photoswitch, the swarming of DNA-conjugated microtubules was controlled and reversible regulation of microtubule swarming was achieved in a repeated manner upon alternate irradiation with UV and visible light. This reversible swarming of microtubules could provide new opportunities for designing complex swarming systems with the ability of multitasking, expediting the development of molecular machines. We demonstrated biomolecular motors driven swarming of microtubules and their dissociation under UV and visible light irradiation, respectively. A photoresponsive molecule, para tert-butyl-substituted azobenzene was incorporated to the backbone of single strand DNA, which functions as a photoswitch to control the swarming of microtubules in a reversible manner. This work is expected to expand the potential applications of biomolecular motors in developing photoregulated molecular machines.
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.
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.
Significance
One cannot help but marvel at the precise organization of microtubule polymers in cellular structures such as the axoneme and the spindle. However, our understanding of the biochemical mechanisms that sculpt these arrays comes largely from in vitro experiments with a small number (one or two) of microtubules. This is somewhat akin to studying the architecture of multilane highways by studying one-lane streets. Here, we directly visualize depolymerizing microtubule arrays at individual microtubule and protofilament resolution using atomic force microscopy. Our results reveal differences in microtubule depolymerase activity and provide insights into how these differences in enzymatic activity on the nanometer scale can result in the differential remodeling of multimicrotubule arrays on the micron-length scale.
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.
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.
The paper presents a sequential use of the AWK and GNU Octave programming languages integrated with Generic Mapping Tools (GMT) for geospatial data analysis. The geographic scope of the research is focused on the Kuril-Kamchatka Trench, north Pacific Ocean. Practical research aim is to analyse and compare bathymetry in the southern and northern part of the trench using digitized cross- section profiles. The initial mapping and geospatial analysis was performed in GMT scripting toolset. The GMT was used for cartographic mapping based on the raster ETOPO1 grid and automatic digitizing of the profiles crossing the trench perpendicularly. Besides visualized map, the processed geodata were received in a numerical form as a complex multi-field table for each segment. These tables were generated by the GMT in its native format and could not be directly processed by the MATLAB/Octave. Therefore, the tables were exported to AWK, a data-driven programming language and a powerful tool for data extraction. The table was then restructured, sorted and reshaped by the AWK script. Because the total amount of profiles overstepped 100 (62 and 52 for the northern and southern trench segments), only selected profiles were visualized. For this purpose, at the next step the modified tables were converted to GNU Octave language for visualizing and plotting selected profiles. Finally, the geomorphology was analysed and two segments compared. The results show that the southern part has deeper bathymetric values, vary in geomorphic structure and has steeper gradient slopes comparing to the north, which is caused by the seismicity, volcanism, geologic and tectonic settings. Three full scripts of GMT, AWK and GNU Octave programming languages are presented for replicability in the Appendix.
Recently we demonstrated swarming of a self-propelled biomolecular motor system microtubule (MT)-kinesin where interactions among thousands of motile MTs were regulated in a highly programmable fashion by using DNA as a processor. However, precise control of this potential system is yet to be achieved to optimize the swarm behavior. In this work, we systematically controlled swarming of MTs on kinesin adhered surface by different physicochemical parameters of MT-kinesin and DNA. Tuning the length of DNA sequences swarming was precisely controlled with thermodynamic and kinetic feasibility. In addition, swarming was regulated using different concentration of DNA crosslinkers. Reversibility of swarming was further controlled by changing the concentration of strand displacement DNA signal allowing dissociation of swarm. The control over the swarm was accompanied by variable stiffness of MTs successfully, providing translational and circular motion. Moreover, the morphology of swarm was also found to be changed not only depending on the stiffness but also body length of MTs. Such detail study of precise control of swarming would provide new insights in developing a promising molecular swarm robotic system with desired functions.
