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Prolongation of the Active Lifetime of a Biomolecular Motor for in Vitro Motility Assay by Using an Inert Atmosphere Arif Md. Rashedul Kabir,† Daisuke Inoue,‡ Akira Kakugo,*,§,|| Akiko Kamei,§ and Jian Ping Gong*,§ †
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Division of Biological Sciences, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan § Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
)
‡
bS Supporting Information ABSTRACT: Over the last few decades, the in vitro motility assay has been performed to probe the biophysical and chemomechanical properties as well as the self-organization process of biomolecular motor systems such as actinmyosin and microtubulekinesin. However, aggression of the reactive oxygen species (ROS) and concomitant termination of the activity of biomolecular motors during investigation remains a drawback of this assay. Despite enzymatic protection that makes use of a combination of glucose, glucose oxidase, and catalase, the active lifetime of biomolecular motors is found to be only a few hours and this short lifetime restricts further study on those systems. We have solved this problem by using a newly developed system of the in vitro motility assay that is conducted in an inert nitrogen gas atmosphere free of ROS. Using microtubulekinesin as a model system we have shown that our system has prolonged the active lifetime of the biomolecular motor until several days and even a week by protecting it from oxidative damage.
’ INTRODUCTION Linear biomolecular motor systems such as actinmyosin and microtubules (MTs)kinesin perform various functions in vivo such as cell motility,1 cytokinesis,2 and cellular transport. The in vitro motility assay3,4 has been widely employed for studying the functions of biomolecular motor systems and unveiled the mechanisms of actinmyosin and MTskinesin interactions, in vivo. During the last few decades, the in vitro motility assay has also provided us with valuable insight into important aspects of biomolecular motor functions.57 More recently, on the basis of the knowledge obtained through the in vitro motility assay, the concept of new devices based on biomolecular motors8 has been developed for serving different purposes such as nanoscale molecular shuttles,9,10 surface imaging,11 force measurements,12 or lab-on-a-chip devices.13,14 However, attack of the reactive oxygen species (ROS) on biomolecular motor systems results in a decrease in their activity and shortens their lifetime during the investigation using the in vitro motility assay. There are some reports detailing the effect of the ROS on the biomolecular motor systems. Vigers et al.15 observed the ROS induced breakage of fluorescent MTs under irradiation of excitation light, both in vivo and in vitro. Dixit and Cyr16 showed that tobacco suspension cells were damaged by ROS when the MTs were visualized by transforming an intrinsically fluorescent protein. Moreover, some photosensitizers were found to destroy MTs1719 but the mechanism still remains impalpable. A combination of glucose, r 2011 American Chemical Society
glucose oxidase, and catalase has been commonly used so far as a scavenger to minimize the oxidative stress on biomolecular motors.20 This scavenger system, however, can work only for a limited period of time (approximately a few hours). Prolonged study on the biomolecular motor systems is considerably restricted which is required to probe the time dependent properties of those systems. For example, it was reported that MTs underwent dynamic self-organization where the morphology of the organized body was found to be dependent on the time length of organization process.2123 Lack of a suitable system which could ensure the longer lifetime and permit for the prolonged study hampered the basic research on biomolecular motors. Moreover, a poor basic understanding on their in vitro stability makes it difficult to develop artificial devices based on biomolecular motor. In this work, we have designed a simple but crucially significant experimental setup named “inert chamber system” (ICS) that enabled us to perform prolonged study using in vitro motility assay which was conducted in an inert nitrogen gas atmosphere free of ROS, as illustrated in Figure 1a. We have employed the fluorescently labeled microtubulesgreen fluorescent proteinfused kinesin (rhodamine MTsGFP- kinesin) as the model Received: June 30, 2011 Revised: October 3, 2011 Published: October 04, 2011 13659
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Figure 1. (a) Schematic illustration of the in vitro motility assay performed inside an inert nitrogen atmosphere. (b) Design of the inert chamber; numbers in the figure indicate distance in millimeters (mm).
biomolecular motor system. By using the ICS, the harmful oxidative stress on the biomolecular motor could be minimized dramatically during the in vitro motility assay, and hence the active lifetime of motor protein kinesin was prolonged until several days or a week depending on our experimental design. By contrast, in the presence of scavengers without ICS, the active lifetime was found to be only a few hours. We have also showed that the use of the ICS can substantially minimize the breakage of fluorescent MTs. The ICS thus offers a powerful means for prolonging the active lifetime of biomolecular motor systems and allows for a long-term investigation. We believe the prolonged active lifetime and increased in vitro stability of biomolecular motor system achieved by using ICS would widen the scope for using in vitro motility assay and enable further investigation of the time dependent properties of the biomolecular motors. At the same time, it will serve as a guideline for the development of the biomolecular motor based artificial nanodevices and increase the potential application of biomolecular motor systems, in vitro.
