Fluctuation in the Sliding Movement of Kinesin-Driven Microtubules Is Regulated Using the Deep-Sea Osmolyte Trimethylamine N-Oxide
- Arif Md. Rashedul Kabir*Arif Md. Rashedul Kabir*Email: [email protected]Faculty of Science, Hokkaido University, Sapporo 060-0810, JapanMore by Arif Md. Rashedul Kabir
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- Tasrina MunmunTasrina MunmunGraduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, JapanMore by Tasrina Munmun
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- Kazuki SadaKazuki SadaFaculty of Science, Hokkaido University, Sapporo 060-0810, JapanGraduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, JapanMore by Kazuki Sada
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- Akira Kakugo*Akira Kakugo*Email: [email protected]Faculty of Science, Hokkaido University, Sapporo 060-0810, JapanGraduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, JapanMore by Akira Kakugo
Copyright © 2022 The Authors. Published by American Chemical Society
Abstract
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.
1. Introduction
ARTICLE SECTIONS
Biomolecular motors and their associated filamentous proteins play a central role in active transportation of materials in living organisms. (1,2) In cooperation with the associated filamentous proteins, biomolecular motors utilize the chemical energy obtained from hydrolysis of adenosine triphosphate (ATP) and perform mechanical work with remarkably high energy efficiency and specific power. (3) Kinesin is a well-studied biomolecular motor that together with microtubules (MTs) comprise one of the major active transport systems in living organisms, which play pivotal roles in many cellular events. (4) MTs are hollow cylindrical protein filaments formed via polymerization of tubulin heterodimers. (5) In active transportation, MTs serve as the tracks along which kinesins carry cargoes to various locations in cells. The biomolecular motor system MT–kinesin has several attractive features such as nanometer scale, high fuel efficiency, and engineering properties, which motivates its utilization in hybrid micro/nanomechanical devices nowadays. (6−8)In vitro gliding assay serves as the platform for most of their applications in artificial environments. In an in vitro gliding assay, MTs are propelled on a substrate by surface-adhered kinesins in the presence of ATP. The in vitro gliding assay has provided valuable insights into important aspects of biomolecular motor functions. (9−11) Consequently, the MT–kinesin-based active transport system has appeared as a key technology in miniaturized micro/nanodevices for serving various purposes. So far, based on the in vitro gliding assay, the MT–kinesin system has been employed in synthetic environments for sensing, (12) nanotransportation and nanostructuring, (13,14) surface imaging, (15) characterizing surface mechanical deformation, (16) force measurement, (17) and molecular robotics. (18)
Although the in vitro gliding assay has been receiving a great deal of attention as an active transport system, controllability of the motion of shuttles (MTs) has been essential for successful applications of the biomolecular motor-based integrated, hybrid nanodevices. Several reports have attempted to control the functions of biomolecular motors in vitro by employing genetic engineering, (19,20) azobenzene-based photoswitches, (21) or tuning physicochemical parameters. (22) However, like any transport system, dispersion is an important metric for biomolecular motor-based active transportation in miniaturized devices. (23) The sensitivity or resolution of any device, based on the active transportation, can be easily affected by the dispersion in transportation. Fluctuation of the velocity of individual transporters, i.e., MTs around their time-average velocity is one of the major contributors to the dispersion in the active transport system constructed from MTs and associated biomolecular motors. (23,24) The motional diffusion coefficient can be employed as a measure of the fluctuation in the velocity of individual shuttles. (23,24) A variation in the velocity of biomolecular motor-driven discrete shuttles along their trajectories may lead to the loss of efficiency; therefore, uniformity in the transportation of the shuttles has been highly desired. However, less attention has been paid to controlling this critical metric of active transportation based on in vitro gliding assay. Consequently, there has been no report to guide on how to regulate the fluctuations in the velocity of kinesin-driven microtubule shuttles in an in vitro gliding assay. Here, we demonstrate a simple strategy to reversibly regulate the fluctuation of the velocity of kinesin-driven MTs using trimethylamine N-oxide (TMAO). TMAO is an osmolyte found in deep-sea animals at high concentrations. (25) TMAO accumulates in the tissue of deep-sea animals and plays protective roles against the protein-destabilizing effects of high temperature, pressure, chemicals, etc. (25−27) Nowadays, TMAO is commercially available and it has been employed in artificial environments for stabilizing microtubules (28) and protecting kinesin from thermal denaturation. (29) In this work, using this natural molecule, we show that without sacrificing the concentration of the fuel (ATP) used in the gliding assay system, the fluctuations in the motility of MTs can be regulated over a broad range using TMAO in a concentration-dependent manner. We have performed in vitro gliding assay of MTs on kinesins in the presence of a wide range of TMAO concentrations (0–2000 mM), where the concentration of the fuel was kept constant at a saturating level (5 mM). We monitored the motility behavior of the gliding MTs and examined the fluctuations in the movement of the MTs in the presence of various TMAO concentrations by analyzing the mean-square deviation of the sliding displacement of MTs from their average. Moreover, we estimated the motional diffusion coefficient of the kinesin-propelled MTs, which is a measure of the fluctuations in MTs’ sliding movement. Our results confirm that TMAO is effective in suppressing the fluctuations in the motility of kinesin-propelled MTs. The motional diffusion coefficient of the gliding MTs decreased upon increasing the TMAO concentration in the gliding assay. We have been able to suppress the motional diffusion coefficient of MTs ∼200-fold by employing 2 M TMAO in the gliding assay. Furthermore, we found that upon elimination of TMAO from the gliding assay, the motional diffusion coefficient of the MTs can be restored to the initial value observed in the absence of TMAO. Therefore, this work offers a facile means to reversibly regulate the fluctuations in the sliding movement of kinesin-propelled MTs in an in vitro gliding assay. Such an ability to regulate the dynamic behavior of biomolecular motor-driven shuttles is expected to foster the applications of biomolecular motors and their associated proteins in nanotechnology, material science, and bioengineering.
2. Materials and Methods
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Chemicals and Buffers
TMAO, purchased from Sigma-Aldrich, was used without further purification. BRB80 buffer was prepared maintaining the final concentration of the components as 80 mM PIPES, 1 mM MgCl2, and 1 mM EGTA. The pH of BRB80 buffer was adjusted to 6.8 using KOH. The BRB80-TMAO imaging solutions contained 5 mM ATP, 1 mM DTT, 2 mM Trolox, 1 mM MgCl2, 10 μM paclitaxel, 0.5 mg/mL casein, 4.5 mg/mL d-glucose, 50 U/mL glucose oxidase, and 50 U/mL catalase.
Purification, Labeling of Tubulin, and Preparation of MTs
Tubulin was purified from the fresh porcine brain using a high-concentration PIPES buffer (1 M PIPES, 20 mM EGTA, 10 mM MgCl2; pH adjusted to 6.8 using KOH) according to a previous report. (30) Atto550-labeled tubulin (RT) was prepared using Atto550 NHS ester (ATTO-TEC, Gmbh) according to a standard technique. (31) The labeling ratio of fluorescence dye to tubulin was ∼1.0, as determined from the absorbance of tubulin at 280 nm and fluorescence dye at 554 nm. MTs were prepared by polymerizing a mixture of RT and nonlabeled tubulin (WT) (RT:WT = 1:1; final tubulin concentration = 40 μM). A total of 4.0 μL of a mixture of RT and WT was mixed with 1 μL of a GTP premix (5 mM GTP, 20 mM MgCl2, 25% DMSO in BRB80) and incubated at 37 °C for 30 min. After polymerization, the MTs were stabilized using paclitaxel (50 μM paclitaxel in DMSO).
Expression and Purification of Motor Protein
A GFP-fused recombinant kinesin-1 construct consisting of the first 465 amino acid residues of human kinesin-1 (K465), an N-terminal histidine tag, and a C-terminal avidin tag was used to propel MTs in the in vitro gliding assay. The expression and purification of the kinesin were done as described in a previously published report. (32)
In Vitro Gliding Assay
A flow cell with dimensions of 9 × 2 × 0.1 mm3 (L × W × H) was assembled from two cover glasses 9 × 18 mm2 and 40 × 50 mm2 (MATSUNAMI) using a double-sided tape as a spacer. First, the flow cell was filled with 5 μL of a 1 mg/mL streptavidin solution (Sigma-Aldrich, S4762) and incubated for 5 min. The flow cell was then washed with wash buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, and ∼0.5 mg/mL casein; pH 6.8). Next, 5 μL of a kinesin solution (800 nM) was introduced into the streptavidin-coated flow cell. The flow cell was then incubated for 5 min to allow the binding of kinesins to the glass surface through interaction with streptavidin. After washing the flow cell with 10 μL of wash buffer, 10 μL of an MT solution (200 nM, paclitaxel stabilized GTP-MTs) was introduced and incubated for 5 min, which was followed by washing with 10 μL of wash buffer. The motility of the MTs was initiated by applying 5 μL of motility buffer containing 5 mM ATP. In the experiments where TMAO was used, 5 μL of motility buffer containing 5 mM ATP and TMAO of prescribed concentrations was infused into the flow cell. The MTs were monitored using a fluorescence microscope within 5 min of ATP buffer addition. All of the experiments were performed at room temperature (∼22 °C).
Microscopy Image Capture and Data Analysis
Samples were illuminated with a 100 W mercury lamp and visualized using an epi-fluorescence microscope (Eclipse Ti; Nikon) equipped with an oil-coupled Plan Apo 60×1.40 objective (Nikon). A filter block with UV-cut specification (TRITC: EX540/25, DM565, BA606/55; Nikon) was used in the optical path of the microscope that allowed visualization of MTs eliminating the UV part of radiation and minimized the harmful effect of UV radiation on samples. Images were captured using a cooled CMOS camera (Neo CMOS; Andor) connected to a PC. To capture images of MTs for several minutes, an ND4 filter (25% transmittance) was inserted into the illuminating light path of the fluorescence microscope to avoid photobleaching. All movies and images captured using the epi-fluorescence microscope were analyzed using an image analysis software (ImageJ 1.46r).
3. Results and Discussion
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We have explored the effect of TMAO on the sliding movement of kinesin-propelled MTs by performing in vitro gliding assay of MTs on a kinesin-coated substrate in which the concentration of TMAO was varied (Figure 1a,b). In brief, a flow cell was prepared on a glass substrate and kinesin motors were adsorbed to the flow cell. MTs, polymerized from tubulin dimers using GTP and stabilized using paclitaxel, were attached to the kinesin-coated surface of the flow cell. The motility of the MTs was initiated by introducing the motility buffer to the flow cell. The concentration of ATP was maintained at a saturating level (5 mM), whereas the concentration of TMAO varied between 0 and 2000 mM. After initiating the motility of MTs, we monitored the gliding MTs using a fluorescence microscope and investigated their motility behavior by examining fluctuations in the movement of individual MTs.
Figure 1
Figure 1. Schematic representation of (a) molecular structure of TMAO and (b) in vitro gliding assay of MTs on a kinesin-coated glass substrate in the presence of TMAO. In the molecular structure of TMAO, the red, blue, black, and white spheres denote the oxygen, nitrogen, carbon, and hydrogen atoms, respectively. “+ATP” indicates the use of ATP in the in vitro gliding assay. (c) Representative data show the instantaneous velocity of an MT with time where the velocity was quantified over a 10 s interval. Abrupt changes in the velocity of MTs with time were observed in the absence of TMAO, which diminished upon increasing the concentration of TMAO.
First, we examined the instantaneous velocity of the MT filaments along their trajectories. As shown by the representative data in Figure 1c, the instantaneous velocity of a single MT along its gliding trajectory fluctuates frequently when TMAO was not used in the gliding assay (Movie S1). On contrary, in the presence of TMAO (e.g., 1200 mM), such abrupt fluctuations in the instantaneous velocity of MTs seems to be diminished (Movie S2), which could be clearly observed in Figure 1c, when the concentration of TMAO was increased further (e.g., 2000 mM). To confirm the suppression of the fluctuation in MTs’ velocity in the presence of TMAO, we plotted the histogram of the instantaneous velocity of MTs over 10 s intervals (Figure 2), where the number of MTs considered was 50 in each condition. In the absence of TMAO, the instantaneous velocity of MTs was found to be distributed over a wide range around the mean value. Upon introducing the TMAO in the gliding assay (e.g., 400 or 800 mM), the range of distribution of the instantaneous velocity of MTs became narrower. The range of the instantaneous velocity of MTs became even smaller upon increasing the TMAO concentration further in the gliding assay (e.g., 1500, 2000 mM). Thus, these results clearly indicate that TMAO affects the dynamic behavior of kinesin-propelled MTs in the gliding assay. It is to note that we also noticed a gradual shift of the histograms of MT velocity toward lower values, which agrees to past reports on kinesin-propelled MTs or myosin-propelled actin filaments. (33,34)
Figure 2
Figure 2. Histograms show the distribution of the instantaneous velocity of MTs in the absence and in the presence of TMAO of various concentrations. The concentration of TMAO is mentioned inside the histograms in each case, and “no TMAO” indicates the absence of TMAO. As the concentration of TMAO in the gliding assay was increased, the histograms became narrower and shifted toward lower velocity values. The red lines represent the fitting of the histograms using the equation of Gaussian distribution.
To quantify the TMAO-mediated diminution of the distribution range of MTs’ instantaneous velocity, we fitted the histograms according to the equation of Gaussian distribution and estimated the mean and standard deviation in each case. The results, shown in Figure S1, reveal that the standard deviation of MT velocity around the mean decreases upon increasing the TMAO concentration in the gliding assay. A similar trend was observed even when we considered the standard deviation obtained from the arithmetic mean of MT velocity, instead of the standard deviation and the mean estimated from Gaussian fitting (Figure S2). To confirm that the observed behavior is not a measurement artifact related to lower velocities of MTs at higher TMAO concentrations, the standard deviation was plotted as a function of the average velocity of MTs for each TMAO concentration (Figures S1 and S2). The standard deviation around the mean velocity of MTs was found to change nonlinearly with MT velocity, which suggests that the narrowing of the distribution range of the instantaneous velocity of MTs is due to the presence of TMAO in the gliding assay, not due to the reduction in MT velocity.
To gain a deeper insight into the dynamic behavior of MTs in the presence of varying concentrations of TMAO, we examined the fluctuations in the movement of individual MTs. We calculated the mean-square deviation of the sliding displacement of MTs from the average with averaging of multiple trajectories, (23,24,35) which when plotted as a function of time produces a diffusion coefficient of the motile MTs according to the following equation
Here, <(ΔXv-flu)2> = <(ΔX(t) – vΔt)2>, where X(t) is the position of MT filaments along their trajectories, ΔX(t) = X(t + Δt) – X(t) is the displacement of the filaments during time Δt, “v” is the mean velocity of MTs, and Dv-flu is the diffusion coefficient of MTs, which is termed in the literature as the motional diffusion coefficient, (23,24) and is a measure of the fluctuations of the sliding movement of MTs propelled by kinesin motors. In calculating the mean-square deviation of the sliding displacement of MTs from the average, we ignored short MTs and considered only the relatively long MTs with a length of >15 μm. This is because the motional diffusion coefficient of such long MTs is independent of their length and in-feed kinesin concentration in the gliding assay. (24) As shown in Figure 3, the mean-square displacement deviation from the average increases linearly with time both in the absence and in the presence of TMAO of various concentrations. In this figure, the solid lines for each data set represent the regression line and the motional diffusion coefficient of MTs is estimated as 0.5 times of the slope of these lines. Our results reveal that the motional diffusion coefficient of MTs gradually decreased with increasing the concentration of TMAO in the gliding assay (Figure 3). The motional diffusion coefficient of MTs was 0.125 ± 0.0047 μm2 /s in the absence of TMAO, which dropped to 6.7 × 10–4 ± 2.2 × 10–5 μm2 /s when 2000 mM TMAO was used. This result confirms that fluctuation in MT velocity has been suppressed by more than 200-fold using TMAO in the gliding assay.
