90 research outputs found
Stabilization of microtubules by encapsulation of the GFP using a Tau-derived peptide
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
Cyclic Tau-derived peptides for stabilization of microtubules
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 is 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
Radial alignment of microtubules through tubulin polymerization in an evaporating droplet
© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Keya, J. J., Kudoh, H., Kabir, A. M. R., Inoue, D., Miyamoto, N., Tani, T., Kakugo, A., & Shikinaka, K. Radial alignment of microtubules through tubulin polymerization in an evaporating droplet. Plos One, 15(4), (2020): e0231352, doi:10.1371/journal.pone.0231352.We report the formation of spherulites from droplets of highly concentrated tubulin solution via nucleation and subsequent polymerization to microtubules (MTs) under water evaporation by heating. Radial alignment of MTs in the spherulites was confirmed by the optical properties of the spherulites observed using polarized optical microscopy and fluorescence microscopy. Temperature and concentration of tubulins were found as important parameters to control the spherulite pattern formation of MTs where evaporation plays a significant role. The alignment of MTs was regulated reversibly by temperature induced polymerization and depolymerization of tubulins. The formation of the MTs patterns was also confirmed at the molecular level from the small angle X-ray measurements. This work provides a simple method for obtaining radially aligned arrays of MTs.Fund receiver: Akira Kakugo Grant-in-Aid for Scientific Research on Innovative Areas (Grant Nos. JP24104004 and 18H05423) and a Grant-in-Aid for Scientific Research (A) (Grant No. 18H03673) from kaken. NO - The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip
Microtubule bundle formation driven by ATP : the effect of concentrations of kinesin, streptavidin and microtubules
Recently, a method was established for the formation of microtubule (MT) assemblies by an active self-organization (AcSO) process, in which MTs were crosslinked during sliding motion on a kinesin-coated surface, and this was coupled with adenosine triphosphate (ATP) hydrolysis. Streptavidin (ST) was the glue used to crosslink biotin-labeled MTs. Although most of the MT assemblies were in the bundle form, they varied in size, shape and motility, depending on the initial conditions used. In this paper, we systematically examined the effects of the concentrations of kinesin, ST and MT on the formation of MT bundles under the initial conditions of the process
Gel machines constructed from chemically cross-linked actins and myosins
We report an ATP fueled soft gel machine reconstructed from muscle proteins of actin and myosin. Chemically cross-linked actin gel filaments, several decade times the length of native actin filaments (F-actin) move along a chemically cross-linked myosin fibrous gel (1 cm long and 50 μm in diameter) with a velocity as high as that of native F-actin, by coupling to ATP hydrolysis. The motility observed in muscle protein-gels suggests that one might reconstruct a soft machine fueled by chemical energy by using actin and myosin molecules as elementary elements
Complete, rapid and reversible regulation of the motility of a nano-biomolecular machine using an osmolyte trimethylamine-N-oxide
Nanoscale transportation in engineered environments is critical towards designing efficient and smart hybrid bio-nanodevices. Biomolecular motors, the smallest natural machines, are promising as actuators as well as sensors in hybrid nanodevices and hold enormous potentials in nanoscale transportation. Highly specific regulation of the activity of biomolecular motors is the key to control such integrated nanodevices. We present a simple method to regulate the activity of a biomolecular motor system, microtubule (MT)-kinesin by using a natural osmolyte trimethylamine-N-oxide (TMAO). Motility of kinesin-driven MTs in an in vitro gliding assay is regulated over a broad spectrum by using TMAO in a concentration dependent manner. The regulation of MT motility is rapid, reversible and repeatable over multiple cycles. Interestingly, the motility of MTs can be completely turned off using TMAO of a relatively high concentration. The halted motility of MTs is fully restored upon elimination of TMAO. Repeated cycles of TMAO addition and removal enable cyclical inhibition and restoration of the motility of MTs. These results demonstrate an ability to control nanoscale motion of a biomolecular motor in an artificial environment. This work facilitates further tunability over functions of biomolecular motors, which in turn will foster their nanotechnological applications, such as in nano-transportation
Effect of microtubule immobilization by glutaraldehyde on kinesin-driven cargo transport
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
Formation of motile assembly of microtubules driven by kinesins
Microtubule (MT) and kinesin are rail and motor proteins that are involved in various moving events of eukaryotic cells in natural systems. In vitro, the sliding motion of microtubules (rail) can be reproduced on a kinesin (motor protein)-coated surface coupled with adenosine triphosphate (ATP) hydrolysis, which is called a "motility assay". Based on this technique, a method was recently established to form MT assemblies by an active self-organization (AcSA) process, in which MTs are crosslinked during a sliding motion on a kinesin-coated surface. Streptavidin (ST) was employed as glue to crosslink biotin-labeled MTs. Various shapes, sizes, and motilities were formed with the AcSA MT assemblies, depending on the initial conditions. In this paper, we briefly review our recent work on the formation of MT assemblies on a kinesin-coated surface
Integration of Motor Proteins – Towards an ATP Fueled Soft Actuator
We present a soft bio-machine constructed from biological motors (actin/myosin). We have found that chemically cross-linked polymer-actin complex gel filaments can move on myosin coated surfaces with a velocity as high as that of native Factin, by coupling to ATP hydrolysis. Additionally, it is shown that the velocity of polymer-actin complex gel depends on the species of polycations binding to the F-actins. Since the design of functional actuators of well-defined size and morphology is important, the structural behavior of polymer-actin complexes has been investigated. Our results show that the morphology and growth size of polymer-actin complex can be controlled by changes in the electrostatic interactions between F-actins and polycations. Our results indicate that bio actuators with desired shapes can be created by using a polymer-actin complex
Controlling the kinetics of interaction between microtubules and kinesins over a wide temperature range using the deep-sea osmolyte trimethylamine N-oxide
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
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