’ EXPERIMENTAL SECTION Preparation of Tubulins and Kinesin. Tubulin was purified from porcine brain using high-concentration PIPES buffer (1 M PIPES, 20 mM EGTA, 10 mM MgCl2; pH adjusted to 6.8 using KOH). Highmolarity PIPES buffer (HMPB) and brain reconstitution buffer 80 mM PIPES (BRB80) were prepared using PIPES from Sigma, and the pH was adjusted using KOH.24 GFP-fused kinesin-1, consisting of the first 560 amino acids (K560-GFP), was prepared by partially modifying the expression and purification methods.25 Preparation of Labeled Tubulins. Rhodamine-labeled tubulin was prepared using tetramethylrhodamine succinimidyl ester (TAMRASE; Invitrogen) according to the standard techniques.26 Rhodaminetubulin was obtained by chemical cross-linking and the labeling ratio was 1.5. This ratio was determined by measuring the absorbance of the protein at 280 nm and that of tetramethylrhodamine at 555 nm.
Motility Assay 1 (Motility Series No. 1 and 4). Rhodaminelabeled MTs were obtained by polymerizing rhodaminetubulin and native tubulin (molar ratio of 1:17; final concentration was 53.5 μM) in a polymerization buffer (80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 1 mM guanosine-50 -triphosphate (GTP), 5% DMSO; pH adjusted to 6.8) incubating at 37 °C for 30 min. The solution containing the MTs was then diluted 10-fold with the stabilizing buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1% DMSO, 10 μM paclitaxel; pH adjusted to 6.8) and gently pipetted 10 times. Hereafter, the concentration of MTs represents the concentration of tubulins. This MTs was again diluted 10-fold with the Pre-MT buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL1 casein, 1 mM DTT, 10 μM paclitaxel, ∼1% DMSO, 4.5 mg mL1 D-glucose, 50 U mL1 glucose oxidase, 50 U mL1 catalase as scavenger; pH 6.8) to a concentration of 535 nM (hereafter MT buffer) just before performing the motility assay. Flow cells were prepared by placing a cover glass (18 18 mm2; Matsunami) on a slide glass (26 76 mm2) equipped with a pair of spacers to form a chamber of approximate dimensions 4 18 0.1 mm3 (W L H). A single layer of Parafilm was used to fix the spacer-separated glasses by heating. The flow cells were filled with 0.2 mg mL1 anti-GFP antibody (Invitrogen) and incubated for 15 min, followed by a wash with 48 μL of WB_1 buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL1 casein; pH adjusted to 6.8 with KOH). After incubating with the WB_1 buffer for 5 min to block the remaining glass surface, 24 μL of KG buffer (622 nM kinesin, ∼80 mM PIPES, 40 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL1 casein, 1 mM DTT, 10 μM paclitaxel; pH 6.8) was introduced, and this was incubated for 10 min to allow kinesin to bind to the antibody. The flow cells were washed with 32 μL of M_BRB buffer (∼80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL1 casein, 1 mM DTT, 10 μM paclitaxel, 1% DMSO, 4.5 mg mL1 D-glucose, 50 U mL1 glucose oxidase, 50 U mL1 catalase; pH 6.8). Next, 24 μL of MT buffer was introduced and incubated for 10 min, followed by washing with 32 μL of M_BRB buffer. Finally, the motility assay was initiated by applying 24 μL of M-buffer (∼80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 0.5 mg mL1 casein, 1 mM DTT, 10 μM paclitaxel, 1% DMSO, 4.5 mg mL1 D-glucose, 50 U mL1 glucose oxidase, 50 U mL1 13660
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Langmuir catalase, 5 mM ATP; pH 6.8). As the ATP recycling system for motility series no. 4, phosphoenolpyruvate (PEP) and pyruvate kinase (PK) (Sigma) were used together mixed with the M-buffer at final concentrations of 2.5 mM and 2 U mL1, respectively. To prevent drying of the sample, extra M-buffer was kept at both ends of the flow cell. First microscopic observation was performed just after the addition of M-buffer.