Figure 3
Figure 3. Mean-square displacement deviation of MTs from the average as a function of time, calculated by “multiple trajectories averaging” over 50 different trajectories of MTs, in the absence and in the presence of different TMAO concentrations. Error bars are not shown here for simplicity. The solid lines indicate the linear regression fit of the data in each condition (left). Change in the motional diffusion coefficient of MTs, propelled by kinesins in an in vitro gliding assay, upon changing the TMAO concentration in the gliding assay (right). Error bar: standard deviation.
Thus, based on these results, it can be confirmed that fluctuations in the sliding movement of MTs are modulated using TMAO in an in vitro gliding assay. From our results, it appears that TMAO suppresses both the velocity of MTs and dispersion in MT velocity. Using TMAO, the uniform motion of MTs seems to be achieved at the expense of their suppressed motion. However, the advantage of using TMAO is that we do not need to decrease the ATP concentration or alter other physicochemical parameters to lower the MT velocity or suppress the dispersion of MT velocity. In addition, we have performed further experiments to confirm whether TMAO plays any direct role in ensuring the uniform motion of MTs. We demonstrated in vitro gliding assay of MTs, in the absence of TMAO, by decreasing the ATP concentration from 5 mM to 50 μM. The average velocity of MTs was 106 ± 18 nm/s using 50 μM ATP, which is close to the velocity of MTs (116 ± 16 nm/s) observed using 1000 mM TMAO and 5 mM ATP. In these conditions, the motional diffusion coefficients of MTs were estimated to be 0.032 ± 0.002 and 0.021 ± 0.001 μm2/s when the average MT velocities were 106 ± 18 nm/s (50 μM ATP, no TMAO) and 116 ± 16 nm/s (5 mM ATP, 1000 mM TMAO), respectively. This result clearly reveals that TMAO plays a direct role as a regulator of dispersion in MT velocity.
According to the literature, TMAO can suppress the motility of kinesin-propelled MTs and myosin-driven actin filaments. (33,34) In these works, the motility of MTs or actin filaments was characterized as the time-average velocity of ensembles, which kept the motility behavior of individual shuttles concealed. Furthermore, any effect of TMAO on the fluctuation in velocity of individual MTs was not investigated in these past works, which has been unraveled by the above results. Recently, the use of TMAO also enabled the regulation of MT motility in a reversible manner. (33) Therefore, we have been motivated to investigate if the fluctuation in velocity of individual MTs can also be reversibly regulated using TMAO. To examine, first we performed a gliding assay of MTs on kinesins in the absence of TMAO; then, we applied 1000 mM TMAO in the flow cell by mixing with ATP buffer. Next, by extensive washing of the flow cell, we eliminated the TMAO from the gliding assay. Our results reveal that upon application of 1000 mM TMAO, the range of distribution of the instantaneous velocity of MTs became narrower, which returned to the initial broader range after elimination of the TMAO from the gliding assay (Figure 4). In each condition, we analyzed the mean-square deviation of MT displacement from the average displacement with “multiple trajectory averaging” (Figure 5), based on which we could also confirm the reversibility. Initially, the motional diffusion coefficient of MTs was 0.13 ± 0.002 μm2/s in the absence of TMAO, which was reduced to 0.02 ± 0.0006 μm2/s using 1000 mM TMAO. After the elimination of TMAO, the motional diffusion coefficient of MTs was returned to 0.12 ± 0.002 μm2/s (Figure 5). Such reversible regulation of the fluctuations in MT motility using TMAO coincides with the reversible regulation of the activity of kinesins using TMAO. (33) Overall, these results confirm that the utilization of TMAO offers a simple means to regulate the fluctuations in the sliding movement of kinesin-propelled MTs in a reversible manner.
Figure 4
Figure 4. Reversible regulation of the distribution of instantaneous velocity of MTs using TMAO. The distribution of MT velocity in the absence of TMAO (top), in the presence of 1 M TMAO (middle), and after elimination of the TMAO, i.e., in the absence of TMAO (bottom). The red lines represent fitting of the histograms using the equation of Gaussian distribution.
Figure 5
Figure 5. Mean-square displacement deviation of MTs from the average as a function of time, calculated by “multiple trajectories averaging” over 50 different trajectories of MTs, in the absence of TMAO, upon application of 1 M TMAO, and upon elimination of the TMAO (left). Error bars are not shown here for simplicity. The solid lines indicate the linear regression fit of the data in each condition. Reversible regulation of the motional diffusion coefficient of MTs using 1 M TMAO (right). Error bar: standard deviation.
The motional diffusion coefficient of biomolecular motor-driven MTs is contributed by two factors: thermally generated fluctuations of MT filaments and fluctuations in active force of the kinesin motors. The motional diffusion coefficient is helpful in predicting friction between MTs and kinesin motors. A large value of diffusion coefficient indicates a small friction force between MTs and motors. (24) The observed decrease in the motional diffusion coefficient of the MTs, in the presence of TMAO, suggests higher friction between MTs and kinesins. (24) Such an increase in friction between shuttles and motor proteins may work as a velocity-limiting factor for the shuttles (36) or motors. (37) Decrease in MT velocity upon increasing the TMAO concentration in the gliding assay (33,38) could be accounted for by such an increase in friction between MTs and kinesins mediated by TMAO. Friction between MTs and kinesins may increase when the transition between mechanical states of kinesin is suppressed. (37) Future investigations are required to understand the effect of TMAO on the mechanochemical cycles and energy efficiency of kinesins.
Although, based on our results, TMAO is found useful in controlling the fluctuations in the sliding movement of MTs, the associated mechanism behind such modulation is unclear at this moment. Despite that, defective motors, motor orientation or attachment geometry of motors to MTs, and stiffness of motors have been suspected to play important roles in the fluctuation in the sliding movement of MTs at saturating ATP concentrations. Recently, an increased frequency of pinning of gliding MTs in the presence of TMAO was reported. (39) Pinning of MTs by defective motors is known to increase the motional diffusion coefficient. (23) However, in our work, we observed a decrease in the motional diffusion coefficient of MTs upon increasing the TMAO concentration in the gliding assay. Moreover, we carefully selected smooth MT trajectories to exclude the effect of MT pinning on our results. Thus, based on these arguments, we can rule out any involvement of pinning of MTs in the altered motional diffusion coefficients of MTs observed in our work. On the other hand, the deep-sea osmolyte TMAO is known to stabilize the biomolecular motor proteins through alteration in their surrounding microenvironments, which is also known to affect the structure of motor proteins. (34) Such alterations in the motor structure may affect the mechanical property, e.g., stiffness of kinesin motors or kinesin’s attachment geometry to MTs, which, in turn, may alter the motional diffusion coefficient of MTs in the gliding assay. (40) Further investigation will be required in future to unravel the mechanism behind TMAO-mediated alteration in fluctuations of the motility of the kinesin-propelled MTs.
Although fluctuation of the velocity of the kinesin-propelled MTs has been regulated using TMAO, the velocity of MTs has also been sacrificed in this process. The decreased velocity of MTs may adversely impact their applications; for example, due to the slower velocity of MTs, a relatively longer time would be required to deliver nanomaterials to a target destination. The experimental conditions should be optimized considering a trade-off between MT velocity and fluctuation in their velocity. In this work, we have investigated the dynamic behavior of kinesin-propelled microtubules in the presence of TMAO without sacrificing the fuel (ATP) concentration. Therefore, we maintained a saturating concentration of ATP (5 mM) in our experiments. On the one hand, this strategy will permit using the available ATP for operating any other biomolecular processes together with our presented experimental system of MT–kinesin. On the other hand, use of a high ATP concentration will allow the kinesins to propel MTs for longer time. Considering the emerging applications of biomolecular motors in molecular machine, molecular robotics, micro/nanodevices, synthetic biology, etc., these two advantages would be important. Furthermore, we have performed in vitro gliding assay of MTs, in the absence of TMAO, by decreasing the ATP concentration from 5 mM to 50 μM. The average velocity of MTs was 106 ± 18 nm/s using 50 μM ATP, which is close to the velocity of MTs (116 ± 16 nm/s) observed using 1000 mM TMAO and 5 mM ATP. In these conditions, the motional diffusion coefficients of MTs were estimated to be 0.032 ± 0.002 and 0.021 ± 0.001 μm2/s when the average MT velocities were 106 ± 18 nm/s (50 μM ATP, no TMAO) and 116 ± 16 nm/s (5 mM ATP, 1000 mM TMAO), respectively. This result confirms that the suppressed velocity of MTs was not the sole factor in controlling uniformity in MT motion and TMAO plays a direct role as a regulator of dispersion in MT velocity.
Finally, it is to note that we have also performed in vitro gliding assay of MTs on kinesin where we decreased the concentration of ATP from 5 to 1 mM, which is still a saturating concentration. (22) Here, the concentration of MgCl2 was 1 mM, whereas the concentration of TMAO was 1000 mM. In this experimental condition, the velocity of MTs was 108 ± 15 nm/s, whereas the velocity of MTs in the absence of TMAO was 313 ± 22 nm/s using 1 mM ATP. This result confirms that the decrease in the gliding velocity of MTs or suppression of fluctuation of the MT velocity in the presence of TMAO, as discussed above, was not due to any decrease in the effective concentration of ATP. It was the TMAO that caused such a decrease in the gliding velocity of MTs, possibly by altering the activity of kinesin motors, confirmation of which requires further investigation in the future.
4. Conclusions
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Utilizing the deep-sea osmolyte TMAO, we have successfully regulated the fluctuation in the sliding movement of kinesin-propelled MTs, in an in vitro gliding assay, in a reversible manner. The motional diffusion coefficient of the motile MTs is tuned over a wide range, even at a saturating fuel concentration, simply by varying the concentration of TMAO. Fluctuations in the motility of kinesin-propelled MTs were reported to be affected by the length of MTs and kinesin density on the substrate. However, any report that could offer an efficient strategy to systematically regulate the fluctuations in motility of MTs was lacking. This work provides such a guideline to control the dynamic behavior of the self-propelled biomolecular motor system MT–kinesin. An ability to control the fluctuations in motility of biomolecular motor-propelled shuttles, which is an important design metric in miniaturized lab-on-a-chip devices, is expected to further advance the applications of biomolecular motors in nanotechnology, materials science, and other related fields. (41,42)
Supporting Information
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c01228.
Additional experimental results on the effect of TMAO on the standard deviation of the MT velocity and standard deviation/average velocity of MTs, mean-square deviation of the displacement of MTs as a function of travel time at various TMAO concentrations, reversible change of the mean-square deviation of the displacement of MTs as a function of travel time at 1000 mM TMAO, and captions for supporting movies (PDF)
Motility of MTs on kinesins, in an in vitro gliding assay, in the absence of TMAO. The field of view is 237.6 μm × 281.6 μm (Movie S1) (MP4)
Motility of MTs on kinesins, in an in vitro gliding assay, in the presence of 1200 mM TMAO. The field of view is 237.6 μm × 281.6 μm (Movie S2) (MP4)
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Author Information
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- Arif Md. Rashedul Kabir - Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan;
https://orcid.org/0000-0001-6367-7690;
- Akira Kakugo - Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan; Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, Japan;https://orcid.org/0000-0002-1591-867X;
- Kazuki Sada - Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan; Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, Japan;https://orcid.org/0000-0001-7348-0533
A.M.R.K. and T.M. contributed equally to this work. A.M.R.K. contributed to conceptualization; A.M.R.K. and A.K. contributed to funding acquisition; T.M. and A.M.R.K. contributed to methodology; T.M. contributed to material preparation and investigation; T.M. and A.M.R.K. contributed to data analysis; A.M.R.K. contributed to project administration and supervision; A.M.R.K. contributed to original draft writing; and A.M.R.K., T.M., K.S., and A.K contributed to review and editing of the manuscript.
Acknowledgments
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This work was supported by a research grant (PK22201017) from the Hirose Foundation and JSPS KAKENHI Grant Numbers JP21K04846 and JP20H05972 awarded to A.M.R.K and JSPS KAKENHI Grant Numbers JP18H05423, JP21H04434, and JP21K19877 awarded to A. K.
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- 4Vale, R. D.; Fletterick, R. J. The design plan of kinesin motors. Annu. Rev. Cell Dev. Biol. 1997, 13, 745– 777, DOI: 10.1146/annurev.cellbio.13.1.745[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXisFSrtQ%253D%253D&md5=d0345a9315a5a8f1a61066e05b610d81The design plan of kinesin motorsVale, Ronald D.; Fletterick, Robert J.Annual Review of Cell and Developmental Biology (1997), 13 (), 745-777CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)A review, with ∼80 refs. The kinesin superfamily comprises a large and structurally diverse group of microtubule-based motor proteins that produce a variety of force-generating activities within cells. This review addresses how the structures of kinesin proteins provide clues as to their biol. functions and motile properties. We discuss structural features common to all kinesin motors, as well as specialized features that enable subfamilies of related motors to carry out specialized activities. We also discuss how the kinesin motor domain uses chem. energy from ATP hydrolysis to move along microtubules.
- 5Kirschner, M.; Mitchison, T. Beyond self-assembly: from microtubules to morphogenesis. Cell 1986, 45, 329– 342, DOI: 10.1016/0092-8674(86)90318-1[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XktVahtbk%253D&md5=c772d87269a329ea68ce7ca4efe86a41Beyond self-assembly: from microtubules to morphogenesisKirschner, Marc; Mitchison, TimCell (Cambridge, MA, United States) (1986), 45 (3), 329-42CODEN: CELLB5; ISSN:0092-8674.A review, with 84 refs., of the spatial organization of microtubules, with emphasis on the biochem. properties of the microtubule polymers themselves. Topics discussed include the GTP hydrolysis activity of tubulin, microtubule dynamics, and the relation of these dynamics to morphogenesis.
- 6van den Heuvel, M. G. L.; Dekker, C. Motor proteins at work for nanotechnology. Science 2007, 317, 333– 336, DOI: 10.1126/science.1139570[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnslGrs7Y%253D&md5=96877d4426278f133b82783e7e0ac9c7Motor Proteins at Work for Nanotechnologyvan den Heuvel, Martin G. L.; Dekker, CeesScience (Washington, DC, United States) (2007), 317 (5836), 333-336CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. The biol. cell is equipped with a variety of mol. machines that perform complex mech. tasks such as cell division or intracellular transport. One can envision employing these biol. motors in artificial environments. The authors review the progress that has been made in using motor proteins for powering or manipulating nanoscale components. In particular, kinesin and myosin biomotors that move along linear biofilaments have been widely explored as active components. Currently realized applications are merely proof-of-principle demonstrations. Yet, the sheer availability of an entire ready-to-use toolbox of nanosized biol. motors is a great opportunity that calls for exploration.