In nature, swarming behavior has evolved repeatedly among motile organisms because it confers a variety of beneficial emergent properties. These include improved information gathering, protection from predators, and resource utilization. Some organisms, e.g., locusts, switch between solitary and swarm behavior in response to external stimuli. Aspects of swarming behavior have been demonstrated for motile supramolecular systems composed of biomolecular motors and cytoskeletal filaments, where cross-linkers induce large scale organization. The capabilities of such supramolecular systems may be further extended if the swarming behavior can be programmed and controlled. Here, we demonstrate that the swarming of DNA-functionalized microtubules (MTs) propelled by surface-adhered kinesin motors can be programmed and reversibly regulated by DNA signals. Emergent swarm behavior, such as translational and circular motion, can be selected by tuning the MT stiffness. Photoresponsive DNA containing azobenzene groups enables switching between solitary and swarm behavior in response to stimulation with visible or ultraviolet light.
In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.
The scanning tunneling microscope is proposed as a method to measure forces as small as 10-18 N. As one application for this concept, we introduce a new type of microscope capable of investigating surfaces of insulators on an atomic scale. The atomic force microscope is a combination of the principles of the scanning tunneling microscope and the stylus profilometer. It incorporates a probe that does not damage the surface. Our preliminary results in air demonstrate a lateral resolution of 30 ÅA and a vertical resolution less than 1 Å.
High-speed atomic force microscopy (HS-AFM) and total internal reflection fluorescence microscopy (TIRFM) have mutually complementary capabilities. Here, we report techniques to combine these microscopy systems so that both microscopy capabilities can be simultaneously used in the full extent. To combine the two systems, we have developed a tip-scan type HS-AFM instrument equipped with a device by which the laser beam from the optical lever detector can track the cantilever motion in the X- and Y-directions. This stand-alone HS-AFM system is mounted on an inverted optical microscope stage with a wide-area scanner. The capability of this combined system is demonstrated by simultaneous HS-AFM∕TIRFM imaging of chitinase A moving on a chitin crystalline fiber and myosin V walking on an actin filament.
In this paper, we study self-organized flocking in a swarm of mobile robots. We present Kobot, a mobile robot platform developed
specifically for swarm robotic studies. We describe its infrared-based short range sensing system, capable of measuring the
distance from obstacles and detecting kin robots, and a novel sensing system called the virtual heading system(VHS) which
uses a digital compass and a wireless communication module for sensing the relative headings of neighboring robots.
We propose a behavior based on heading alignment and proximal control that is capable of generating self-organized flocking
in a swarm of Kobots. By self-organized flocking we mean that a swarm of mobile robots, initially connected via proximal sensing,
is able to wander in an environment by moving as a coherent group in open space and to avoid obstacles as if it were a “super-organism”.
We propose a number of metrics to evaluate the quality of flocking. We use a default set of behavioral parameter values that
can generate acceptable flocking in robots, and analyze the sensitivity of the flocking behavior against changes in each of
the parameters using the metrics that were proposed. We show that the proposed behavior can generate flocking in a small group
of physical robots in a closed arena as well as in a swarm of 1000 simulated robots in open space. We vary the three main
characteristics of the VHS, namely: (1)the amount and nature of noise in the measurement of heading, (2)the number of VHS
neighbors, and (3)the range of wireless communication. Our experiments show that the range of communication is the main factor
that determines the maximum number of robots that can flock together and that the behavior is highly robust against the other
two VHS characteristics. We conclude by discussing this result in the light of related theoretical studies in statistical
physics.
The design and structure of a self-assembly modular robot (Sambot) are presented in this paper. Each module has its own autonomous mobility and can connect with other modules to form robotic structures with different manipulation abilities. Sambot has a versatile, robust, and flexible structure. The computing platform provided for each module is distributed and consists of a number of interlinked microcontrollers. The interaction and connectivity between different modules is achieved through infrared sensors and Zigbee wireless communication in discrete state and control area network bus communication in robotic configuration state. A new mechanical design is put forth to realize the autonomous motion and docking of Sambots. It is a challenge to integrate actuators, sensors, microprocessors, power units, and communication elements into a highly compact and flexible module with the overall size of 80 mm × 80 mm × 102 mm. The work describes represents a mature development in the area of self-assembly distributed robotics.