Motility Assay 2 (Motility Series No. 2, 3, 5, 6 and Active Lifetime Assay). Motility assay series no. 2, 3, 5, and 6 and active lifetime assay were performed inside an inert nitrogen gas atmosphere using the ICS. The ICS consists of three main parts: nitrogen gas container (cylinder), humidity supporter, and the chamber as shown schematically in Figure 1a. Design of the chamber is shown in Figure 1b. The chamber has two parts: one is the base plate made of stainless steel, and the other one is the cover plate made of poly(methyl methacrylate) (PMMA) (see the Supporting Information, Figure 1). The base plate contains the inlet and outlet placed horizontally, which facilitates the passage of nitrogen gas through the chamber and allows the flow rate of nitrogen gas to be controlled. The base plate also contains a hole which allows the 60 objective and the bottom surface of the flow cell to come in close contact during observation by fluorescence microscope. The method for the preparation of flow cell was almost the same as described in the “motility assay 1” section, except that the size of the slide glass was 50 40 mm2. The volume of the flow cell was 4 18 0.1 mm3, for which 90 min incubation of the flow cell (after applying the sample) in the nitrogen gas atmosphere inside the inert chamber (prior to observation by fluorescence microscope) was found to be sufficient for ensuring good motility of MTs. To observe good motility of MTs in the case of flow cells bigger than 4 18 0.1 mm3, a longer incubation time was required. Compositions of WB_1, KG-buffer, M_BRB, MT-buffer, and M-buffer were the same as described in the section “motility assay 1” unless mentioned otherwise. First, the flow cells were filled with 0.2 mg mL1 anti-GFP antibody (Invitrogen) and incubated for 15 min, followed by washing with 48 μL of WB_1 buffer. After incubation with the WB_1 buffer for 5 min, 24 μL of KG buffer was introduced, followed by 10 min incubation. The flow cells were then washed with 32 μL of M_BRB buffer with scavenger (for motility series 2 and 5) and without scavenger (for motility series 3, 6 and active lifetime assay). Next, 24 μL of MT buffer (with/without scavenger) was introduced in respective flow cells, and flow cells were incubated for 10 min, followed by washing with 32 μL of M_BRB buffer (with/without scavenger). Finally, the motility assay was initiated by applying 24 μL of M-buffer (with/without scavenger). For motility series 5, 6 and active lifetime assay, phosphoenolpyruvate (PEP) and pyruvate kinase (PK) (Sigma) were used at final concentrations of 2.5 mM and 2 U mL1, respectively, together mixed with the M-buffer as the ATP recycling system. After preparation, flow cells were placed manually inside the inert chamber covering the entire area of the hole at the base plate. The compression spring (see the Supporting Information, Figure 1) was then placed upon the flow cell, and the chamber was closed. Three main parts of the ICS (nitrogen gas container, humidity supporter, and inert chamber) were then connected (series connection) by tube (PharMed BPT). After that, humid nitrogen gas was passed through the ICS at a flow rate of 10 cm3 s1, keeping the outlet open to 50% (inlet was kept 100% open) and thus the existing oxygen was gradually removed out from the chamber. During the experiment, the humidity inside the inert chamber was maintained at more than ∼90%. Finally, after passing nitrogen gas for 90 min through the chamber, it was mounted on the stage of a fluorescence microscope by placing the hole of the base plate of inert chamber and the 60 objective facing each other. Prior to this, the 60 objective was coupled with nondrying immersion oil (Cargille Laboratories). Finally, microscopic observation was done at 2225 °C, and nitrogen gas was kept passing continuously until the experiment was finished. The ICS was found to work properly for the above-mentioned volume of the flow cell and incubation time, when the outlet was opened 50%. A longer
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incubation inside the nitrogen gas atmosphere prior to microscopic observation would be required if the outlet is kept open less than 50%. In the active lifetime assay (ALTA; see Results and Discussion), methyl cellulose was used mixed with the M-buffer at a final concentration of 0.06% (w/v) and new MT buffer was added to the flow cell from time to time. For this, we brought the flow cell out of the chamber and added new MT buffer and M-buffer. The flow cell was then again fixed inside the chamber and incubated in nitrogen atmosphere.
Motility Assay 3 (Investigation of Static Lifetime of Motor Protein) (i) Flow cells were prepared as described in the section “motility assay 2”. After application of KG buffer, flow cells were incubated at room temperature inside an inert nitrogen atmosphere (sample) and also in an ambient atmosphere (control) for a prescribed time period. At room temperature, the motility of MTs was initiated by applying M-buffer after introduction of MT buffer and M_BRB buffer with scavenger (for control) and without scavenger (for sample); and microscopic observation was done as described in sections “motility assay 1” and “motility assay 2”. (ii) KG buffer was directly incubated at room temperature inside an an inert nitrogen atmosphere (not as a flow cell) for a prescribed time, and after that incubated kinesin was used for motility assay by preparing a new flow cell as described in the section “motility assay 2”.
Study on Photobleaching Effect and Determination of Dissolved Oxygen Concentration. For studying the photobleaching of samples in the presence of scavengers, flow cells were prepared as described in the section “motility assay 1”. Flow cells were continuously illuminated by radiation under fluorescence microscope, and fluorescence intensity values of GFP-kinesin and rhodamine-MTs were recorded with time. In the case of the photobleaching study using ICS (without scavengers), first flow cells were prepared as described in the section “motility assay 2”. Then flow cells were placed inside the inert chamber, and the chamber was closed. Nitrogen gas was then passed through the chamber for 90 min prior to the irradiation. Exposure time and light source intensity were kept same in all the cases mentioned above. Dissolved oxygen (DO) concentrations inside the motility assay systems, both using an inert chamber system (without scavengers) and using scavengers (without ICS), were determined by a luminescent dissolved oxygen probe (HACH-HQ40d). Prior to the determination of DO, the probe was calibrated in water-saturated air. Due to geometrical difficulty, it was not possible to measure the DO directly inside the flow cell. Instead, we measured the DO in the presence of scavengers, inside a small beaker (open system) maintaining exactly the same condition of the flow cell used for motility assay (as described in the section “motility assay 1”). However, in the case of ICS, first a flow cell was prepared and placed inside the inert chamber as described in the section “motility assay 2”. Then nitrogen gas was kept passing through the chamber and the concentration of DO was measured with time inside a closed humidifier by placing it at the outlet position of the chamber. All the measurements were performed at 2225 °C. Each measurement was performed at least three times for confirming the reliability of data. Microscopic Image Capture. For studying the motility of MTs and photobleaching of MTs and kinesin, samples were illuminated with a 100 W mercury lamp and visualized by epifluorescence microscopy using an oil-coupled Plan Apo 60 1.40 objective (Nikon). Filter blocks with UV-cut specification (TRITC: EX540/25, DM565, BA606/55; GFP-HQ: EX455-485, DM495, BA500-545; Nikon) were used in the optical path of the microscope that allowed the visualization of samples but eliminated the UV part of radiation and minimized the harmful effect of UV radiation on samples. Images were captured using a cooled-CCD camera (Cascade II, Nippon Roper) connected to a PC. To capture a field of view for more than several minutes, ND filters (ND4, 25% transmittance) were inserted into the illuminating light path of the fluorescence microscope to avoid photobleaching of rhodamine-labeled 13661
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Table 1. Experimental Conditions for Different Motility Assay Seriesa motility
scavengers
series no.