- 7Jia, Y.; Li, J. Molecular assembly of rotary and linear motor proteins. Acc. Chem. Res. 2019, 52, 1623– 1631, DOI: 10.1021/acs.accounts.9b00015[ACS Full Text
], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXkvFWku7Y%253D&md5=7e1466038e239f9376b18a176ffc5b0cMolecular Assembly of Rotary and Linear Motor ProteinsJia, Yi; Li, JunbaiAccounts of Chemical Research (2019), 52 (6), 1623-1631CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Mol. machines are an important and emerging frontier in research encompassing interdisciplinary subjects of chem., physics, biol., and nanotechnol. Although there has been major interest in creating synthetic mol. machines, research on natural mol. machines is also crucial. Biomol. motors are natural mol. machines existing in nearly every living systems. They play a vital role in almost every essential process ranging from intracellular transport to cell division, muscle contraction and the biosynthesis of ATP that fuels life processes. The construction of biomol. motor-based biomimetic systems can help not only to deeply understand the mechanisms of motor proteins in the biol. process but also to push forward the development of bionics and biomol. motor-based devices or nanomachines. From combination of natural biomol. motors with supramol. chem., great opportunities could emerge toward the development of intelligent mol. machines and biodevices. In this Account, the authors describe the efforts to design and reconstitute biomol. motor-based active biomimetic systems, in particular, the combination of motor proteins with layer-by-layer (LbL) assembled cellular structures. They are divided into two parts: (i) reconstitution of rotary mol. motor FOF1-ATPase, which is coated on the surface of LbL assembled microcapsules or multilayers and synthesizes ATP through creating a proton gradient; (ii) mol. assembly of linear mol. motors, the kinesin-based active biomimetic systems, which are coated on a planar surface or LbL assembled tubular structure and drive the movement of microtubules. LbL assembled structures offer motor proteins with an environment that resembles the natural cell. This enables high activity and optimized function of the motor proteins. The assembled biomol. motors can mimic their functionalities from the natural system. In addn., LbL assembly provides facile integration of functional components into motor protein-based active biomimetic systems and achieves the manipulation of FOF1-ATPase and kinesin. For FOF1-ATPase, the light-driven proton gradient and controlled ATP synthesis are highlighted. For kinesin, the strategies used for the direction and velocity control of kinesin-based mol. shuttles are discussed. The authors hope this research can inspire new ideas and propel the actual applications of biomol. motor-based devices in the future. - 8Hess, H. Engineering applications of biomolecular motors. Annu. Rev. Biomed. Eng. 2011, 13, 429– 450, DOI: 10.1146/annurev-bioeng-071910-124644[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFCit7rM&md5=7fac9da4248e5be62047a541b6d4a458Engineering applications of biomolecular motorsHess, HenryAnnual Review of Biomedical Engineering (2011), 13 (), 429-450CODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews Inc.)A review. Biomol. motors, in particular motor proteins from the kinesin and myosin families, can be used to explore engineering applications of mol. motors in general. Their outstanding performance enables the exptl. study of hybrid systems, where bio-inspired functions such as sensing, actuation, and transport rely on the nanoscale generation of mech. force. Scaling laws and theor. studies demonstrate the optimality of biomol. motor designs and inform the development of synthetic mol. motors.
- 9Harada, Y.; Noguchi, A.; Kishino, A.; Yanagida, T. Sliding movement of single actin filaments on one-headed myosin filaments. Nature 1987, 326, 805– 808, DOI: 10.1038/326805a0[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXitV2kt7o%253D&md5=7ee287fd278954280675780996e20551Sliding movement of single actin filaments on one-headed myosin filamentsHarada, Yoshie; Noguchi, Akira; Kishino, Akiyoshi; Yanagida, ToshioNature (London, United Kingdom) (1987), 326 (6115), 805-8CODEN: NATUAS; ISSN:0028-0836.The myosin mol. consists of 2 heads, each of which contain an enzymic active site and an actin-binding site. The fundamental problem of whether the 2 heads function independently or cooperatively during muscle contraction was studied by an assay system in which sliding movements of fluorescently labeled, single actin filaments along myosin filaments can be obsd. directly. Direct measurement of the sliding of single actin filaments along 1-headed myosin filaments are reported in which the d. of heads was varied over a wide range. The results show that cooperative interaction between the 2 heads of myosin is not essential for inducing the sliding movement of actin filaments.
- 10Toyoshima, Y. Y.; Kron, S. J.; Spudich, J. A. The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assay. Proc. Natl. Acad. Sci. 1990, 87, 7130– 7134, DOI: 10.1073/pnas.87.18.7130[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXmt1ymsLY%253D&md5=9913f44f6c94d6059cbc769015a90323The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assayToyoshima, Yoko Y.; Kron, Stephen J.; Spudich, James A.Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (18), 7130-4CODEN: PNASA6; ISSN:0027-8424.Chemomech. coupling in muscle contraction may be due to swinging crossbridges, such that a change in the angle at which the myosin head binds to the actin filament is tightly coupled to release of products of ATP hydrolysis. This model would limit the step size, the unit displacement of actin produced by a single ATP hydrolysis, to less than twice the chord length of the myosin head. Recent measurements have found the step size to be significantly larger than this geometric limit, bringing into question any direct correspondence between the crossbridge and ATP-hydrolysis cycles. The rate of ATP hydrolysis due to actin sliding movement was measured in an in vitro motility assay consisting of purified actin and purified myosin. An apparent myosin step size was calcd. well within the geometric limit set by the size of the myosin head. These data are consistent with tight coupling between myosin crossbridge movement and ATP hydrolysis.
- 11Uyeda, T. Q.; Abramson, P. D.; Spudich, J. A. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc. Natl. Acad. Sci. 1996, 93, 4459– 4464, DOI: 10.1073/pnas.93.9.4459[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XivVKqtLY%253D&md5=d19fd6188a063200d490e1d677cc7abbThe neck region of the myosin motor domain acts as a lever arm to generate movementUyeda, Taro Q. P.; Abramson, Paul D.; Spudich, James A.Proceedings of the National Academy of Sciences of the United States of America (1996), 93 (9), 4459-4464CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long α-helical portion of the heavy chain. The swinging neck-lever model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The ΔRLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The ΔBLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.
- 12Fischer, T.; Agarwal, A.; Hess, H. A smart dust biosensor powered by kinesin motors. Nat. Nanotechnol. 2009, 4, 162– 166, DOI: 10.1038/nnano.2008.393[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXis12nsL4%253D&md5=36eac4027e98cde3a472a2addd78224eA smart dust biosensor powered by kinesin motorsFischer, Thorsten; Agarwal, Ashutosh; Hess, HenryNature Nanotechnology (2009), 4 (3), 162-166CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Biosensors can be miniaturized by injecting smaller vols. into micro- and nano-fluidic devices or immersing increasingly sophisticated particles, i.e., smart dust, into the sample. The term, smart dust, originally referred to mm3 wireless semi-conducting sensors which could invisibly monitor the environment in buildings and public spaces; it also included functional μm-sized porous Si particles used to monitor even smaller environments. The principal challenge in designing smart dust biosensors is integrating transport functions with energy supply in the device. A hybrid micro-device powered by ATP and relying on antibody-functionalized micro-tubules and kinesin motors to transport target analytes into a detection region is described. The transport step replaces the wash step in traditional double-antibody sandwich assays. Due to their small size and autonomous function, it is envisioned that large nos. of smart dust biosensors could be inserted into organisms or distributed in the environment for remote sensing.
- 13Bachand, G. D.; Rivera, S. B.; Boal, A. K.; Gaudioso, J.; Liu, J.; Bunker, B. C. Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins. Nano Lett. 2004, 4, 817– 821, DOI: 10.1021/nl049811h[ACS Full Text], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXisVyitLk%253D&md5=74898ec172a5204c765880fdf04c59b3Assembly and Transport of Nanocrystal CdSe Quantum Dot Nanocomposites Using Microtubules and Kinesin Motor ProteinsBachand, George D.; Rivera, Susan B.; Boal, Andrew K.; Gaudioso, Jennifer; Liu, Jun; Bunker, Bruce C.Nano Letters (2004), 4 (5), 817-821CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Nature has evolved dynamic, non-equil. mechanisms for assembling hierarchical complexes of nanomaterials. A crit. element to many of these assembly mechanisms involves the active and directed transport of materials by biomol. motor proteins such as kinesin. In the present work, nanocrystal quantum dots (nQDs) were assembled and organized using microtubule (MT) filaments as nanoscale scaffolds. NQD d. and localization were systematically evaluated by varying the concn. and distribution of functional groups within the MT structure. Confining nQD attachment to a central region within the MT enabled unaffected interaction with kinesin necessary to support active transport of nQD-MT composites. This active transport system will be further refined to control the optical properties of a surface by regulating the collective organization of nQD-MT composites.
- 14Bachand, G. D.; Rivera, S. B.; Carroll-Portillo, A.; Hess, H.; Bachand, M. Active capture and transport of virus particles using a biomolecular motor-driven, nanoscale antibody sandwich assay. Small 2006, 2, 381– 385, DOI: 10.1002/smll.200500262[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhs1aqsLo%253D&md5=33656a1ef85741449a0da49d9971bc49Active capture and transport of virus particles using a biomolecular motor-driven, nanoscale antibody sandwich assayBachand, George D.; Rivera, Susan B.; Carroll-Portillo, Amanda; Hess, Henry; Bachand, MarleneSmall (2006), 2 (3), 381-385CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Virus particles are captured and transported using kinesin-driven, antibody-functionalized microtubules. The functionalization was achieved through covalent crosslinking, which consequently enhanced the microtubule stability. The capture and transport of the virus particles was subsequently demonstrated in gliding motility assays in which antibody-coated microtubules functioned as capture elements, and antibody-coated microspheres served as fluorescent reporters (see Figure).
- 15Hess, H.; Clemmens, J.; Howard, J.; Vogel, V. Surface imaging by self-propelled nanoscale probes. Nano Lett. 2002, 2, 113– 116, DOI: 10.1021/nl015647b[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXovF2mtbY%253D&md5=f3bbe03f84bb90b85d15c1514e55d8c3Surface imaging by self-propelled nanoscale probesHess, Henry; Clemmens, John; Howard, Jonathon; Vogel, ViolaNano Letters (2002), 2 (2), 113-116CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)A new approach to image microscopic surface properties is introduced. Information about surface properties such as topog. is obtained by repeated acquisition of an optical signal from a large no. of microscopic, self-propelled probes moving on random paths across a surface. These self-propelled probes sample the surface in a statistical process in contrast to the deterministic, linear sampling performed by a scanning probe microscope. This method is exptl. demonstrated using microtubules as probes, which are moved by the motor protein kinesin.
- 16Inoue, D.; Nitta, T.; Kabir, A. M. R.; Sada, K.; Gong, J. P.; Konagaya, A.; Kakugo, A. Sensing surface mechanical deformation using active probes driven by motor proteins. Nat. Commun. 2016, 7, 12557 DOI: 10.1038/ncomms12557[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1amtLrM&md5=a8a291a690f1a13623409383a1fd3433Sensing surface mechanical deformation using active probes driven by motor proteinsInoue, Daisuke; Nitta, Takahiro; Kabir, Arif Md. Rashedul; Sada, Kazuki; Gong, Jian Ping; Konagaya, Akihiko; Kakugo, AkiraNature Communications (2016), 7 (), 12557CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Studying mech. deformation at the surface of soft materials has been challenging due to the difficulty in sepg. surface deformation from the bulk elasticity of the materials. Here, we introduce a new approach for studying the surface mech. deformation of a soft material by utilizing a large no. of self-propelled microprobes driven by motor proteins on the surface of the material. Information about the surface mech. deformation of the soft material is obtained through changes in mobility of the microprobes wandering across the surface of the soft material. The active microprobes respond to mech. deformation of the surface and readily change their velocity and direction depending on the extent and mode of surface deformation. This highly parallel and reliable method of sensing mech. deformation at the surface of soft materials is expected to find applications that explore surface mechanics of soft materials and consequently would greatly benefit the surface science.
- 17Hess, H.; Howard, J.; Vogel, V. A piconewton forcemeter assembled from microtubules and kinesins. Nano Lett. 2002, 2, 1113– 1115, DOI: 10.1021/nl025724i[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhs1enuw%253D%253D&md5=c7751c9c2a49ea4de9f18275ac63328dA piconewton forcemeter assembled from microtubules and kinesinsHess, Henry; Howard, Jonathon; Vogel, ViolaNano Letters (2002), 2 (10), 1113-1115CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)A forcemeter capable of detecting piconewton forces was assembled from nanoscale building blocks, using a cantilevered microtubule as a beam of known stiffness, which is loaded by a second microtubule transported by kinesin motor proteins adsorbed to the surface and connected to the cantilevered microtubule by the link whose strength is to be detd. Due to the loading rate of less than 1 pN/s, this forcemeter is ideally suited to study the strength of biol. receptor/ligand pairs, such as streptavidin/biotin.
- 18Keya, J. J.; Suzuki, R.; Kabir, A. M. R.; Inoue, D.; Asanuma, H.; Sada, K.; Hess, H.; Kuzuya, A.; Kakugo, A. DNA-assisted swarm control in a biomolecular motor system. Nat. Commun. 2018, 9, 453 DOI: 10.1038/s41467-017-02778-5[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvlvFGisA%253D%253D&md5=36fd4c5b686e854308f74e26d8a654a6DNA-assisted swarm control in a biomolecular motor systemKeya Jakia Jannat; Suzuki Ryuhei; Sada Kazuki; Kakugo Akira; Kabir Arif Md Rashedul; Inoue Daisuke; Sada Kazuki; Kakugo Akira; Asanuma Hiroyuki; Hess Henry; Kakugo Akira; Kuzuya AkinoriNature communications (2018), 9 (1), 453 ISSN:.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.
- 19Liu, H.; Schmidt, J. J.; Bachand, G. D.; Rizk, S. S.; Looger, L. L.; Hellinga, H. W.; Montemagno, C. D. Control of a biomolecular motor-powered nanodevice with an engineered chemical switch. Nat. Mater. 2002, 1, 173– 177, DOI: 10.1038/nmat761[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptV2qsL0%253D&md5=fe4435470ba89d1169ffd0114340cda7Control of a biomolecular motor-powered nanodevice with an engineered chemical switchLiu, Haiqing; Schmidt, Jacob J.; Bachand, George D.; Rizk, Shahir S.; Looger, Loren L.; Hellinga, Homme W.; Montemagno, Carlo D.Nature Materials (2002), 1 (3), 173-177CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)A mutant F1-ATPase motor contg. a metal-binding site that functions as a zinc-dependent, reversible on/off switch has been constructed and analyzed. Repeated cycles of zinc addn. and removal by chelation result in inhibition and restoration, resp., of both ATP hydrolysis and motor rotation of the mutant, but not of the wild-type F1 fragment. The results demonstrate the ability to engineer chem. regulation into a biomol. motor and represent a crit. step towards controlling integrated nanomech. devices at the single-mol. level.