The authors present general considerations and simple models for the operation of isothermal motors at small scales, in asymmetric environments. Their work is inspired by recent observations on the behavior of molecular motors in the biological realm, where chemical energy is converted into mechanical energy. A generic Onsager-like description of the linear (close to equilibrium) regime is presented, which exhibits structural differences from the usual Carnot engines. Turning to more explicit models for a single motor, the authors show the importance of the time scales involved and of the spatial dependence of the motor's chemical activity. Considering the situation in which a large collection of such motors operates together. The authors exhibit new features among which are dynamical phase transitions formally similar to paramagnetic-ferromagnetic and liquid-vapor transitions.
During the search for a new nest site, use of an inhibitory signal enables honeybees to reach a decision.
New methods based on surfaces or beads have allowed measurement of properties of single DNA molecules in very accurate ways. Theoretical coarse grained models have been developed to understand the behavior of single stranded and double stranded DNA. These models have been shown to be accurate and relatively simple for very short systems of 6-8 base pairs near surfaces. Comparatively less is known about the influence of a surface on the secondary structures of longer molecules important to many technologies. Surface fields due to either applied potentials and/or dielectric boundaries are not in current surface mounted coarse grained models. To gain insight into longer and surface mounted sequences we parameterized a discretized worm-like chain model. Each link is considered a sphere of 6 base pairs in length for dsDNA, and 1.5 bases for ssDNA (requiring an always even number of spheres). For this demonstration of the model, the chain is tethered to a surface by a fixed length, non-interacting 0.536 nm linker. Configurational sampling was achieved via Monte-Carlo simulation. Our model successfully reproduces end to end distance averages from experimental results, in agreement with polymer theory and all atom simulations. Our average tilt results are also in agreement with all atom simulations for the case of dense systems.
The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450-650 kHz) and small spring constants (150-280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 x 100 pixel(2) image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm).
Cellular factors tightly regulate the architecture of bundles of filamentous cytoskeletal proteins, giving rise to assemblies with distinct morphologies and physical properties, and a similar control of the supramolecular organization of nanotubes and nanorods in synthetic materials is highly desirable. However, it is unknown what principles determine how macromolecular interactions lead to assemblies with defined morphologies. In particular, electrostatic interactions between highly charged polyelectrolytes, which are ubiquitous in biological and synthetic self-assembled structures, are poorly understood. We have used a model system consisting of microtubules (MTs) and multivalent cations to examine how microscopic interactions can give rise to distinct bundle phases in biological polyelectrolytes. The structure of these supramolecular assemblies was elucidated on length scales from subnanometer to micrometer with synchrotron x-ray diffraction, transmission electron microscopy, and differential interference contrast microscopy. Tightly packed hexagonal bundles with controllable diameters were observed for large trivalent, tetravalent, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of 2D dynamic assemblies of MTs with linear, branched, and loop topologies. This new bundle phase is an experimental example of nematic membranes. The morphologically distinct MT assemblies give insight into general features of bundle formation and may be used as templates for miniaturized materials with applications in nanotechnology and biotechnology.
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Recent progress in molecular machines or molecular devices, as exemplified by the 2016 Nobel Prize in Chemistry, has greatly accelerated the development of molecular robots through the fusion of various fields. An actuator is a critical component of molecular robots as an actuator is required to generate motion by the molecular robots by transferring the energy obtained from an internal or external source. To date, various attempts have been undertaken, based on bioengineering or synthetic chemistry, to design and fabricate actuators for molecular robots. In this chapter, the application of various natural and synthetic molecules as the actuator of molecular robots is described. The topics covered in this chapter will be the fabrication of molecular robots using reconstructed linear biomolecular motors, application of peptides as the molecular actuator, cell-sized liposomes containing acting, rotary biomolecular motors, bacterial flagellar motor, F1FO ATP synthase and their prospects as molecular actuators. Synthetic or supramolecular actuators for molecular robots such as water-soluble gels and inorganic layered crystals will be also discussed.