a
inert chamber
ATP recycling
system
system
1 2
O O
O
3
O
4
O
O
5
O
O
O
6
O
O
Circle indicates “yes” and cross indicates “no”.
MTs and GFP-kinesins. In this work, the power of the incident radiation on GFP-kinesin and rhodamine-MTs were ∼330 and ∼480 μW, respectively (measured via Neo Ark PM233 Power Meter, Nihon Kagaku Eng.). The motions of MTs were analyzed using image analysis software (Image Pro Plus 6.1, Media Cybernetics). Image Analysis for Motility Assays. Movies of the MTs motility assay captured by fluorescence microscope were analyzed using image analysis software (Image Pro Plus 6.1, Media Cybernetics). MTs that showed linear sliding motion over a distance of more than ∼0.5 μm, which corresponded to 2 pixels in digital data, were judged to be motile MTs. To measure the velocity of MTs, the track of the MTs in the six sequential images with 10 s intervals was manually detected using the software. The measured velocity was the average of the velocity of 50 individual filaments, unless mentioned otherwise; MTs filaments very short in length (less than 1 μm) were not considered for analyses.
’ RESULTS AND DISCUSSION We investigated the effects of the inert atmosphere, scavengers, and the ATP recycling system on the active lifetime of kinesin and MTs by performing motility assays under the six different experimental conditions outlined in Table 1 and monitoring the motion of MTs with time, which is a direct measure of the fineness of the MTskinesin system. We started counting the time after the addition of ATP to the motility assay system, and for those series wherein we used ICS, first microscopic observation was performed after 90 min of ATP addition; but for those series wherein we did not use ICS, first observation was done after 5 min of ATP addition. During our observation by fluorescence microscope, samples were illuminated with a 100 W mercury lamp and ND filters (25% transmittance) were inserted into the illuminating light path of the fluorescence microscope. The use of the ATP recycling system (see Experimental Section) allowed us to follow the motion of MTs over a kinesin-coated surface for a longer time period, ensuring a constant supply of energy through ATP. As the very initial step, we performed a motility assay without using scavengers and ICS where damage of kinesin (confirmed by the static MTs that showed no motility) and severe breakage of MTs were observed within a few seconds of illumination (see the Supporting Information, Movie 1); hence, this series could not be considered for further analysis. Inclusion of commonly used scavengers in the motility assay buffer allowed MTs to show motility by preventing oxidative damage of MTs and kinesin (see the Supporting Information, Movie 2). However, with the passage of time, severe breakage of MTs was observed, and eventually the fragmented MTs lost their motility (see the Supporting Information, Movie 3). After 210 min of ATP addition, although many broken MTs fragments were visible, most of those MTs showed
Figure 2. Effects of different experimental conditions on the moving ability of microtubules. Motility assays were performed using only scavengers (series 1); scavengers and ICS (series 2); only ICS (series 3); scavengers and ATP recycling system (series 4); scavengers, ICS, and ATP recycling system (series 5); and ICS and ATP recycling system (series 6). Error bar: standard deviation.