- 20Konishi, K.; Uyeda, T. Q.; Kubo, T. Genetic engineering of a Ca2+ dependent chemical switch into the linear biomotor kinesin. FEBS Lett. 2006, 580, 3589– 3594, DOI: 10.1016/j.febslet.2006.05.037[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmtVOmsbk%253D&md5=8810143a5de2830e07cb4b5eb33374fdGenetic engineering of a Ca2+ dependent chemical switch into the linear biomotor kinesinKonishi, Kaoru; Uyeda, Taro Q. P.; Kubo, TaiFEBS Letters (2006), 580 (15), 3589-3594CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca2+-calmodulin, the assocn. of the engineered kinesin with microtubules showed a steep Ca2+-dependency in ATPase activity at Ca2+ concns. of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca2+-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca2+-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.
- 21Amrutha, A. S.; Kumar, S. K. R.; Tamaoki, N. Azobenzene-based photoswitches facilitating reversible regulation of kinesin and myosin motor systems for nanotechnological applications. ChemPhotoChem 2019, 3, 337– 346, DOI: 10.1002/cptc.201900037[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnslehsbk%253D&md5=57a4459a09f7c6d5ca6c96a037791e8aAzobenzene-Based Photoswitches Facilitating Reversible Regulation of Kinesin and Myosin Motor Systems for Nanotechnological ApplicationsAmrutha, Ammathnadu S.; Sunil Kumar, K. R.; Tamaoki, NobuyukiChemPhotoChem (2019), 3 (6), 337-346CODEN: CHEMYH ISSN:. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The past decade has witnessed several significant efforts made towards implementing the biomol. functions of motor proteins for nanotechnol. applications. However, employing motor protein systems in artificial environments demands for a precise and flexible spatiotemporal control mechanism. Light being an ideal external stimulus offers major advantages, allowing better spatial and temporal resoln. compared to other diffusion-based approaches. Robust photochromic properties of azobenzene make it a prime candidate that could be incorporated into biol. active mols. to realize two-way regulation of motor protein function. In this Minireview, we summarize the design strategies of azobenzene-based photoswitches for reversibly regulating kinesin- and myosin-driven shuttle movements and briefly discuss photoisomerization-induced modulation in motor protein activity.
- 22Böhm, K. J.; Stracke, R.; Unger, E. Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters. Cell Biol. Int. 2000, 24, 335– 341, DOI: 10.1006/cbir.1999.0515[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXktVKis78%253D&md5=aab30aecbe9a5a52e83dcbea1a126c0fSpeeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parametersBohm, K. J.; Stracke, R.; Unger, E.Cell Biology International (2000), 24 (6), 335-341CODEN: CBIIEV; ISSN:1065-6995. (Academic Press)So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg2+, and kinesin concns., pH and ionic strength, we tried to find conditions that favor microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg2+/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temp. elevation or lowering the no. of kinesin mols. bound to the glass surface. The highest mean gliding velocity (1.8 μm/s±0.09 μm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favorable factors (2.5 m m ATP, 12.5 m m Mg2+, 37°, 450 kinesin mols./μm2). (c) 2000 Academic Press.
- 23Nitta, T.; Hess, H. Dispersion in active transport by kinesin-powered molecular shuttles. Nano Lett. 2005, 5, 1337– 1342, DOI: 10.1021/nl050586t[ACS Full Text], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkvFSrsLw%253D&md5=6c0600f67346d60121f0ea3c690b2263Dispersion in Active Transport by Kinesin-Powered Molecular ShuttlesNitta, Takahiro; Hess, HenryNano Letters (2005), 5 (7), 1337-1342CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Active transport driven by mol. motors is a key technol. for the continued miniaturization of lab-on-a-chip devices, because it is expected to enable nanofluidic devices with channel diams. of less than 1 μm and total channel lengths on the order of 1 mm. An important metric for a transport mechanism employed in an analytic device is dispersion, because it critically affects the sensitivity and resoln. Here, the authors investigate the mechanisms responsible for the dispersion of a swarm of "mol. shuttles", consisting of functionalized microtubules propelled by surface-adhered kinesin motor proteins. Using a simple model and measurements of the path persistence length, motional diffusion coeff., and the distribution of av. velocities, the authors found that, at the time scale relevant in the envisioned nanobiodevices, variations in the time-averaged velocities between shuttles will make a stronger contribution to the dispersion of the swarm than both the fluctuations around the time-averaged velocity of an individual shuttle and the fluctuations in path length due to wiggling within the channel. Overall, the dispersion of such mol. shuttles is comparable to the dispersion of a sample plug transported by electroosmotic flow.
- 24Imafuku, Y.; Toyoshima, Y. Y.; Tawada, K. Fluctuation in the microtubule sliding movement driven by kinesin in vitro. Biophys. J. 1996, 70, 878– 886, DOI: 10.1016/S0006-3495(96)79631-5[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlslCqsQ%253D%253D&md5=9f28da461e8c2d86ccc9d63dcdf022d5Fluctuation in the microtubule sliding movement driven by kinesin in vitroImafuku, Yasuhiro; Toyoshima, Yoko Y.; Tawada, KatsuhisaBiophysical Journal (1996), 70 (2), 878-86CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)The authors studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calcg. the mean-square displacement deviation from the av. as a function of time, the authors obtained motional diffusion coeffs. for microtubules and analyzed the dependence of the coeffs. on microtubule length. The analyses suggest that (1) the motional diffusion coeff. consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coeff. of a filament in Brownian movement is) and another that is independent of the length, and (2) the length-dependent term decreases with increasing kinesin concn. This latter term almost vanishes within the length range the authors studied at high kinesin concns. From the length-dependence relation, the authors evaluated the friction coeff. for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coeff. obsd. at sufficiently high kinesin concns. indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.
- 25Kelly, R. H.; Yancey, P. H. High contents of trimethylamine oxide correlating with depth in deep-sea teleost fishes, skates, and decapod crustaceans. Biol. Bull. 1999, 196, 18– 25, DOI: 10.2307/1543162[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhs12gsrw%253D&md5=930ac9880a36e382ce691fdbf0c7f04dHigh contents of trimethylamine oxide correlating with depth in deep-sea teleost fishes, skates, and decapod crustaceansKelly, Robert H.; Yancey, Paul H.Biological Bulletin (Woods Hole, Massachusetts) (1999), 196 (1), 18-25CODEN: BIBUBX; ISSN:0006-3185. (Marine Biological Laboratory)In muscles of shallow-living marine animals, the osmolyte trimethylamine N-oxide (TMAO) is reportedly found (in millimoles of TMAO per kg of tissue wet wt.) at 30-90 in shrimp, 5-50 in crabs, 61-181 in skates, and 10-70 in most teleost fish. Recently our lab. reported higher levels (83-211 mmol/kg), correlating with habitat depth, in deep-sea gadiform teleosts. We now report the same trend in muscles of other animals, collected off the coast of Oregon from bathyal (1800-2000 m) and abyssal plain (2850 m) sites. TMAO contents (mmol/kg) were as follows: zoarcid teleosts, 103 (bathyal) and 197 (abyssal); scorpaenid teleosts, 32 (shallow) and 141 (bathyal); rajid skates, 215 (bathyal) and 244 (abyssal); caridean shrimp, 76 (shallow), 203 (bathyal), and 299 (abyssal); Chionoecetes crabs, 22 (shallow) and 164 (bathyal). Deep squid, clams, and anemones also had higher contents than shallow species. Osmoconformers showed compensation between TMAO and other osmolytes. Urea contents (typically 300 mmol/kg in shallow elasmobranchs) in skates were 214 (bathyal) and 136 (abyssal). Glycine contents in shrimp were 188 (shallow) and 52 (abyssal). High TMAO contents may reflect diet, reduce osmoregulatory costs, increase buoyancy, or counteract destabilization of proteins by pressure.
- 26Yancey, P. H.; Clark, M. E.; Hand, S. C.; Bowlus, R. D.; Somero, G. N. Living with water stress: evolution of osmolyte systems. Science 1982, 217, 1214– 1222, DOI: 10.1126/science.7112124[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XlsFyisbw%253D&md5=5695fde25a5ca2912c3fd3277c656f3cLiving with water stress: evolution of osmolyte systemsYancey, Paul H.; Clark, Mary E.; Hand, Steven C.; Bowlus, R. David; Somero, George N.Science (Washington, DC, United States) (1982), 217 (4566), 1214-22CODEN: SCIEAS; ISSN:0036-8075.A review and discussion with 80 refs. Striking convergent evolution is found in the properties of the org. osmotic solute (osmolyte) systems obsd. in bacteria, plants, and animals. Polyhydric alcs., free amino acids and their derivs., and combinations of urea and methylamines which comprise org. osmolyte systems, confer selective advantage. They are compatible with macromol. structure and function at high or variable (or both) osmolyte concns., and greatly reduce needs for modifying proteins to function in concd. intracellular solns. Osmolyte compatibility is proposed to result from the absence of osmolyte interactions with substrates and cofactors, and the nonperturbing of favorable effects of osmolytes on macromol.-solvent interactions.
- 27Lidbury, I.; Murrell, J. C.; Chen, Y. Trimethylamine N-oxide metabolism by abundant marine heterotrophic bacteria. Proc. Natl. Acad. Sci. 2014, 111, 2710– 2715, DOI: 10.1073/pnas.1317834111[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXivFamu7Y%253D&md5=de541c431e68a6126a4dbab17345c37aTrimethylamine N-oxide metabolism by abundant marine heterotrophic bacteriaLidbury, Ian; Murrell, J. Colin; Chen, YinProceedings of the National Academy of Sciences of the United States of America (2014), 111 (7), 2710-2715CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Trimethylamine N-oxide (TMAO) is a common osmolyte found in a variety of marine biota and has been detected at nanomolar concns. in oceanic surface waters. TMAO can serve as an important nutrient for ecol. important marine heterotrophic bacteria, particularly the SAR11 clade and marine Roseobacter clade (MRC). However, the enzymes responsible for TMAO catabolism and the membrane transporter required for TMAO uptake into microbial cells have yet to be identified. The authors show here that the enzyme TMAO demethylase (Tdm) catalyzes the first step in TMAO degrdn. This enzyme represents a large group of proteins with an uncharacterized domain (DUF1989). The function of TMAO demethylase in a representative from the SAR11 clade (strain HIMB59) and in a representative of the MRC (Ruegeria pomeroyi DSS-3) was confirmed by heterologous expression of tdm (the gene encoding Tdm) in Escherichia coli. In R. pomeroyi, mutagenesis expts. confirmed that tdm is essential for growth on TMAO. The authors also identified a unique ATP-binding cassette transporter (TmoXWV) found in a variety of marine bacteria and exptl. confirmed its specificity for TMAO through marker exchange mutagenesis and lacZ reporter assays of the promoter for genes encoding this transporter. Both Tdm and TmoXWV are particularly abundant in natural seawater assemblages and actively expressed, as indicated by a no. of recent metatranscriptomic and metaproteomic studies. These data suggest that TMAO represents a significant, yet overlooked, nutrient for marine bacteria.
- 28Bachand, G. D.; Jain, R.; Ko, R.; Bouxsein, N. F.; VanDelinder, V. Inhibition of microtubule depolymerization by osmolytes. Biomacromolecules 2018, 19, 2401– 2408, DOI: 10.1021/acs.biomac.7b01799[ACS Full Text], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFGnt7o%253D&md5=53e1b16f653a0e95c8fea72589cf56acInhibition of microtubule depolymerization by osmolytesBachand, George D.; Jain, Rishi; Ko, Randy; Bouxsein, Nathan F.; Van Delinder, VirginiaBiomacromolecules (2018), 19 (7), 2401-2408CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)Microtubule dynamics play a crit. role in the normal physiol. of eukaryotic cells as well as a no. of cancers and neurodegenerative disorders. The polymn./depolymn. of microtubules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-assocd. proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here we describe the ability of the osmolytes polyethylene glycol (PEG) and trimethylamine-N-oxide (TMAO) to inhibit the depolymn. of individual microtubule filaments for extended periods of time (up to 30 days). We further show that PEG stabilizes microtubules against both temp.- and calcium-induced depolymn. Our results collectively suggest that the obsd. inhibition may be related to combination of the kosmotropic behavior and excluded vol./osmotic pressure effects assocd. with PEG and TMAO. Taken together with prior studies, our data suggest that the physiochem. properties of the local environment can regulate microtubule depolymn. and may potentially play an important role in in vivo microtubule dynamics.
- 29Chase, K.; Doval, F.; Vershinin, M. Enhanced stability of kinesin-1 as a function of temperature. Biochem. Biophys. Res. Commun. 2017, 493, 1318– 1321, DOI: 10.1016/j.bbrc.2017.09.172[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1aisbbN&md5=48f99ee38fbc3c2ce85eab4aedeaae4dEnhanced stability of kinesin-1 as a function of temperatureChase, K.; Doval, F.; Vershinin, M.Biochemical and Biophysical Research Communications (2017), 493 (3), 1318-1321CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Kinesin-1 is a mechanochem. enzyme which mediates long distance intracellular cargo transport along microtubules in a wide variety of eukaryotic cells. Kinesin is also relatively easy to purify and shows robust function in vitro, leading to numerous proposals for using the kinesin-1/microtubule system for nanoscale transport in engineered devices. However, kinesin in vitro shows signs of degrdn. at ∼30 °C which severely limits its usability in biomimetic engineering. Notably, kinesin-1 functions robustly in animal cells at body temps. as high as 40 °C which suggests that kinesin functioning can be stabilized beyond what is obsd. in vitro. The present study investigated the effect of trimethylamine N-oxide (TMAO) as a potential heat-protecting agent for kinesin function and microtubule stability. We show that at a concn. of 200 mM, TMAO can indeed stabilize kinesin-based motility up to a little over 50 °C and that such motility can be sustained for extended periods of time. Our results suggest that intracellular crowding (mimicked in vitro by TMAO) can indeed stabilize kinesin-1 at high temps. and helps resolve a long standing discrepancy between thermal stability of kinesin-1 obsd. in vivo and in vitro. Moreover, when considered together with our previous report that kinesin-1 can function well down to near-freezing conditions, this study establishes kinesin-1/microtubule motility as a thermally viable engineering platform.
- 30Castoldi, M.; Popov, A. V. Purification of brain tubulin through two cycles of polymerization–depolymerization in a high-molarity buffer. Protein Expression Purif. 2003, 32, 83– 88, DOI: 10.1016/S1046-5928(03)00218-3[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXosVKlu74%253D&md5=73a6d137c85393afdbaa4dbfc50ed059Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity bufferCastoldi, Mirco; Popov, Andrei V.Protein Expression and Purification (2003), 32 (1), 83-88CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier Science)Microtubules can be assembled in vitro from purified α/β tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymn.-depolymn., followed by ion-exchange chromatog. to remove any contaminating microtubule-assocd. proteins and motors. Here, we show that only two cycles of polymn.-depolymn. of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and prodn. of a high-concn. tubulin soln. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatog.-based procedures.