High-speed atomic force microscopy (AFM) is a versatile method that can visualize proteins and protein systems on the nanometer scale and at a temporal resolution of 100 ms. The application to microtubules can not only reveal structural information with single-tubulin resolution but can also extract mechanical information and allows to study single motor proteins walking on microtubules, among others. This chapter provides a step-by-step guide from microtubule polymerization to successful observation with high-speed AFM.
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.
Microtubules are biopolymers composed of tubulin and play diverse roles in a wide variety of biological processes such as cell division, migration and intracellular transport in eukaryotic cells. To perform their functions, microtubules are mechanically stressed and, thereby, susceptible to structural defects. Local variations in mechanical properties caused by these defects modulate their biological functions, including binding and transportation of microtubule-associated proteins. Therefore, assessing the local mechanical properties of microtubules and analyzing their dynamic response to mechanical stimuli provides insight to fundamental processes. It is, however, not trivial to control defect formation, gather mechanical information at the same time, and subsequently image the result at a high temporal resolution at the molecular level with minimal delay. In this work, we describe the so-called in-line force curve mode based on high-speed atomic force microscopy. This method is directly applied to create defects in microtubules at the level of tubulin dimers and monitor the following dynamic processes around the defects. Further, force curves taken during defect formation provide quantitative mechanical information to estimate the bonding energy between tubulin dimers.
Self-assembly is the autonomous organization of components into patterns or structures without human intervention. Self-assembling processes are common throughout nature and technology. They involve components from the molecular (crystals) to the planetary (weather systems) scale and many different kinds of interactions. The concept of self-assembly is used increasingly in many disciplines, with a different flavor and emphasis in each.
Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being considered a model system to experimentally investigate various aspects of group behavior and pattern formation by self-propelled objects. In the in vitro gliding assay, cytoskeletal filaments F-actin or microtubules are driven by the surface immobilized associated biomolecular motors myosin or dynein respectively. Although the F-actin/myosin or microtubule/dynein system was found to be promising in understanding the collective motion and pattern formation by self-propelled objects, the most widely used biomolecular motor system microtubule/kinesin could not be successfully employed so far in this regard. Failure in exhibiting collective motion by kinesin driven microtubules is attributed to the intrinsic properties of kinesin, which was speculated to affect the behavior of individual gliding microtubules and mutual interactions among them. In this work, for the first time, we have demonstrated the collective motion of kinesin driven microtubules by regulating the mutual interaction among the gliding microtubules, by employing a depletion force among them. Proper regulation of the mutual interaction among the gliding microtubules through the employment of the depletion force was found to allow the exhibition of collective motion and stream pattern formation by the microtubules. This work offers a universal means for demonstrating the collective motion using the in vitro gliding assay of biomolecular motor systems and will help obtain a meticulous understanding of the fascinating coordinated behavior and pattern formation by self-propelled objects.
Microtubule (MT)–kinesin, a biomolecular motor system, is a promising candidate for construction of artificial biomachines for a variety of nanotechnology applications. An active self-organization (AcSO) method involving a specific streptavidin (St)–biotin (Bt) interaction has been developed to assemble MTs into a highly ordered structure by exploiting their motility on a kinesin coated surface. Dynein is another biomolecular motor that moves along the MTs in the opposite direction from kinesin. Dynein has not yet been used to demonstrate the AcSO of MTs. In this study, we report the first successful demonstration of the AcSO of MTs on a dynein-coated surface to produce ring-shaped MT assemblies similar to those of kinesin. We found that ring-shaped
MT assemblies obtained on dynein showed equal clockwise and counterclockwise rotational motion. This work will enrich the building blocks for designing future oriented motor protein-based artificial devices.