no motility. This indicated that a loss of activity of kinesin, rather than that of MTs, was largely responsible for the impairment of the MTskinesin system. Performing all of the six motility assay series, it was revealed that, within the same time period after ATP addition, different series of motility assays showed different percentages of moving MTs as shown in Figure 2. For the motility assay performed using only scavengers, the number of moving MTs decreased sharply with time and this damage could be prevented successfully using the ICS (see the Supporting Information, Movies 4 and 5). Thus, the scavengers used in the motility assays were not capable of protecting MTs and kinesin for a prolonged time period. When the ATP recycling system was included, the number of motile MTs decreased more rapidly with time compared to the case where the motility assay was performed using only scavengers. However, changing the impact of ATP recycling system by changing the concentration of ATP and PEP/NADH was found to bring no noticeable change in the extent of damage and lifetime of the MTskinesin system (data not shown). This damage could be prevented by performing the motility assay using the ICS, where the damage occurred much more slowly compared to the motility assay performed without the ICS. Thus, although initially it seemed that the combination of ATP recycling system and scavengers might have a harmful effect on the active lifetime of kinesin and MTs, it was the oxygen that caused such damage to MTs and kinesin. Comparing the results of series no. 2 “motility assay using scavengers and ICS” and series no. 5 “motility assay using scavengers, ICS, and ATP recycling system” shown in Figure 2, it could be noticed that the percentage of motile MTs at any time within the observed time length was consistently higher in the former series than in the latter one, although the velocitytime profile (discussed later) showed the opposite trend. In addition to this, it was observed that performing the motility assay using the ICS and the ATP recycling system, but without scavengers, dramatically increased the percentage of motile MTs. Thus, the combination of scavengers and ATP recycling system itself may not be harmful for the 13662
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Figure 3. Effect of scavengers, oxygen, and ICS on motion of MTs in (a) motility series no. 1, 2, and 3; (b) motility series no. 4, 5, and 6. Reason for the unavailability of data: damage of MT-kinesin system (series 1 and 4) and incubation of sample inside inert chamber (series 2, 3, 5, and 6).
MTskinesin system. For those series wherein ICS (with/ without ATP recycling system) but no scavengers was used, a high percentage of motile MTs was observed even after 8 h of ATP addition, and all these data helped us to conclude that it was mainly the oxygen and partly the scavengers that caused the damage the motor protein system. At the same time, the ICS appeared as a very good substitute for commonly used scavengers which can also prevent damage to biomolecular motors and MTs for a prolonged time period. Next, we investigated the effect of the scavengers, oxygen, and inert nitrogen atmosphere on the motion of motile MTs. Figure 3a shows the comparative histogram obtained from motility assays
performed at three different experimental conditions (series no. 1, series no. 2, and series no. 3), wherein no ATP recycling system was used. The use of the ICS prevented the decrease both in the number of moving filaments and also their velocity with time. Figure 3b shows that similar results were derived from the motility assay series performed using the ATP recycling system (series no. 4, series no. 5, and series no. 6). Thus, it is clear that oxygen is harmful and a nitrogen-rich inert environment is much better for fluorescence imaging and prolonged motor activity. The velocitytime profile (Figure 4) obtained from different motility assay series further indicates the effectiveness of the ICS in prolonging the active lifetime of kinesin and MTs, and also in 13663
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Figure 4. Change in motion of motile MTs with time during motility assays performed using only scavengers (series 1); scavengers and ICS (series 2); only ICS (series 3); scavengers and ATP recycling system (series 4); scavengers, ICS, and ATP recycling system (series 5); and ICS and ATP recycling system (series 6). Error bar: standard deviation.
minimizing the harmful effect of oxygen during in vitro motility assay. Considering the results obtained from the motility series 4 as shown in Figures 3b and 4, it is clear that the ATP recycling system has no role in ensuring the prolonged motility of MTs when the activity of motor protein is lost. Thus, the high activity of the MTskinesin system turned out to be a prerequisite for harvesting the advantage of using the ATP recycling system in prolonged investigation, which was achieved in other motility series, for example, series number 6 (Figures 3b and 4). From the comparison between results of motility series 2 (scavenger and ICS) and series 3 (only ICS) as shown in Figure 4, it could be observed that the average velocity of MTs was consistently higher at any time in the latter series, provided that the average initial length of MTs was almost similar in both cases (series 2: 21.52 ( 0.14 μm; series 3: 23.64 ( 0.12 μm). Similar results could be observed when comparing series 5 and 6, and these differences might have arisen from the harmful effect of scavengers on the MTskinesin system. It could be noted that, from the results of all the motility assay series where ICS was used, a decrease in the density of MTs filaments on the kinesin coated surface with time was observed (Figure 5). As the ICS could ensure high activity of kinesin, such a decrease in the density of MTs might have originated from the very high activity of kinesin. Although the decrease in MTs density with time was observed in all of the motility series 2, 3, 5, and 6, this phenomenon was severe in the case of series 5. However, both the motility series 5 and 6 used the ATP recycling system, but no scavenger was used in series 6 and a difference in the extent of decrease in MTs density with time could be noticed. Similar difference is also found if the results of motility series 5 and 2 are compared. Thus, the observed difference in the extent of decrease in MTs density with time might be due to the difference in the activity of kinesin inside the inert atmosphere. At the same time, we also noticed a decrease in fluorescence intensity of GFPkinesin with time (see the Supporting Information, Figure 2), probably owing to desorption of kinesin (or kinesin-anti GFP) from the glass surface. The decrease in density