- 31Peloquin, J.; Komarova, Y.; Borisy, G. Conjugation of fluorophores to tubulin. Nat. Methods 2005, 2, 299– 303, DOI: 10.1038/nmeth0405-299[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisFejtLc%253D&md5=3636fb218713abcab73a959131c558d5Conjugation of fluorophores to tubulinPeloquin, John; Komarova, Yulia; Borisy, GaryNature Methods (2005), 2 (4), 299-303CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The use of fluorescently labeled proteins as probes of the structure and function of living cells began with the study of the components of the cytoskeleton and has become a powerful approach for studying the distribution and dynamics of these components. Primary components of the cytoskeleton such as tubulin, actin and myosin are abundant and can be purified relatively easily in hundreds of milligram quantities from animal tissues. Fluorophores contg. amine or thiol reactive groups are used to selectively modify lysine or cysteine residues, resp. This protocol describes the fluorescent conjugation of tubulin isolated from brain tissue.
- 32Fujimoto, K.; Kitamura, M.; Yokokawa, M.; Kanno, I.; Kotera, H.; Yokokawa, R. Colocalization of quantum dots by reactive molecules carried by motor proteins on polarized microtubule arrays. ACS Nano 2013, 7, 447– 455, DOI: 10.1021/nn3045038[ACS Full Text], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVahs77N&md5=adf4185e38e52015279614f69e8fbc5aColocalization of Quantum Dots by Reactive Molecules Carried by Motor Proteins on Polarized Microtubule ArraysFujimoto, Kazuya; Kitamura, Masuto; Yokokawa, Masatoshi; Kanno, Isaku; Kotera, Hidetoshi; Yokokawa, RyujiACS Nano (2013), 7 (1), 447-455CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The field of microfluidics has drastically contributed to downscale the size of benchtop expts. to the dimensions of a chip without compromising results. However, further miniaturization and the ability to directly manipulate individual mols. require a platform that permits organized mol. transport. The motor proteins and microtubules that carry out orderly intracellular transport are ideal for driving in vitro nanotransport. Here, a reconstruction of the cellular kinesin/dynein-microtubule system in nanotracks provides a mol. total anal. system (MTAS) to control massively parallel chem. reactions. The mobility of kinesin and a microtubule dissocn. method enable orientation of a microtubule in an array for directed transport of reactive mols. carried by kinesin or dynein. The binding of glutathione S-transferase (GST) to glutathione (GSH) and the binding of streptavidin to biotin are visualized as colocalizations of quantum dots (Q-dots) when motor motilities bring them into contact. The organized nanotransport demonstrated here suggests the feasibility of using the authors' platform to perform parallel biochem. reactions focused at the mol. level.
- 33Munmun, T.; Kabir, A. M. R.; Sada, K.; Kakugo, A. Complete, rapid and reversible regulation of the motility of a nano-biomolecular machine using an osmolyte trimethylamine-N-oxide. Sens. Actuators, B 2020, 304, 127231 DOI: 10.1016/j.snb.2019.127231[Crossref], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFCmsrrM&md5=c8fcf88d3b6dca1b29ee762a6e62d29dComplete, rapid and reversible regulation of the motility of a nano-biomolecular machine using an osmolyte trimethylamine-N-oxideMunmun, Tasrina; Kabir, Arif Md. Rashedul; Sada, Kazuki; Kakugo, AkiraSensors and Actuators, B: Chemical (2020), 304 (), 127231CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)Nanoscale transportation in engineered environments is crit. towards designing efficient and smart hybrid bio-nanodevices. Biomol. 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 biomol. motors is the key to control such integrated nanodevices. We present a simple method to regulate the activity of a biomol. 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 concn. 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 concn. The halted motility of MTs is fully restored upon elimination of TMAO. Repeated cycles of TMAO addn. and removal enable cyclical inhibition and restoration of the motility of MTs. These results demonstrate an ability to control nanoscale motion of a biomol. motor in an artificial environment. This work facilitates further tunability over functions of biomol. motors, which in turn will foster their nanotechnol. applications, such as in nano-transportation.
- 34Kumemoto, R.; Yusa, K.; Shibayama, T.; Hatori, K. Trimethylamine N-oxide suppresses the activity of the actomyosin motor. Biochim. Biophys. Acta, Gen. Subj. 1820, 2012, 1597– 1604, DOI: 10.1016/j.bbagen.2012.06.006
- 35Qian, H.; Sheetz, M. P.; Elson, E. L. Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys. J. 1991, 60, 910– 921, DOI: 10.1016/S0006-3495(91)82125-7[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK38%252FnsFyqsw%253D%253D&md5=074789e42fc9976b328088a2ed4971a1Single particle tracking. Analysis of diffusion and flow in two-dimensional systemsQian H; Sheetz M P; Elson E LBiophysical journal (1991), 60 (4), 910-21 ISSN:0006-3495.Analysis of the trajectories of small particles at high spatial and temporal resolution using video enhanced contrast microscopy provides a powerful approach to characterizing the mechanisms of particle motion in living cells and in other systems. We present here the theoretical basis for the analysis of these trajectories for particles undergoing random diffusion and/or systematic transport at uniform velocity in two-dimensional systems. The single particle tracking method, based on observations of the trajectories of individual particles, is compared with methods that characterize the motions of a large collection of particles such as fluorescence photobleaching recovery. Determination of diffusion coefficients or transport velocities either from correlation of positions or of velocities of the particles is discussed. A result of practical importance is an analysis of the dependence of the expected statistical uncertainty of these determinations on the number of position measurements. This provides a way of judging the accuracy of the diffusion coefficients and transport velocities obtained using this approach.
- 36Tawada, K.; Sekimoto, K. Protein friction exerted by motor enzymes through a weak-binding interaction. J. Theor. Biol. 1991, 150, 193– 200, DOI: 10.1016/S0022-5193(05)80331-5[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmtVWgtbs%253D&md5=aa21e75cebf986f8df7d6845d672bb98Protein friction exerted by motor enzymes through a weak-binding interactionTawada, Katsuhisa; Sekimoto, KenJournal of Theoretical Biology (1991), 150 (2), 193-200CODEN: JTBIAP; ISSN:0022-5193.Recently R. D. Vale et al. (1989) reported an observation of the one-dimensional Brownian movement of microtubules bound to flagellar dynein through a weak-binding interaction. In this study, a theor. model of this phenomenon is proposed. The model consists of a rigid microtubule assocd. with a no. of elastic dynein heads through a weak-binding interaction at equil. The model implies that (1) the Brownian motion of the microtubule is not directly driven by the at. collision of the solvent particles, but is driven by the thermally-generated structural fluctuations of the dynein heads which interact with the microtubule; (2) dynein heads through a weak-binding interaction exert a frictional drag force on the sliding motion of the microtubule and the drag force is proportional to the sliding velocity the same as in hydrodynamic viscous friction. This protein friction, with such viscous-like characteristics, may well play a role as a velocity-limiting factor in the normal ATP-induced sliding movement of motile proteins.
- 37Bormuth, V.; Varga, V.; Howard, J.; Schäffer, E. Protein friction limits diffusive and directed movements of kinesin motors on microtubules. Science 2009, 325, 870– 873, DOI: 10.1126/science.1174923[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXps1Ogtr8%253D&md5=0fe1c7ebf58a66d30f2898a457a5be3fProtein friction limits diffusive and directed movements of kinesin motors on microtubulesBormuth, Volker; Varga, Vladimir; Howard, Jonathon; Schaeffer, ErikScience (Washington, DC, United States) (2009), 325 (5942), 870-873CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Friction limits the operation of macroscopic engines and is crit. to the performance of micromech. devices. We report measurements of friction in a biol. nanomachine. Using optical tweezers, we characterized the frictional drag force of individual kinesin-8 motor proteins interacting with their microtubule tracks. At low speeds and with no energy source, the frictional drag was related to the diffusion coeff. by the Einstein relation. At higher speeds, the frictional drag force increased nonlinearly, consistent with the motor jumping 8 nm between adjacent tubulin dimers along the microtubule, and was asym., reflecting the structural polarity of the microtubule. We argue that these frictional forces arise from breaking bonds between the motor domains and the microtubule, and they limit the speed and efficiency of kinesin.
- 38Munmun, T.; Kabir, A. M. R.; Katsumoto, Y.; Sada, K.; Kakugo, A. Controlling the kinetics of interaction between microtubules and kinesins over a wide temperature range using the deep-sea osmolyte trimethylamine N-oxide. Chem. Commun. 2020, 56, 1187– 1190, DOI: 10.1039/C9CC09324A[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVyntL3N&md5=d0992dafb66c9a4057e1656c40e4778dControlling the kinetics of interaction between microtubules and kinesins over a wide temperature range using the deep-sea osmolyte trimethylamine N-oxideMunmun, Tasrina; Kabir, Arif Md. Rashedul; Katsumoto, Yukiteru; Sada, Kazuki; Kakugo, AkiraChemical Communications (Cambridge, United Kingdom) (2020), 56 (8), 1187-1190CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Trimethylamine N-oxide is found to be effective in regulating the interaction between microtubules and kinesins over a wide temp. range. The lifetime of the motility of microtubules on kinesins at high temps. is prolonged using trimethylamine N-oxide. The activation energy of microtubule motility is increased by trimethylamine N-oxide. Prolonged operation at high temps. decreased the activation energy of MT motility despite the increase in concn. of trimethylamine N-oxide.
- 39VanDelinder, V.; Sickafoose, I.; Imam, Z. I.; Ko, R.; Bachand, G. D. The effects of osmolytes on in vitro kinesin-microtubule motility assays. RSC Adv. 2020, 10, 42810– 42815, DOI: 10.1039/D0RA08148E[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVSks7vM&md5=68d4365c5e6d8c6aa563ae40e388e478The effects of osmolytes on in vitro kinesin-microtubule motility assaysVanDelinder, Virginia; Sickafoose, Ian; Imam, Zachary I.; Ko, Randy; Bachand, George D.RSC Advances (2020), 10 (70), 42810-42815CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)The gliding motility of microtubule filaments has been used to study the biophys. properties of kinesin motors, as well as being used in a variety of nanotechnol. applications. While microtubules are generally stabilized in vitro with paclitaxel (Taxol), osmolytes such as polyethylene glycol (PEG) and trimethylamine N-oxide (TMAO) are also able to inhibit depolymn. over extended periods of time. High concns. of TMAO have also been reported to reversibly inhibit kinesin motility of paclitaxel-stabilized microtubules. Here, we examd. the effects of the osmolytes PEG, TMAO, and glycerol on stabilizing microtubules during gliding motility on kinesin-coated substrates. As previously obsd., microtubule depolymn. was inhibited in a concn. dependent manner by the addn. of the different osmolytes. Kinesin-driven motility also exhibited concn. dependent effects with the addn. of the osmolytes, specifically reducing the velocity, increasing rates of pinning, and altering trajectories of the microtubules. These data suggest that there is a delicate balance between the ability of osmolytes to stabilize microtubules without inhibiting motility. Overall, these findings provide a more comprehensive understanding of how osmolytes affect the dynamics of microtubules and kinesin motors, and their interactions in crowded environments.
- 40Palacci, H.; Idan, O.; Armstrong, M. J.; Agarwal, A.; Nitta, T.; Hess, H. Velocity fluctuations in kinesin-1 gliding motility assays originate in motor attachment geometry variations. Langmuir 2016, 32, 7943– 7950, DOI: 10.1021/acs.langmuir.6b02369[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFKrtrbI&md5=6b9a72b06c5a12b38cba1cfb4c862c9dVelocity Fluctuations in Kinesin-1 Gliding Motility Assays Originate in Motor Attachment Geometry VariationsPalacci, Henri; Idan, Ofer; Armstrong, Megan J.; Agarwal, Ashutosh; Nitta, Takahiro; Hess, HenryLangmuir (2016), 32 (31), 7943-7950CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Motor proteins such as myosin and kinesin play a major role in cellular cargo transport, muscle contraction, cell division, and engineered nanodevices. Quantifying the collective behavior of coupled motors is crit. to our understanding of these systems. An excellent model system is the gliding motility assay, where hundreds of surface-adhered motors propel one cytoskeletal filament such as an actin filament or a microtubule. The filament motion can be obsd. using fluorescence microscopy, revealing fluctuations in gliding velocity. These velocity fluctuations have been previously quantified by a motional diffusion coeff., which Sekimoto and Tawada explained as arising from the addn. and removal of motors from the linear array of motors propelling the filament as it advances, assuming that different motors are not equally efficient in their force generation. A computational model of kinesin head diffusion and binding to the microtubule allowed us to quantify the heterogeneity of motor efficiency arising from the combination of anharmonic tail stiffness and varying attachment geometries assuming random motor locations on the surface and an absence of coordination between motors. Knowledge of the heterogeneity allows the calcn. of the proportionality const. between the motional diffusion coeff. and the motor d. The calcd. value (0.3) is within a std. error of our measurements of the motional diffusion coeff. on surfaces with varying motor densities calibrated by landing rate expts. This allowed us to quantify the loss in efficiency of coupled mol. motors arising from heterogeneity in the attachment geometry.
- 41Tsitkov, S., Hess, H. Design of active nanosystems incorporating biomolecular motors. In Out-of-Equilibrium (Supra) molecular Systems and Materials, 1st ed.; Giuseppone, N., Walther, A., Eds.; Wiley-VCH: Weinheim, Germany, 2021; pp 379– 422.
- 42Saper, G.; Hess, H. Synthetic systems powered by biological molecular motors. Chem. Rev. 2020, 120, 288– 309, DOI: 10.1021/acs.chemrev.9b00249[ACS Full Text], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslektr%252FN&md5=e9b4499656ef83ef272964d21edb71dcSynthetic Systems Powered by Biological Molecular MotorsSaper, Gadiel; Hess, HenryChemical Reviews (Washington, DC, United States) (2020), 120 (1), 288-309CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Biol. mol. motors (or biomol. motors for short) are nature's soln. to the efficient conversion of chem. energy to mech. movement. In biol. systems, these fascinating mols. are responsible for movement of mols., organelles, cells, and whole animals. In engineered systems, these motors can potentially be used to power actuators and engines, shuttle cargo to sensors, and enable new computing paradigms. Here, we review the progress in the past decade in the integration of biomol. motors into hybrid nanosystems. After briefly introducing the motor proteins kinesin and myosin and their assocd. cytoskeletal filaments, we review recent work aiming for the integration of these biomol. motors into actuators, sensors, and computing devices. In some systems, the creation of mech. work and the processing of information become intertwined at the mol. scale, creating a fascinating type of "active matter". We discuss efforts to optimize biomol. motor performance, construct new motors combining artificial and biol. components, and contrast biomol. motors with current artificial mol. motors. A recurrent theme in the work of the past decade was the induction and utilization of collective behavior between motile systems powered by biomol. motors, and we discuss these advances. The exertion of external control over the motile structures powered by biomol. motors has remained a topic of many studies describing exciting progress. Finally, we review the current limitations and challenges for the construction of hybrid systems powered by biomol. motors and try to ascertain if there are theor. performance limits. Engineering with biomol. motors has the potential to yield com. viable devices, but it also sharpens our understanding of the design problems solved by evolution in nature. This increased understanding is valuable for synthetic biol. and potentially also for medicine.