A dynamical mean field theory is used to predict the end-monomer mean square displacement of single-stranded DNA and finally estimate two important parameters—the persistence length lplp and the length per base ldld. Both parameters are set free, and finally reach optimum values by fitting the theoretical data to the experimental data of Shusterman et al. [R. Shusterman, S. Alon, T. Gavrinyov, O. Krichevsky, Monomer dynamics in double- and single-stranded DNA polymers, Phys. Rev. Lett. 92 (2004) 048303]. Three optimization methods, global optimization, individual optimization and selected optimization are performed with the Monte Carlo method. All the optimization methods can faithfully reproduce the experimental data. In selected optimization for 2400 and 6700 bases ssDNA, lp=2.223nm and ld=0.676nm are obtained. The theoretical results show a larger persistence length for ssDNA than ordinary synthetic polymers, and the obtained length per base is larger than the reported value obtained from single molecule force measurements. The lplp and ldld obtained from mean field theory complement the current data previously measured for different salt concentrations in solution.
A study was conducted to demonstrate the mechanisms of biomolecular motors that drive the assembly and disassembly of the composite ring structure. Biotinylated microtubles were polymerized, to serve as the primary scaffold for binding streptavidin-coated quantum dots (SQDs) and assembling the nanocomposite rings. It was found that the microtubles contribute considerable mechanical strain energy to the nanocomposite ring, based on its relatively rigid nature. It was observed that the energy-dissipating component involves the chemo-mechanical transduction, which is associated with kinesin biomolecular motors in the form of ATP hydrolysis and the generation of ~40 pN nm of mechanical work, with an overall efficiency of ~50%. A kinesin-1 was purified from Drosophila melanogaster and used in gliding mobility assay, to assemble the nanocomposite rings.
In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide-field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide-field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.
Glutaraldehyde-cross-linked microtubules were investigated as substrates for kinesin motility. Microtubules, formed in vitro from chicken brain tubulin, were stabilized with Taxol and chemically fixed with glutaraldehyde. The degree of tubulin monomer cross-linking as a function of time and glutaraldehyde concentration was characterized using polyacrylamide gel electrophoresis. Atomic force microscopy of fixed microtubules indicated that the cross-linking is sufficient to stabilize the gross structure of the microtubules against air drying or a distilled water challenge. Kinesin movement on immobilized, fixed microtubules was determined using a kinesin-coated bead motility assay observed with differential interference contrast microscopy. Within measurement error, kinesin bead movement velocities were independent of the degree of microtubule cross-linking. Binding affinity, however, decreased with increased cross-linking. Although air- and water-challenged microtubules did not support kinesin motility, a dilute suspension of glutaraldehyde-fixed microtubules in buffer supported kinesin motility for at least 2 days without any substantial degradation of activity. Fixed microtubules may be useful for several applications, including affinity purification of microtubule-associated proteins and motility measurements under extreme conditions of temperature and other variables.
Division of labor is one of the most basic and widely studied aspects of colony behavior in social insects. Studies of division of labor are concerned with the integration of individual worker behavior into colony level task organization and with the question of how regulation of division of labor may contribute to colony efficiency.
Here we describe and critique the current models concerned with the proximate causes of division of labor in social insects. The models have identified various proximate mechanisms to explain division of labor, based on both internal and external factors. On the basis of these factors, we suggest a classification of the models. We first describe the different types of models and then review the empirical evidence supporting them.
The models to date may be considered preliminary and exploratory; they have advanced our understanding by suggesting possible mechanisms for division of labor and by revealing how individual and colony-level behavior may be related. They suggest specific hypotheses that can be tested by experiment and so may lead to the development of more powerful and integrative explanatory models.