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of MTs filaments on the kinesin coated surface with time might have a relation with such desorption of kinesin or antibody, although we have no direct evidence right now to support this statement and this would be investigated in future work. The use of the ICS suppressed photobleaching of rhodamine MTs and GFPkinesins (see the Supporting Information, Figures 35). Breakage of fluorescently labeled MTs with time could also be minimized by employing the inert atmosphere (see the Supporting Information, Figure 6). In the motility assay performed using only scavengers, the average length of MTs decreased to 45% and 16% of their initial values (at 5 min) after 100 and 210 min of ATP addition, respectively. In the presence of the ATP recycling system, the damage was similarly severe. However, in the motility assay performed using scavengers and ICS, the average length of MTs remained at 88% and 57% of their initial values (at 100 min) after 210 and 480 min, respectively. In the motility assay performed using only the ICS, the average length of MTs was 92% and 73% of their initial values (at 100 min) after 210 and 480 min, respectively. We also determined the concentration of dissolved oxygen (DO) inside the system both with and without the ICS. Scavengers decreased the DO concentration rapidly just after the addition to the system; but with time the DO concentration was found to increase (at 5 min: lower than the detectable limit of the probe; at 3 h: 0.1 ppm; and at 5 h: 0.15 ppm). However, using the ICS without scavengers, the DO concentration inside the motility system decreased beyond the detectable limit of the probe within 30 min of passing nitrogen gas, and this condition remained same over prolonged time (5, 10 h). While optimizing the working conditions of ICS, we found that it was required to pass the nitrogen gas for at least 1 h to observe good motility of MTs in the absence of scavengers (for a flow cell with volume as mentioned in the Experimental Section, “motility assay 2”). However, here we found that within 30 min the DO concentration decreased at a value less than 0.1 ppm. This indicates that DO concentration at the “ppb” level is also strong enough to damage the motor protein system, particularly under the fluorescence microscope, and that is the reason why commonly used scavengers cannot warrant a prolonged investigation. On the contrary, using ICS the prolonged lifetime of motor proteins, reduced breakage of MTs and suppressed photobleaching of MTs and kinesin are achieved at the expense of very low oxygen concentration inside the motility assay system. Although in addition to the use of scavengers, sealing of the flow cell with vacuum grease is another method used in the in vitro motility assay; in our study, we also checked its applicability and found that this method failed to maintain the hydration state in the flow cell for longer time, and similar to the scavengers it was also unable to secure a prolonged observation under fluorescence microscope as was achieved by using the ICS. Having successfully monitored the motion of MTs for approximately 8 h by using the ICS and ATP recycling system, we investigated the length of time for which the ICS could save the biomolecular motor. We performed an active lifetime assay (ALTA) in the presence of methyl cellulose that prevented the decrease in the density of MTs on the glass surface with time.27 We followed the motion of MTs until it was possible to monitor and found that MTs continued to move on the kinesin-coated surface even after 88 h, although the motion became much slower. Figure 6 shows the outcome of the ALTA. Initially, the percentage of motile MTs and their average velocity were slightly lower than those observed in the motility assay performed using the ICS and ATP recycling system in the absence of methyl 13664
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Figure 5. Fluorescence microscopic images showing the effect of the reactive oxygen species and ICS on the breakage of microtubules (MTs) with time during motility assays performed using (a) only scavengers (series 1); (b) scavengers and ATP recycling system (series 4); (c) scavengers and ICS (series 2); (d) only ICS (series 3); (e) scavengers, ICS, and ATP recycling system (series 5); and (f) ICS and ATP recycling system (series 6). Scale bar: 20 μm.
cellulose (Figure 4). This might be due to the presence of methyl cellulose, because we found a concentration-dependent effect of methyl cellulose on the motility of MTs (see the Supporting Information, Figure 7). During the ALTA, the percentage of motile MTs was quite high until 50 h, after which a declining trend was observed. On the other hand, the average velocity of MTs increased with time until 74 h and then decreased drastically at 88 h when it was difficult to find any motile MT. The initial rising trend of average velocity with time could be accounted for by desorption of kinesin (or kinesin-antibody) from the glass surface. B€ohm et al.28 reported that the motion of MTs depended
on the kinesin density on the surface; there existed an optimum density of kinesin that could generate maximum velocity, and also a critical kinesin density below which no motion of MTs was observed. To confirm whether the observed change in velocity of MTs during the ALTA was caused by a decrease in kinesin density on the surface or by damage to the kinesin, we performed a number of investigations. First, we determined the GFPkinesin concentration that could produce fluorescence intensity under microscope similar to that observed at 88 h during the ALTA. We maintained the exposure time and light source intensity similar to 13665
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Figure 6. Summary of the active lifetime assay (ALTA): (a) Comparative histogram obtained from the result of 88 h motility assay; (b) change in velocity of motile MTs with time (inset shows the change in percentage of motile MTs with time). Error bar: standard deviation.