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Abstract
Figure 1
Figure 1. Schematic representation of (a) molecular structure of TMAO and (b) in vitro gliding assay of MTs on a kinesin-coated glass substrate in the presence of TMAO. In the molecular structure of TMAO, the red, blue, black, and white spheres denote the oxygen, nitrogen, carbon, and hydrogen atoms, respectively. “+ATP” indicates the use of ATP in the in vitro gliding assay. (c) Representative data show the instantaneous velocity of an MT with time where the velocity was quantified over a 10 s interval. Abrupt changes in the velocity of MTs with time were observed in the absence of TMAO, which diminished upon increasing the concentration of TMAO.
Figure 2
Figure 2. Histograms show the distribution of the instantaneous velocity of MTs in the absence and in the presence of TMAO of various concentrations. The concentration of TMAO is mentioned inside the histograms in each case, and “no TMAO” indicates the absence of TMAO. As the concentration of TMAO in the gliding assay was increased, the histograms became narrower and shifted toward lower velocity values. The red lines represent the fitting of the histograms using the equation of Gaussian distribution.
Figure 3
Figure 3. Mean-square displacement deviation of MTs from the average as a function of time, calculated by “multiple trajectories averaging” over 50 different trajectories of MTs, in the absence and in the presence of different TMAO concentrations. Error bars are not shown here for simplicity. The solid lines indicate the linear regression fit of the data in each condition (left). Change in the motional diffusion coefficient of MTs, propelled by kinesins in an in vitro gliding assay, upon changing the TMAO concentration in the gliding assay (right). Error bar: standard deviation.
Figure 4
Figure 4. Reversible regulation of the distribution of instantaneous velocity of MTs using TMAO. The distribution of MT velocity in the absence of TMAO (top), in the presence of 1 M TMAO (middle), and after elimination of the TMAO, i.e., in the absence of TMAO (bottom). The red lines represent fitting of the histograms using the equation of Gaussian distribution.
Figure 5
Figure 5. Mean-square displacement deviation of MTs from the average as a function of time, calculated by “multiple trajectories averaging” over 50 different trajectories of MTs, in the absence of TMAO, upon application of 1 M TMAO, and upon elimination of the TMAO (left). Error bars are not shown here for simplicity. The solid lines indicate the linear regression fit of the data in each condition. Reversible regulation of the motional diffusion coefficient of MTs using 1 M TMAO (right). Error bar: standard deviation.
References
ARTICLE SECTIONSThis article references 42 other publications.
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- 9Harada, Y.; Noguchi, A.; Kishino, A.; Yanagida, T. Sliding movement of single actin filaments on one-headed myosin filaments. Nature 1987, 326, 805– 808, DOI: 10.1038/326805a0[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXitV2kt7o%253D&md5=7ee287fd278954280675780996e20551Sliding movement of single actin filaments on one-headed myosin filamentsHarada, Yoshie; Noguchi, Akira; Kishino, Akiyoshi; Yanagida, ToshioNature (London, United Kingdom) (1987), 326 (6115), 805-8CODEN: NATUAS; ISSN:0028-0836.The myosin mol. consists of 2 heads, each of which contain an enzymic active site and an actin-binding site. The fundamental problem of whether the 2 heads function independently or cooperatively during muscle contraction was studied by an assay system in which sliding movements of fluorescently labeled, single actin filaments along myosin filaments can be obsd. directly. Direct measurement of the sliding of single actin filaments along 1-headed myosin filaments are reported in which the d. of heads was varied over a wide range. The results show that cooperative interaction between the 2 heads of myosin is not essential for inducing the sliding movement of actin filaments.
- 10Toyoshima, Y. Y.; Kron, S. J.; Spudich, J. A. The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assay. Proc. Natl. Acad. Sci. 1990, 87, 7130– 7134, DOI: 10.1073/pnas.87.18.7130[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXmt1ymsLY%253D&md5=9913f44f6c94d6059cbc769015a90323The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assayToyoshima, Yoko Y.; Kron, Stephen J.; Spudich, James A.Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (18), 7130-4CODEN: PNASA6; ISSN:0027-8424.Chemomech. coupling in muscle contraction may be due to swinging crossbridges, such that a change in the angle at which the myosin head binds to the actin filament is tightly coupled to release of products of ATP hydrolysis. This model would limit the step size, the unit displacement of actin produced by a single ATP hydrolysis, to less than twice the chord length of the myosin head. Recent measurements have found the step size to be significantly larger than this geometric limit, bringing into question any direct correspondence between the crossbridge and ATP-hydrolysis cycles. The rate of ATP hydrolysis due to actin sliding movement was measured in an in vitro motility assay consisting of purified actin and purified myosin. An apparent myosin step size was calcd. well within the geometric limit set by the size of the myosin head. These data are consistent with tight coupling between myosin crossbridge movement and ATP hydrolysis.
- 11Uyeda, T. Q.; Abramson, P. D.; Spudich, J. A. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc. Natl. Acad. Sci. 1996, 93, 4459– 4464, DOI: 10.1073/pnas.93.9.4459[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XivVKqtLY%253D&md5=d19fd6188a063200d490e1d677cc7abbThe neck region of the myosin motor domain acts as a lever arm to generate movementUyeda, Taro Q. P.; Abramson, Paul D.; Spudich, James A.Proceedings of the National Academy of Sciences of the United States of America (1996), 93 (9), 4459-4464CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long α-helical portion of the heavy chain. The swinging neck-lever model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The ΔRLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The ΔBLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.
- 12Fischer, T.; Agarwal, A.; Hess, H. A smart dust biosensor powered by kinesin motors. Nat. Nanotechnol. 2009, 4, 162– 166, DOI: 10.1038/nnano.2008.393[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXis12nsL4%253D&md5=36eac4027e98cde3a472a2addd78224eA smart dust biosensor powered by kinesin motorsFischer, Thorsten; Agarwal, Ashutosh; Hess, HenryNature Nanotechnology (2009), 4 (3), 162-166CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Biosensors can be miniaturized by injecting smaller vols. into micro- and nano-fluidic devices or immersing increasingly sophisticated particles, i.e., smart dust, into the sample. The term, smart dust, originally referred to mm3 wireless semi-conducting sensors which could invisibly monitor the environment in buildings and public spaces; it also included functional μm-sized porous Si particles used to monitor even smaller environments. The principal challenge in designing smart dust biosensors is integrating transport functions with energy supply in the device. A hybrid micro-device powered by ATP and relying on antibody-functionalized micro-tubules and kinesin motors to transport target analytes into a detection region is described. The transport step replaces the wash step in traditional double-antibody sandwich assays. Due to their small size and autonomous function, it is envisioned that large nos. of smart dust biosensors could be inserted into organisms or distributed in the environment for remote sensing.
- 13Bachand, G. D.; Rivera, S. B.; Boal, A. K.; Gaudioso, J.; Liu, J.; Bunker, B. C. Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins. Nano Lett. 2004, 4, 817– 821, DOI: 10.1021/nl049811h[ACS Full Text], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXisVyitLk%253D&md5=74898ec172a5204c765880fdf04c59b3Assembly and Transport of Nanocrystal CdSe Quantum Dot Nanocomposites Using Microtubules and Kinesin Motor ProteinsBachand, George D.; Rivera, Susan B.; Boal, Andrew K.; Gaudioso, Jennifer; Liu, Jun; Bunker, Bruce C.Nano Letters (2004), 4 (5), 817-821CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Nature has evolved dynamic, non-equil. mechanisms for assembling hierarchical complexes of nanomaterials. A crit. element to many of these assembly mechanisms involves the active and directed transport of materials by biomol. motor proteins such as kinesin. In the present work, nanocrystal quantum dots (nQDs) were assembled and organized using microtubule (MT) filaments as nanoscale scaffolds. NQD d. and localization were systematically evaluated by varying the concn. and distribution of functional groups within the MT structure. Confining nQD attachment to a central region within the MT enabled unaffected interaction with kinesin necessary to support active transport of nQD-MT composites. This active transport system will be further refined to control the optical properties of a surface by regulating the collective organization of nQD-MT composites.
- 14Bachand, G. D.; Rivera, S. B.; Carroll-Portillo, A.; Hess, H.; Bachand, M. Active capture and transport of virus particles using a biomolecular motor-driven, nanoscale antibody sandwich assay. Small 2006, 2, 381– 385, DOI: 10.1002/smll.200500262[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhs1aqsLo%253D&md5=33656a1ef85741449a0da49d9971bc49Active capture and transport of virus particles using a biomolecular motor-driven, nanoscale antibody sandwich assayBachand, George D.; Rivera, Susan B.; Carroll-Portillo, Amanda; Hess, Henry; Bachand, MarleneSmall (2006), 2 (3), 381-385CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Virus particles are captured and transported using kinesin-driven, antibody-functionalized microtubules. The functionalization was achieved through covalent crosslinking, which consequently enhanced the microtubule stability. The capture and transport of the virus particles was subsequently demonstrated in gliding motility assays in which antibody-coated microtubules functioned as capture elements, and antibody-coated microspheres served as fluorescent reporters (see Figure).
- 15Hess, H.; Clemmens, J.; Howard, J.; Vogel, V. Surface imaging by self-propelled nanoscale probes. Nano Lett. 2002, 2, 113– 116, DOI: 10.1021/nl015647b[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXovF2mtbY%253D&md5=f3bbe03f84bb90b85d15c1514e55d8c3Surface imaging by self-propelled nanoscale probesHess, Henry; Clemmens, John; Howard, Jonathon; Vogel, ViolaNano Letters (2002), 2 (2), 113-116CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)A new approach to image microscopic surface properties is introduced. Information about surface properties such as topog. is obtained by repeated acquisition of an optical signal from a large no. of microscopic, self-propelled probes moving on random paths across a surface. These self-propelled probes sample the surface in a statistical process in contrast to the deterministic, linear sampling performed by a scanning probe microscope. This method is exptl. demonstrated using microtubules as probes, which are moved by the motor protein kinesin.
- 16Inoue, D.; Nitta, T.; Kabir, A. M. R.; Sada, K.; Gong, J. P.; Konagaya, A.; Kakugo, A. Sensing surface mechanical deformation using active probes driven by motor proteins. Nat. Commun. 2016, 7, 12557 DOI: 10.1038/ncomms12557[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1amtLrM&md5=a8a291a690f1a13623409383a1fd3433Sensing surface mechanical deformation using active probes driven by motor proteinsInoue, Daisuke; Nitta, Takahiro; Kabir, Arif Md. Rashedul; Sada, Kazuki; Gong, Jian Ping; Konagaya, Akihiko; Kakugo, AkiraNature Communications (2016), 7 (), 12557CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Studying mech. deformation at the surface of soft materials has been challenging due to the difficulty in sepg. surface deformation from the bulk elasticity of the materials. Here, we introduce a new approach for studying the surface mech. deformation of a soft material by utilizing a large no. of self-propelled microprobes driven by motor proteins on the surface of the material. Information about the surface mech. deformation of the soft material is obtained through changes in mobility of the microprobes wandering across the surface of the soft material. The active microprobes respond to mech. deformation of the surface and readily change their velocity and direction depending on the extent and mode of surface deformation. This highly parallel and reliable method of sensing mech. deformation at the surface of soft materials is expected to find applications that explore surface mechanics of soft materials and consequently would greatly benefit the surface science.
- 17Hess, H.; Howard, J.; Vogel, V. A piconewton forcemeter assembled from microtubules and kinesins. Nano Lett. 2002, 2, 1113– 1115, DOI: 10.1021/nl025724i[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhs1enuw%253D%253D&md5=c7751c9c2a49ea4de9f18275ac63328dA piconewton forcemeter assembled from microtubules and kinesinsHess, Henry; Howard, Jonathon; Vogel, ViolaNano Letters (2002), 2 (10), 1113-1115CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)A forcemeter capable of detecting piconewton forces was assembled from nanoscale building blocks, using a cantilevered microtubule as a beam of known stiffness, which is loaded by a second microtubule transported by kinesin motor proteins adsorbed to the surface and connected to the cantilevered microtubule by the link whose strength is to be detd. Due to the loading rate of less than 1 pN/s, this forcemeter is ideally suited to study the strength of biol. receptor/ligand pairs, such as streptavidin/biotin.
- 18Keya, J. J.; Suzuki, R.; Kabir, A. M. R.; Inoue, D.; Asanuma, H.; Sada, K.; Hess, H.; Kuzuya, A.; Kakugo, A. DNA-assisted swarm control in a biomolecular motor system. Nat. Commun. 2018, 9, 453 DOI: 10.1038/s41467-017-02778-5[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvlvFGisA%253D%253D&md5=36fd4c5b686e854308f74e26d8a654a6DNA-assisted swarm control in a biomolecular motor systemKeya Jakia Jannat; Suzuki Ryuhei; Sada Kazuki; Kakugo Akira; Kabir Arif Md Rashedul; Inoue Daisuke; Sada Kazuki; Kakugo Akira; Asanuma Hiroyuki; Hess Henry; Kakugo Akira; Kuzuya AkinoriNature communications (2018), 9 (1), 453 ISSN:.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.
- 19Liu, H.; Schmidt, J. J.; Bachand, G. D.; Rizk, S. S.; Looger, L. L.; Hellinga, H. W.; Montemagno, C. D. Control of a biomolecular motor-powered nanodevice with an engineered chemical switch. Nat. Mater. 2002, 1, 173– 177, DOI: 10.1038/nmat761[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptV2qsL0%253D&md5=fe4435470ba89d1169ffd0114340cda7Control of a biomolecular motor-powered nanodevice with an engineered chemical switchLiu, Haiqing; Schmidt, Jacob J.; Bachand, George D.; Rizk, Shahir S.; Looger, Loren L.; Hellinga, Homme W.; Montemagno, Carlo D.Nature Materials (2002), 1 (3), 173-177CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)A mutant F1-ATPase motor contg. a metal-binding site that functions as a zinc-dependent, reversible on/off switch has been constructed and analyzed. Repeated cycles of zinc addn. and removal by chelation result in inhibition and restoration, resp., of both ATP hydrolysis and motor rotation of the mutant, but not of the wild-type F1 fragment. The results demonstrate the ability to engineer chem. regulation into a biomol. motor and represent a crit. step towards controlling integrated nanomech. devices at the single-mol. level.
- 20Konishi, K.; Uyeda, T. Q.; Kubo, T. Genetic engineering of a Ca2+ dependent chemical switch into the linear biomotor kinesin. FEBS Lett. 2006, 580, 3589– 3594, DOI: 10.1016/j.febslet.2006.05.037[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmtVOmsbk%253D&md5=8810143a5de2830e07cb4b5eb33374fdGenetic engineering of a Ca2+ dependent chemical switch into the linear biomotor kinesinKonishi, Kaoru; Uyeda, Taro Q. P.; Kubo, TaiFEBS Letters (2006), 580 (15), 3589-3594CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca2+-calmodulin, the assocn. of the engineered kinesin with microtubules showed a steep Ca2+-dependency in ATPase activity at Ca2+ concns. of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca2+-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca2+-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.