Three protein motors have been unambiguously identified as rotary engines: the bacterial flagellar motor and the two motors that constitute ATP synthase (F(0)F(1) ATPase). Of these, the bacterial flagellar motor and F(0) motors derive their energy from a transmembrane ion-motive force, whereas the F(1) motor is driven by ATP hydrolysis. Here, we review the current understanding of how these protein motors convert their energy supply into a rotary torque.
Mastering supramolecular self-assembly to a similar degree as nature has achieved on a subcellular scale is critical for the efficient fabrication of complex nanoscopic and mesoscopic structures. We demonstrate that active, molecular-scale transport powered by biomolecular motors can be utilized to drive the self-assembly of mesoscopic structures that would not form in the absence of active transport. In the presented example, functionalized microtubules transported by surface-immobilized kinesin motors cross-link via biotin/streptavidin bonds and form extended linear and circular mesoscopic structures, which move in the presence of ATP. The self-assembled structures are oriented, exhibit large internal strains, and are metastable while the biomolecular motors are active. The integration of molecular motors into the self-assembly process overcomes the trade-off between stability and complexity in thermally activated molecular self-assembly.
A variety of bifunctional crosslinking agents have been explored for stabilizing microtubule shuttles used for the active transport of nanomaterials in artificial environments. Crosslinking agents that target amine residues form intertubulin crosslinks that produce crosslinked microtubules (CLMTs) with structural and functional lifetimes that can be up to four times as long as those achieved with taxol stabilization. Such CLMTs are stable at temperatures down to -10 degrees C, are resistant to depolymerization induced by metal ions such as Ca2+, and yet continue to be adsorbed and transported by self-assembled monolayers containing the motor protein kinesin. However, crosslinkers that target cysteine residues depolymerize the MTs, probably by interfering with the guanosine triphosphate binding site. The impact of crosslink attributes, including terminal group chemistry, chain length, crosslink density, and specific location on the tubulin surface, on microtubule stability and functionality are discussed.
- G M Whitesides
- B Grzybowski
G. M. Whitesides, B. Grzybowski, Science 2002, 295, 2418.
81
- S N Beshers
- J H Fewell
S. N. Beshers, J. H. Fewell, Annu. Rev. Entomol. 2001, 46, 413.
- J E Niven
J. E. Niven, Science 2012, 335, 43.
- A Kakugo
A. Kakugo, Sci. Rep. 2018, 8, 1.
- H Sada
- A Hess
- A Kuzuya
- Kakugo
Sada, H. Hess, A. Kuzuya, A. Kakugo, Nat. Commun. 2018, 9, 4.
91
- D Bachand
D. Bachand, Adv. Mater. 2008, 20, 4476.
- K Sada
- A Konagaya
- A Kakugo
K. Sada, A. Konagaya, A. Kakugo, Nanoscale 2015, 7, 18054.
- C R Wilson
- Safinya
Wilson, C. R. Safinya, Proc. Natl. Acad. Sci. U. S. A. 2004, 101,
10
16099.
- G Binnig
- C F Quate
G. Binnig, C. F. Quate, Handb. Phys. Med. Biol. 1986, 56, 5.
12
18
- C Ganser
- T Uchihashi
C. Ganser, T. Uchihashi, Methods Mol. Biol. 2022, 2430, 337.
19
22
- T Igarashi
- Ando
Igarashi, T. Ando, Rev. Sci. Instrum. 2013, 84.
- Kakugo
Kakugo, Methods Mol. Biol. 2022, 2430, 47-59.
- Q Chi
- G Wang
- J Jiang
Q. Chi, G. Wang, J. Jiang, Phys. A Stat. Mech. its Appl. 2013, 392,
35
1072.
- E H K Stelzer
- R Microsc
- Soc
E. H. K. Stelzer, R. Microsc. Soc. J. Microsc. 1998, 189, 15.