that maintained in the ALTA and found that 31 nM kinesin could produce similar fluorescence intensity, whereas we used 622 nM kinesin for ALTA. Moreover, this 31 nM kinesin resulted in a very poor motion of MTs (0.08 μm/s ( 0.004 μm/s) when we performed motility assay keeping the flow cell volume identical to that used in the ALTA. Thus, as reported by B€ohm et al.,28 motility generation requires a minimal (critical) kinesin density on the surface, below which no motion of MTs could be observed. In our ALTA, the GFP-fluorescence intensity at 88 h corresponded to a kinesin concentration that appeared to produce a kinesin density very close to the critical density required for motility of MTs. Thus, the poor motion observed at 88 h might have been caused by desorption of kinesin (or kinesin-antibody) from the glass surface with time, and not due to any oxidative damage of the kinesin. In addition, during the ALTA, the fluorescence intensity of GFP-kinesin decreased gradually with time (see the Supporting Information, Figure 8), and this further confirms the desorption of kinesin (or kinesin-antibody) from the surface. Next, we performed a static lifetime investigation, as described in the Experimental Section. After incubation of the flow cell inside an inert environment for 88 h and subsequent motility assay, the observed GFP-fluorescence intensity was 2384 au, although the motion of MTs was very close to that observed at 88 h in the ALTA (0.19 ( 0.03 μm/s). This further indicates that the poor motion observed at 88 h in ALTA was probably due to desorption of kinesin from the surface. Finally, we directly incubated some KG buffer (containing the same concentration of kinesin used in the ALTA) instead of a flow cell in an oxygen free nitrogen environment. After 88 h incubation, we performed a motility assay using this KG buffer preparing a new flow cell. We observed very good motility of MTs (0.56 ( 0.004 μm/s), and also GFP-fluorescence intensity was very close (3386 au) to that observed at the initial time of the ALTA. This further confirms our theory that the poor motion of MTs observed at 88 h in the ALTA was caused by the low density of kinesin resulting from desorption from the surface with time, and not by any oxidative damage to the kinesin. Moreover, the observed decrease of the GFP-fluorescence intensity value during the ALTA was caused by desorption of kinesin (or kinesin-antibody) from the surface,
and not by damage to kinesin on irradiation under fluorescence microscope. Desorption of kinesin (or kinesin-antibody) from the glass surface with time might be prevented by appropriate modification of the glass surface, which we did not perform in the present work. We also performed motility assay using KG buffer incubated for 8 days inside inert environment where most of the MTs were found in static condition and only a few MT filaments (5%) showed motility (0.13 ( 0.02 μm/s). This suggested that ICS could save kinesin for almost 8 days, and damage of kinesin also might have occurred between 88 h and 8 days. The reason for the damage that occurred between 88 h and 8 days is not clear at this moment. In the case of motility assay performed using kinesin incubated for 88 h under ambient conditions, no motion of MTs was observed and the GFP-fluorescence intensity (1611 au) was lower than that observed in the motility assay performed using kinesin incubated for 88 h in an inert environment. This further confirmed the effectiveness of the inert atmosphere in prolonging the lifetime of kinesin. In our study, we also observed the effect of dissolved oxygen on the lifetime of theMTkinesin system. For this, different flow cells were kept in ambient light and dark conditions after the ATP addition and were observed after 5 h. In both cases, severe breakage of MTs and very slow motion were observed compared to that observed after 5 min of ATP addition. Brunner et al.29 also reported such breakage of MTs on glass surface even in the dark. However, using ICS neither such severe breakage of MTs nor the slowdown of their motion was observed after 5 h of ATP addition (both in ambient light and dark). As ICS protected MTs from breakage and slowdown of motion even in the dark, this might be the dissolved oxygen in buffer that caused such damage of the MTkinesin system observed during the motility assay without ICS. Finally, we verified the effectiveness of the ICS by performing motility assay using a flow cell made from materials other than glass. Although polydimethylsiloxane (PDMS) has been shown to have excellent biocompatibility in cell-patterning30 and proteinstamping31 applications, it was found to limit the lifetime of the MTskinesin system in an in vitro motility assay. Brunner et al.29 reported that, even in the presence of scavengers, use of PDMS as a substrate for in vitro motility assay could completely 13666
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Langmuir disintegrate MTs within 30 s of illumination. We performed a motility assay over a PDMS surface (method for performing motility assay was similar to that described in the Experimental Section “motility assay 2”) by using the ICS (without scavenger) and observed very good motion of MTs (0.48 ( 0.006 μm/s) and no damage to MTs or kinesin during the 10 min observation time. On the other hand, without the ICS (with scavengers), complete disintegration and loss of motility of MTs was observed within a few seconds of illumination. In fact, owing to its very high oxygen affinity and permeability, PDMS acted as a reservoir of oxygen. During observation by fluorescence microscope, the oxygen was gradually released from the PDMS into the buffer solution. Upon irradiation, this molecular oxygen caused damage to the MTskinesin system producing ROS, even in the presence of scavengers. A complete prepurge of PDMS inside the ICS successfully removed the molecular oxygen from PDMS and thus saved the MTskinesin system from damage.