- 21Amrutha, A. S.; Kumar, S. K. R.; Tamaoki, N. Azobenzene-based photoswitches facilitating reversible regulation of kinesin and myosin motor systems for nanotechnological applications. ChemPhotoChem 2019, 3, 337– 346, DOI: 10.1002/cptc.201900037[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnslehsbk%253D&md5=57a4459a09f7c6d5ca6c96a037791e8aAzobenzene-Based Photoswitches Facilitating Reversible Regulation of Kinesin and Myosin Motor Systems for Nanotechnological ApplicationsAmrutha, Ammathnadu S.; Sunil Kumar, K. R.; Tamaoki, NobuyukiChemPhotoChem (2019), 3 (6), 337-346CODEN: CHEMYH ISSN:. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The past decade has witnessed several significant efforts made towards implementing the biomol. functions of motor proteins for nanotechnol. applications. However, employing motor protein systems in artificial environments demands for a precise and flexible spatiotemporal control mechanism. Light being an ideal external stimulus offers major advantages, allowing better spatial and temporal resoln. compared to other diffusion-based approaches. Robust photochromic properties of azobenzene make it a prime candidate that could be incorporated into biol. active mols. to realize two-way regulation of motor protein function. In this Minireview, we summarize the design strategies of azobenzene-based photoswitches for reversibly regulating kinesin- and myosin-driven shuttle movements and briefly discuss photoisomerization-induced modulation in motor protein activity.
- 22Böhm, K. J.; Stracke, R.; Unger, E. Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters. Cell Biol. Int. 2000, 24, 335– 341, DOI: 10.1006/cbir.1999.0515[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXktVKis78%253D&md5=aab30aecbe9a5a52e83dcbea1a126c0fSpeeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parametersBohm, K. J.; Stracke, R.; Unger, E.Cell Biology International (2000), 24 (6), 335-341CODEN: CBIIEV; ISSN:1065-6995. (Academic Press)So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg2+, and kinesin concns., pH and ionic strength, we tried to find conditions that favor microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg2+/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temp. elevation or lowering the no. of kinesin mols. bound to the glass surface. The highest mean gliding velocity (1.8 μm/s±0.09 μm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favorable factors (2.5 m m ATP, 12.5 m m Mg2+, 37°, 450 kinesin mols./μm2). (c) 2000 Academic Press.
- 23Nitta, T.; Hess, H. Dispersion in active transport by kinesin-powered molecular shuttles. Nano Lett. 2005, 5, 1337– 1342, DOI: 10.1021/nl050586t[ACS Full Text], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkvFSrsLw%253D&md5=6c0600f67346d60121f0ea3c690b2263Dispersion in Active Transport by Kinesin-Powered Molecular ShuttlesNitta, Takahiro; Hess, HenryNano Letters (2005), 5 (7), 1337-1342CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Active transport driven by mol. motors is a key technol. for the continued miniaturization of lab-on-a-chip devices, because it is expected to enable nanofluidic devices with channel diams. of less than 1 μm and total channel lengths on the order of 1 mm. An important metric for a transport mechanism employed in an analytic device is dispersion, because it critically affects the sensitivity and resoln. Here, the authors investigate the mechanisms responsible for the dispersion of a swarm of "mol. shuttles", consisting of functionalized microtubules propelled by surface-adhered kinesin motor proteins. Using a simple model and measurements of the path persistence length, motional diffusion coeff., and the distribution of av. velocities, the authors found that, at the time scale relevant in the envisioned nanobiodevices, variations in the time-averaged velocities between shuttles will make a stronger contribution to the dispersion of the swarm than both the fluctuations around the time-averaged velocity of an individual shuttle and the fluctuations in path length due to wiggling within the channel. Overall, the dispersion of such mol. shuttles is comparable to the dispersion of a sample plug transported by electroosmotic flow.
- 24Imafuku, Y.; Toyoshima, Y. Y.; Tawada, K. Fluctuation in the microtubule sliding movement driven by kinesin in vitro. Biophys. J. 1996, 70, 878– 886, DOI: 10.1016/S0006-3495(96)79631-5[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlslCqsQ%253D%253D&md5=9f28da461e8c2d86ccc9d63dcdf022d5Fluctuation in the microtubule sliding movement driven by kinesin in vitroImafuku, Yasuhiro; Toyoshima, Yoko Y.; Tawada, KatsuhisaBiophysical Journal (1996), 70 (2), 878-86CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)The authors studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calcg. the mean-square displacement deviation from the av. as a function of time, the authors obtained motional diffusion coeffs. for microtubules and analyzed the dependence of the coeffs. on microtubule length. The analyses suggest that (1) the motional diffusion coeff. consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coeff. of a filament in Brownian movement is) and another that is independent of the length, and (2) the length-dependent term decreases with increasing kinesin concn. This latter term almost vanishes within the length range the authors studied at high kinesin concns. From the length-dependence relation, the authors evaluated the friction coeff. for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coeff. obsd. at sufficiently high kinesin concns. indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.
- 25Kelly, R. H.; Yancey, P. H. High contents of trimethylamine oxide correlating with depth in deep-sea teleost fishes, skates, and decapod crustaceans. Biol. Bull. 1999, 196, 18– 25, DOI: 10.2307/1543162[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhs12gsrw%253D&md5=930ac9880a36e382ce691fdbf0c7f04dHigh contents of trimethylamine oxide correlating with depth in deep-sea teleost fishes, skates, and decapod crustaceansKelly, Robert H.; Yancey, Paul H.Biological Bulletin (Woods Hole, Massachusetts) (1999), 196 (1), 18-25CODEN: BIBUBX; ISSN:0006-3185. (Marine Biological Laboratory)In muscles of shallow-living marine animals, the osmolyte trimethylamine N-oxide (TMAO) is reportedly found (in millimoles of TMAO per kg of tissue wet wt.) at 30-90 in shrimp, 5-50 in crabs, 61-181 in skates, and 10-70 in most teleost fish. Recently our lab. reported higher levels (83-211 mmol/kg), correlating with habitat depth, in deep-sea gadiform teleosts. We now report the same trend in muscles of other animals, collected off the coast of Oregon from bathyal (1800-2000 m) and abyssal plain (2850 m) sites. TMAO contents (mmol/kg) were as follows: zoarcid teleosts, 103 (bathyal) and 197 (abyssal); scorpaenid teleosts, 32 (shallow) and 141 (bathyal); rajid skates, 215 (bathyal) and 244 (abyssal); caridean shrimp, 76 (shallow), 203 (bathyal), and 299 (abyssal); Chionoecetes crabs, 22 (shallow) and 164 (bathyal). Deep squid, clams, and anemones also had higher contents than shallow species. Osmoconformers showed compensation between TMAO and other osmolytes. Urea contents (typically 300 mmol/kg in shallow elasmobranchs) in skates were 214 (bathyal) and 136 (abyssal). Glycine contents in shrimp were 188 (shallow) and 52 (abyssal). High TMAO contents may reflect diet, reduce osmoregulatory costs, increase buoyancy, or counteract destabilization of proteins by pressure.
- 26Yancey, P. H.; Clark, M. E.; Hand, S. C.; Bowlus, R. D.; Somero, G. N. Living with water stress: evolution of osmolyte systems. Science 1982, 217, 1214– 1222, DOI: 10.1126/science.7112124[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XlsFyisbw%253D&md5=5695fde25a5ca2912c3fd3277c656f3cLiving with water stress: evolution of osmolyte systemsYancey, Paul H.; Clark, Mary E.; Hand, Steven C.; Bowlus, R. David; Somero, George N.Science (Washington, DC, United States) (1982), 217 (4566), 1214-22CODEN: SCIEAS; ISSN:0036-8075.A review and discussion with 80 refs. Striking convergent evolution is found in the properties of the org. osmotic solute (osmolyte) systems obsd. in bacteria, plants, and animals. Polyhydric alcs., free amino acids and their derivs., and combinations of urea and methylamines which comprise org. osmolyte systems, confer selective advantage. They are compatible with macromol. structure and function at high or variable (or both) osmolyte concns., and greatly reduce needs for modifying proteins to function in concd. intracellular solns. Osmolyte compatibility is proposed to result from the absence of osmolyte interactions with substrates and cofactors, and the nonperturbing of favorable effects of osmolytes on macromol.-solvent interactions.
- 27Lidbury, I.; Murrell, J. C.; Chen, Y. Trimethylamine N-oxide metabolism by abundant marine heterotrophic bacteria. Proc. Natl. Acad. Sci. 2014, 111, 2710– 2715, DOI: 10.1073/pnas.1317834111[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXivFamu7Y%253D&md5=de541c431e68a6126a4dbab17345c37aTrimethylamine N-oxide metabolism by abundant marine heterotrophic bacteriaLidbury, Ian; Murrell, J. Colin; Chen, YinProceedings of the National Academy of Sciences of the United States of America (2014), 111 (7), 2710-2715CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Trimethylamine N-oxide (TMAO) is a common osmolyte found in a variety of marine biota and has been detected at nanomolar concns. in oceanic surface waters. TMAO can serve as an important nutrient for ecol. important marine heterotrophic bacteria, particularly the SAR11 clade and marine Roseobacter clade (MRC). However, the enzymes responsible for TMAO catabolism and the membrane transporter required for TMAO uptake into microbial cells have yet to be identified. The authors show here that the enzyme TMAO demethylase (Tdm) catalyzes the first step in TMAO degrdn. This enzyme represents a large group of proteins with an uncharacterized domain (DUF1989). The function of TMAO demethylase in a representative from the SAR11 clade (strain HIMB59) and in a representative of the MRC (Ruegeria pomeroyi DSS-3) was confirmed by heterologous expression of tdm (the gene encoding Tdm) in Escherichia coli. In R. pomeroyi, mutagenesis expts. confirmed that tdm is essential for growth on TMAO. The authors also identified a unique ATP-binding cassette transporter (TmoXWV) found in a variety of marine bacteria and exptl. confirmed its specificity for TMAO through marker exchange mutagenesis and lacZ reporter assays of the promoter for genes encoding this transporter. Both Tdm and TmoXWV are particularly abundant in natural seawater assemblages and actively expressed, as indicated by a no. of recent metatranscriptomic and metaproteomic studies. These data suggest that TMAO represents a significant, yet overlooked, nutrient for marine bacteria.
- 28Bachand, G. D.; Jain, R.; Ko, R.; Bouxsein, N. F.; VanDelinder, V. Inhibition of microtubule depolymerization by osmolytes. Biomacromolecules 2018, 19, 2401– 2408, DOI: 10.1021/acs.biomac.7b01799[ACS Full Text], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFGnt7o%253D&md5=53e1b16f653a0e95c8fea72589cf56acInhibition of microtubule depolymerization by osmolytesBachand, George D.; Jain, Rishi; Ko, Randy; Bouxsein, Nathan F.; Van Delinder, VirginiaBiomacromolecules (2018), 19 (7), 2401-2408CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)Microtubule dynamics play a crit. role in the normal physiol. of eukaryotic cells as well as a no. of cancers and neurodegenerative disorders. The polymn./depolymn. of microtubules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-assocd. proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here we describe the ability of the osmolytes polyethylene glycol (PEG) and trimethylamine-N-oxide (TMAO) to inhibit the depolymn. of individual microtubule filaments for extended periods of time (up to 30 days). We further show that PEG stabilizes microtubules against both temp.- and calcium-induced depolymn. Our results collectively suggest that the obsd. inhibition may be related to combination of the kosmotropic behavior and excluded vol./osmotic pressure effects assocd. with PEG and TMAO. Taken together with prior studies, our data suggest that the physiochem. properties of the local environment can regulate microtubule depolymn. and may potentially play an important role in in vivo microtubule dynamics.
- 29Chase, K.; Doval, F.; Vershinin, M. Enhanced stability of kinesin-1 as a function of temperature. Biochem. Biophys. Res. Commun. 2017, 493, 1318– 1321, DOI: 10.1016/j.bbrc.2017.09.172[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1aisbbN&md5=48f99ee38fbc3c2ce85eab4aedeaae4dEnhanced stability of kinesin-1 as a function of temperatureChase, K.; Doval, F.; Vershinin, M.Biochemical and Biophysical Research Communications (2017), 493 (3), 1318-1321CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Kinesin-1 is a mechanochem. enzyme which mediates long distance intracellular cargo transport along microtubules in a wide variety of eukaryotic cells. Kinesin is also relatively easy to purify and shows robust function in vitro, leading to numerous proposals for using the kinesin-1/microtubule system for nanoscale transport in engineered devices. However, kinesin in vitro shows signs of degrdn. at ∼30 °C which severely limits its usability in biomimetic engineering. Notably, kinesin-1 functions robustly in animal cells at body temps. as high as 40 °C which suggests that kinesin functioning can be stabilized beyond what is obsd. in vitro. The present study investigated the effect of trimethylamine N-oxide (TMAO) as a potential heat-protecting agent for kinesin function and microtubule stability. We show that at a concn. of 200 mM, TMAO can indeed stabilize kinesin-based motility up to a little over 50 °C and that such motility can be sustained for extended periods of time. Our results suggest that intracellular crowding (mimicked in vitro by TMAO) can indeed stabilize kinesin-1 at high temps. and helps resolve a long standing discrepancy between thermal stability of kinesin-1 obsd. in vivo and in vitro. Moreover, when considered together with our previous report that kinesin-1 can function well down to near-freezing conditions, this study establishes kinesin-1/microtubule motility as a thermally viable engineering platform.
- 30Castoldi, M.; Popov, A. V. Purification of brain tubulin through two cycles of polymerization–depolymerization in a high-molarity buffer. Protein Expression Purif. 2003, 32, 83– 88, DOI: 10.1016/S1046-5928(03)00218-3[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXosVKlu74%253D&md5=73a6d137c85393afdbaa4dbfc50ed059Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity bufferCastoldi, Mirco; Popov, Andrei V.Protein Expression and Purification (2003), 32 (1), 83-88CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier Science)Microtubules can be assembled in vitro from purified α/β tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymn.-depolymn., followed by ion-exchange chromatog. to remove any contaminating microtubule-assocd. proteins and motors. Here, we show that only two cycles of polymn.-depolymn. of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and prodn. of a high-concn. tubulin soln. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatog.-based procedures.
- 31Peloquin, J.; Komarova, Y.; Borisy, G. Conjugation of fluorophores to tubulin. Nat. Methods 2005, 2, 299– 303, DOI: 10.1038/nmeth0405-299[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisFejtLc%253D&md5=3636fb218713abcab73a959131c558d5Conjugation of fluorophores to tubulinPeloquin, John; Komarova, Yulia; Borisy, GaryNature Methods (2005), 2 (4), 299-303CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The use of fluorescently labeled proteins as probes of the structure and function of living cells began with the study of the components of the cytoskeleton and has become a powerful approach for studying the distribution and dynamics of these components. Primary components of the cytoskeleton such as tubulin, actin and myosin are abundant and can be purified relatively easily in hundreds of milligram quantities from animal tissues. Fluorophores contg. amine or thiol reactive groups are used to selectively modify lysine or cysteine residues, resp. This protocol describes the fluorescent conjugation of tubulin isolated from brain tissue.