’ CONCLUSIONS In conclusion, we have successfully overcome a limitation of the in vitro motility assay. As reported previously, fluorescence microscopy induces the photochemical reaction that produces ROS which then causes great damage to biomolecules under investigation using in vitro motility assay.16,32 However, in our experiment, we have also observed damage of the biomolecular motor system even in the dark, and a similar phenomenon was reported by Brunner et al.,29 too. Decrease in the activity and shortening of the lifetime of biomolecular motor systems could be attributed to the harmful effect of oxygen and commonly used scavengers. In this report, we have shown that the use of the inert atmosphere combined with the traditional in vitro motility assay can successfully solve these problems and prolong the active lifetime of kinesin until 88 h and even a week, increasing its in vitro stability. The breakage of MTs and photobleaching of MTs and kinesin were also substantially minimized. The use of the ICS combining with the traditional in vitro motility assay showed a predominant effect on the lifetime of biomolecular motor system compared with the case where scavengers were used. We believe that this is the first successful demonstration of a long-term in vitro motility assay. Our results would accelerate the in vitro application of biomolecular motors and foster the development toward biomolecular motor based artificial devices. The knowledge obtained would also be crucial to micro- and nanofabrication, and to achieve a unified perspective on engineering challenges in the design of hybrid devices based on biomolecular motors. At the same time, the usage of the in vitro motility assay technique will be increased tremendously to elucidate a detailed time dependent biophysical response of biomolecular motor systems, necessary to unveil their detailed in vivo life cycle scenario. Further development of this ICS can probably offer a means to control the extent of oxidative stress during the in vitro motility assay. In that case, it would be possible to study the detailed correlation among the oxidative damage, biological and biomechanical properties of biomolecular motor systems, important for achieving a meticulous description of the effect of ROS on biomolecular motor systems, in vivo. On the other hand, the ROS are also believed to be involved in different diseases, including atherosclerosis, Parkinson’s disease, heart failure, myocardial infarction, Alzheimer’s disease (AD), and schizophrenia; additionally, a direct link of the dysfunction of MTskinesin system to some neurodegenerative diseases has also been
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reported.3335 Our results probably would allow for a greater understanding of different neurodegenerative diseases linked to the dysfunction of biomolecular motor systems such as MTs kinesin and thus of the treatment and future drug therapy for such diseases.
’ ASSOCIATED CONTENT
bS
Supporting Information. Figures showing the detailed experimental procedure regarding the use of ICS, data on effect of ICS on photobleaching of MTs and kinesin and breakage of MTs, effect of methyl cellulose on MTs motility, desorption of kinesin from the surface, and movies of different motility assay series (the time is accelerated 100-fold, i.e., 1 s movie time equals to 100 s experimental time). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected] (A.K.);
[email protected]. ac.jp (J.P.G.). Telephone/fax: +81-11-706-2774.
’ ACKNOWLEDGMENT We would like to thank Mr. Yasutaka Sasaki of the Technical Division, Faculty of Science, Hokkaido University, for his cooperation in constructing the Inert Chamber. This research was financially supported by the Ministry of Education, Science, Sports, and Culture of Japan (Grant-in-Aid of Specially Promoted Scientific Research) and PRESTO (Japan Science and Technology Agency). ’ REFERENCES (1) Cooper, J. A. Annu. Rev. Physiol. 1991, 53, 585–605. (2) Umeda, M.; Emoto, K. Chem. Phys. Lipids 1999, 101, 81–91. (3) Kron, S. J.; Toyoshima, Y. Y.; Uyeda, T. Q.; Spudich, J. A. Methods Enzymol. 1991, 196, 399–416. (4) Kron, S. J.; Spudich, J. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6272–6276. (5) Harada, Y.; Noguchi, A.; Kishino, A.; Yanagida, T. Nature 1987, 326, 805–808. (6) Toyoshima, Y. Y.; Kron, S. J.; Spudich, J. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7130–7134. (7) Uyeda, T. Q.; Abramson, P. D.; Spudich, J. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4459–4464. (8) Kakugo, A.; Sugimoto, S.; Gong, J. P.; Osada, Y. Adv. Mater. 2002, 14, 1124–1126. (9) Turner, D. C.; Chang, C.; Fang, K.; Brandow, S. L.; Murphy, D. B. Biophys. J. 1995, 69, 2782–2789. (10) Suzuki, H.; Oiwa, K.; Yamada, A.; Sakakibara, H.; Nakayama, H.; Mashiko, S. Jpn. J. Appl. Phys. 1995, 34, 3937–3941. (11) Hess, H.; Clemmens, J.; Howard, J.; Vogel, V. Nano Lett. 2002, 2, 113–116. (12) Hess, H.; Howard, J.; Vogel, V. A. Nano Lett. 2002, 2, 1113– 1115. (13) Martin, G. N.; Heuvel, V. D.; Dekker, C. Science 2007, 317, 333–336. (14) Goel, A.; Vogel, V. Nat. Nanotechnol. 2008, 3, 465–475. (15) Vigers, G. P. A.; Coue, M.; McIntosh, J. R. J. Cell. Biol. 1988, 107, 1011–1024. (16) Dixit, R.; Cyr, R. Plant J. 2003, 36, 280–290. (17) Sporn, L. A.; Foster, T. H. Cancer Res. 1992, 52, 3443–3448. 13667
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