- 32Fujimoto, K.; Kitamura, M.; Yokokawa, M.; Kanno, I.; Kotera, H.; Yokokawa, R. Colocalization of quantum dots by reactive molecules carried by motor proteins on polarized microtubule arrays. ACS Nano 2013, 7, 447– 455, DOI: 10.1021/nn3045038[ACS Full Text], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVahs77N&md5=adf4185e38e52015279614f69e8fbc5aColocalization of Quantum Dots by Reactive Molecules Carried by Motor Proteins on Polarized Microtubule ArraysFujimoto, Kazuya; Kitamura, Masuto; Yokokawa, Masatoshi; Kanno, Isaku; Kotera, Hidetoshi; Yokokawa, RyujiACS Nano (2013), 7 (1), 447-455CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The field of microfluidics has drastically contributed to downscale the size of benchtop expts. to the dimensions of a chip without compromising results. However, further miniaturization and the ability to directly manipulate individual mols. require a platform that permits organized mol. transport. The motor proteins and microtubules that carry out orderly intracellular transport are ideal for driving in vitro nanotransport. Here, a reconstruction of the cellular kinesin/dynein-microtubule system in nanotracks provides a mol. total anal. system (MTAS) to control massively parallel chem. reactions. The mobility of kinesin and a microtubule dissocn. method enable orientation of a microtubule in an array for directed transport of reactive mols. carried by kinesin or dynein. The binding of glutathione S-transferase (GST) to glutathione (GSH) and the binding of streptavidin to biotin are visualized as colocalizations of quantum dots (Q-dots) when motor motilities bring them into contact. The organized nanotransport demonstrated here suggests the feasibility of using the authors' platform to perform parallel biochem. reactions focused at the mol. level.
- 33Munmun, T.; Kabir, A. M. R.; Sada, K.; Kakugo, A. Complete, rapid and reversible regulation of the motility of a nano-biomolecular machine using an osmolyte trimethylamine-N-oxide. Sens. Actuators, B 2020, 304, 127231 DOI: 10.1016/j.snb.2019.127231[Crossref], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFCmsrrM&md5=c8fcf88d3b6dca1b29ee762a6e62d29dComplete, rapid and reversible regulation of the motility of a nano-biomolecular machine using an osmolyte trimethylamine-N-oxideMunmun, Tasrina; Kabir, Arif Md. Rashedul; Sada, Kazuki; Kakugo, AkiraSensors and Actuators, B: Chemical (2020), 304 (), 127231CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)Nanoscale transportation in engineered environments is crit. towards designing efficient and smart hybrid bio-nanodevices. Biomol. 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 biomol. motors is the key to control such integrated nanodevices. We present a simple method to regulate the activity of a biomol. 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 concn. 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 concn. The halted motility of MTs is fully restored upon elimination of TMAO. Repeated cycles of TMAO addn. and removal enable cyclical inhibition and restoration of the motility of MTs. These results demonstrate an ability to control nanoscale motion of a biomol. motor in an artificial environment. This work facilitates further tunability over functions of biomol. motors, which in turn will foster their nanotechnol. applications, such as in nano-transportation.
- 34Kumemoto, R.; Yusa, K.; Shibayama, T.; Hatori, K. Trimethylamine N-oxide suppresses the activity of the actomyosin motor. Biochim. Biophys. Acta, Gen. Subj. 1820, 2012, 1597– 1604, DOI: 10.1016/j.bbagen.2012.06.006
- 35Qian, H.; Sheetz, M. P.; Elson, E. L. Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys. J. 1991, 60, 910– 921, DOI: 10.1016/S0006-3495(91)82125-7[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK38%252FnsFyqsw%253D%253D&md5=074789e42fc9976b328088a2ed4971a1Single particle tracking. Analysis of diffusion and flow in two-dimensional systemsQian H; Sheetz M P; Elson E LBiophysical journal (1991), 60 (4), 910-21 ISSN:0006-3495.Analysis of the trajectories of small particles at high spatial and temporal resolution using video enhanced contrast microscopy provides a powerful approach to characterizing the mechanisms of particle motion in living cells and in other systems. We present here the theoretical basis for the analysis of these trajectories for particles undergoing random diffusion and/or systematic transport at uniform velocity in two-dimensional systems. The single particle tracking method, based on observations of the trajectories of individual particles, is compared with methods that characterize the motions of a large collection of particles such as fluorescence photobleaching recovery. Determination of diffusion coefficients or transport velocities either from correlation of positions or of velocities of the particles is discussed. A result of practical importance is an analysis of the dependence of the expected statistical uncertainty of these determinations on the number of position measurements. This provides a way of judging the accuracy of the diffusion coefficients and transport velocities obtained using this approach.
- 36Tawada, K.; Sekimoto, K. Protein friction exerted by motor enzymes through a weak-binding interaction. J. Theor. Biol. 1991, 150, 193– 200, DOI: 10.1016/S0022-5193(05)80331-5[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmtVWgtbs%253D&md5=aa21e75cebf986f8df7d6845d672bb98Protein friction exerted by motor enzymes through a weak-binding interactionTawada, Katsuhisa; Sekimoto, KenJournal of Theoretical Biology (1991), 150 (2), 193-200CODEN: JTBIAP; ISSN:0022-5193.Recently R. D. Vale et al. (1989) reported an observation of the one-dimensional Brownian movement of microtubules bound to flagellar dynein through a weak-binding interaction. In this study, a theor. model of this phenomenon is proposed. The model consists of a rigid microtubule assocd. with a no. of elastic dynein heads through a weak-binding interaction at equil. The model implies that (1) the Brownian motion of the microtubule is not directly driven by the at. collision of the solvent particles, but is driven by the thermally-generated structural fluctuations of the dynein heads which interact with the microtubule; (2) dynein heads through a weak-binding interaction exert a frictional drag force on the sliding motion of the microtubule and the drag force is proportional to the sliding velocity the same as in hydrodynamic viscous friction. This protein friction, with such viscous-like characteristics, may well play a role as a velocity-limiting factor in the normal ATP-induced sliding movement of motile proteins.
- 37Bormuth, V.; Varga, V.; Howard, J.; Schäffer, E. Protein friction limits diffusive and directed movements of kinesin motors on microtubules. Science 2009, 325, 870– 873, DOI: 10.1126/science.1174923[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXps1Ogtr8%253D&md5=0fe1c7ebf58a66d30f2898a457a5be3fProtein friction limits diffusive and directed movements of kinesin motors on microtubulesBormuth, Volker; Varga, Vladimir; Howard, Jonathon; Schaeffer, ErikScience (Washington, DC, United States) (2009), 325 (5942), 870-873CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Friction limits the operation of macroscopic engines and is crit. to the performance of micromech. devices. We report measurements of friction in a biol. nanomachine. Using optical tweezers, we characterized the frictional drag force of individual kinesin-8 motor proteins interacting with their microtubule tracks. At low speeds and with no energy source, the frictional drag was related to the diffusion coeff. by the Einstein relation. At higher speeds, the frictional drag force increased nonlinearly, consistent with the motor jumping 8 nm between adjacent tubulin dimers along the microtubule, and was asym., reflecting the structural polarity of the microtubule. We argue that these frictional forces arise from breaking bonds between the motor domains and the microtubule, and they limit the speed and efficiency of kinesin.
- 38Munmun, T.; Kabir, A. M. R.; Katsumoto, Y.; Sada, K.; Kakugo, A. Controlling the kinetics of interaction between microtubules and kinesins over a wide temperature range using the deep-sea osmolyte trimethylamine N-oxide. Chem. Commun. 2020, 56, 1187– 1190, DOI: 10.1039/C9CC09324A[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVyntL3N&md5=d0992dafb66c9a4057e1656c40e4778dControlling the kinetics of interaction between microtubules and kinesins over a wide temperature range using the deep-sea osmolyte trimethylamine N-oxideMunmun, Tasrina; Kabir, Arif Md. Rashedul; Katsumoto, Yukiteru; Sada, Kazuki; Kakugo, AkiraChemical Communications (Cambridge, United Kingdom) (2020), 56 (8), 1187-1190CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Trimethylamine N-oxide is found to be effective in regulating the interaction between microtubules and kinesins over a wide temp. range. The lifetime of the motility of microtubules on kinesins at high temps. is prolonged using trimethylamine N-oxide. The activation energy of microtubule motility is increased by trimethylamine N-oxide. Prolonged operation at high temps. decreased the activation energy of MT motility despite the increase in concn. of trimethylamine N-oxide.
- 39VanDelinder, V.; Sickafoose, I.; Imam, Z. I.; Ko, R.; Bachand, G. D. The effects of osmolytes on in vitro kinesin-microtubule motility assays. RSC Adv. 2020, 10, 42810– 42815, DOI: 10.1039/D0RA08148E[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVSks7vM&md5=68d4365c5e6d8c6aa563ae40e388e478The effects of osmolytes on in vitro kinesin-microtubule motility assaysVanDelinder, Virginia; Sickafoose, Ian; Imam, Zachary I.; Ko, Randy; Bachand, George D.RSC Advances (2020), 10 (70), 42810-42815CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)The gliding motility of microtubule filaments has been used to study the biophys. properties of kinesin motors, as well as being used in a variety of nanotechnol. applications. While microtubules are generally stabilized in vitro with paclitaxel (Taxol), osmolytes such as polyethylene glycol (PEG) and trimethylamine N-oxide (TMAO) are also able to inhibit depolymn. over extended periods of time. High concns. of TMAO have also been reported to reversibly inhibit kinesin motility of paclitaxel-stabilized microtubules. Here, we examd. the effects of the osmolytes PEG, TMAO, and glycerol on stabilizing microtubules during gliding motility on kinesin-coated substrates. As previously obsd., microtubule depolymn. was inhibited in a concn. dependent manner by the addn. of the different osmolytes. Kinesin-driven motility also exhibited concn. dependent effects with the addn. of the osmolytes, specifically reducing the velocity, increasing rates of pinning, and altering trajectories of the microtubules. These data suggest that there is a delicate balance between the ability of osmolytes to stabilize microtubules without inhibiting motility. Overall, these findings provide a more comprehensive understanding of how osmolytes affect the dynamics of microtubules and kinesin motors, and their interactions in crowded environments.
- 40Palacci, H.; Idan, O.; Armstrong, M. J.; Agarwal, A.; Nitta, T.; Hess, H. Velocity fluctuations in kinesin-1 gliding motility assays originate in motor attachment geometry variations. Langmuir 2016, 32, 7943– 7950, DOI: 10.1021/acs.langmuir.6b02369[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFKrtrbI&md5=6b9a72b06c5a12b38cba1cfb4c862c9dVelocity Fluctuations in Kinesin-1 Gliding Motility Assays Originate in Motor Attachment Geometry VariationsPalacci, Henri; Idan, Ofer; Armstrong, Megan J.; Agarwal, Ashutosh; Nitta, Takahiro; Hess, HenryLangmuir (2016), 32 (31), 7943-7950CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)Motor proteins such as myosin and kinesin play a major role in cellular cargo transport, muscle contraction, cell division, and engineered nanodevices. Quantifying the collective behavior of coupled motors is crit. to our understanding of these systems. An excellent model system is the gliding motility assay, where hundreds of surface-adhered motors propel one cytoskeletal filament such as an actin filament or a microtubule. The filament motion can be obsd. using fluorescence microscopy, revealing fluctuations in gliding velocity. These velocity fluctuations have been previously quantified by a motional diffusion coeff., which Sekimoto and Tawada explained as arising from the addn. and removal of motors from the linear array of motors propelling the filament as it advances, assuming that different motors are not equally efficient in their force generation. A computational model of kinesin head diffusion and binding to the microtubule allowed us to quantify the heterogeneity of motor efficiency arising from the combination of anharmonic tail stiffness and varying attachment geometries assuming random motor locations on the surface and an absence of coordination between motors. Knowledge of the heterogeneity allows the calcn. of the proportionality const. between the motional diffusion coeff. and the motor d. The calcd. value (0.3) is within a std. error of our measurements of the motional diffusion coeff. on surfaces with varying motor densities calibrated by landing rate expts. This allowed us to quantify the loss in efficiency of coupled mol. motors arising from heterogeneity in the attachment geometry.
- 41Tsitkov, S., Hess, H. Design of active nanosystems incorporating biomolecular motors. In Out-of-Equilibrium (Supra) molecular Systems and Materials, 1st ed.; Giuseppone, N., Walther, A., Eds.; Wiley-VCH: Weinheim, Germany, 2021; pp 379– 422.
- 42Saper, G.; Hess, H. Synthetic systems powered by biological molecular motors. Chem. Rev. 2020, 120, 288– 309, DOI: 10.1021/acs.chemrev.9b00249[ACS Full Text], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslektr%252FN&md5=e9b4499656ef83ef272964d21edb71dcSynthetic Systems Powered by Biological Molecular MotorsSaper, Gadiel; Hess, HenryChemical Reviews (Washington, DC, United States) (2020), 120 (1), 288-309CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Biol. mol. motors (or biomol. motors for short) are nature's soln. to the efficient conversion of chem. energy to mech. movement. In biol. systems, these fascinating mols. are responsible for movement of mols., organelles, cells, and whole animals. In engineered systems, these motors can potentially be used to power actuators and engines, shuttle cargo to sensors, and enable new computing paradigms. Here, we review the progress in the past decade in the integration of biomol. motors into hybrid nanosystems. After briefly introducing the motor proteins kinesin and myosin and their assocd. cytoskeletal filaments, we review recent work aiming for the integration of these biomol. motors into actuators, sensors, and computing devices. In some systems, the creation of mech. work and the processing of information become intertwined at the mol. scale, creating a fascinating type of "active matter". We discuss efforts to optimize biomol. motor performance, construct new motors combining artificial and biol. components, and contrast biomol. motors with current artificial mol. motors. A recurrent theme in the work of the past decade was the induction and utilization of collective behavior between motile systems powered by biomol. motors, and we discuss these advances. The exertion of external control over the motile structures powered by biomol. motors has remained a topic of many studies describing exciting progress. Finally, we review the current limitations and challenges for the construction of hybrid systems powered by biomol. motors and try to ascertain if there are theor. performance limits. Engineering with biomol. motors has the potential to yield com. viable devices, but it also sharpens our understanding of the design problems solved by evolution in nature. This increased understanding is valuable for synthetic biol. and potentially also for medicine.
Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c01228.
Additional experimental results on the effect of TMAO on the standard deviation of the MT velocity and standard deviation/average velocity of MTs, mean-square deviation of the displacement of MTs as a function of travel time at various TMAO concentrations, reversible change of the mean-square deviation of the displacement of MTs as a function of travel time at 1000 mM TMAO, and captions for supporting movies (PDF)
Motility of MTs on kinesins, in an in vitro gliding assay, in the absence of TMAO. The field of view is 237.6 μm × 281.6 μm (Movie S1) (MP4)
Motility of MTs on kinesins, in an in vitro gliding assay, in the presence of 1200 mM TMAO. The field of view is 237.6 μm × 281.6 μm (Movie S2) (MP4)
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