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MICROTUBULE GLIDING at the BOUNDARY of KINESIN and DYNEIN PATTERNED SURFACE Junya Ikuta1*, Nagendra K

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MICROTUBULE GLIDING at the BOUNDARY of KINESIN and DYNEIN PATTERNED SURFACE Junya Ikuta1*, Nagendra K MICROTUBULE GLIDING AT THE BOUNDARY OF KINESIN AND DYNEIN PATTERNED SURFACE Junya Ikuta1*, Nagendra K. Kamisetty1, Hirofumi Shintaku1, Hidetoshi Kotera1 and Ryuji Yokokawa1,2 1Kyoto University, JAPAN and 2JST-PRESTO, JAPAN ABSTRACT We constructed a molecular platform to evaluate a tug-of-war between motor proteins, kinesin and dynein. It is the molecular system using the gliding assay, where microtubules (MTs) glide on immobilized motors. In this particular study, we selectively patterned kinesin and dynein on a substrate so as to attach MTs at the boundary between the two motor proteins. We could evaluate the time course of MT position and velocity at the boundary. For the analysis of the number of kinesin and dynein bound to MT, we measured the density of kinesin and dynein on a substrate. We monitored the velocity change of a MT according to the numbers of kinesin and dynein molecules propelling the MT. Force balance between two segments of the MT pulled by kinesin or dynein varies due to the increase or decrease of motors bound. Therefore, we are able to conclude that the velocity change was caused by the force balance between the segments. In conclusion, we could propose a new method to evaluate a tug-of-war between kinesin and dynein. KEYWORDS: Motor protein, Kinesin, Dynein, Microtubule, tug-of-war, Molecular system INTRODUCTION Conventional kinesin and cytoplasmic dynein move along MT in the direction of MT’s plus end or minus end, respectively. They carry intracellular materials along MTs. Thereby, kinesin and dynein have a role of transporting intracellular materials. Many cargos are transported bi-directionally by the cooperative movements of both kinesin and dynein [1]. It is considered that cargos change their moving directions through the counter directional force generated by kinesin and dynein, which is known as tug-of-war [2]. For the past decade, many researches about a tug-of-war have been carried out [3][4]. However, there are a few researches which evaluated relation between cargo’s motility and the numbers of kinesin and dynein of a tug-of-war in vitro [5]. For investigating how a single motor protein or multiple motors cooperatively carry a cargo in vivo, it is important to reconstruct their molecular system in vitro and to evaluate their motility. As a typical method of evaluating it in vitro, there is a gliding assay format, in which microtubules glide on the motor protein-immobilized substrate [6]. We constructed the molecular system to evaluate a tug-of-war between kinesin and dynein (Fig. 1). Figure 1: Schematic of the molecular system of a tug-of-war between kinesin and dynein using the gliding assay. EXPERIMENTAL Selective patterning of kinesin and dynein: For selective coating of kinesin and dynein, we prepared Au and glass patterned substrate. We immobilized kinesin on glass surface with non-specific bindings and dynein on Au surface with Au-thiol and biotin-avidin bindings [7]. A hundred-µm line-and-space of Au/Cr was patterned on a fused silica substrate with thermal deposition followed by lift-off process (Fig. 2). For immobilizing biotin on the Au-coated surface, the substrate was immersed in a 20 µM solution of biotin-self-assembled monolayer (biotin-SAM) for 12 hours. According to the following procedure 1-4, kinesins were immobilized on the bare glass, and dyneins were immobilized on the Au-coated surface by sequential Figure 2: Au/Cr line and space on the Figure 3: Sequential fluorescence images of PMMTs gliding. White arrow fused silica substrate. Bright area is heads indicate PMMT’s leading ends. Bright ends and dark ends indicate Au-coated surface, and dark area is minus or plus ends, respectively. a) On the glass surface. b) On Au-coated bare glass surface. surface. 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1454 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany protein injections. 1) Pluronic (2 mg/ml) was coated to inhibit the non-specific bindings on the Au-coated surface. 2) Kinesin (0.2 mg/ml) was non-specifically immobilized on the bare glass surface. 3) Streptavidin (0.5 mg/ml) was immobilized on the biotin-SAM. 4) Biotinylated dynein (62 µg/ml) was immobilized on the streptavidin. After selective patterning, polarity marked microtubules (PMMTs) were introduced to kinesin- and dynein-coated surface, and we observed their movement on each surface (Fig. 3). Measurement of the motor protein’s density on kinesin- or dynein-coated surface: We can evaluate the average distance between motors immobilized on the substrate, <d>, from the trajectory of short MT’s motion. When a MT is longer than <d>, the MT constantly binds to multiple motors. Therefore, the MT keeps gliding in a smooth fashion without sudden direction changes. In contrast, a MT comparable to <d> occasionally binds to a single motor. It means the short MT displays sharp pivoting around the motor and glides irregularly. Heuvel et al. [8] proposed a method of measuring <d> with the average distance between MT pivots, <S>. 2 2 dL d L L d S e 1 (1) 3 dL L d Here, L is the length of MT. For measuring S, we observed short MTs gliding on the kinesin- or dynein-coated surface (Fig. 4) and estimated the trajectory and curvature of them. When a MT makes pivot turns, curvature of the MT results in higher value than that of a longer MT that glides smoothly. We defined the maximum curvature of a smoothly gliding MT as the threshold value that is used to judge if a MT had a chance to bind to a single motor. We evaluated curvature of a short MT if it exceed the threshold value. If it does so, we concluded that the MT made pivot turns, since its gliding was supported by a motor at that position. Thereby, we calculated the average value of <S> between pivot turns. Due to the finite optical resolution of the microscope, fluorescence images consist of a convolution of the point-spread-function (PSF) with the actual MT size. L is overestimated when it is measured by the intensity line scans in fluorescence images. Therefore, we deconvoluted the intensity line scans of fluorescence images from PSF. Thereby, we got the intensity profile of the actual MT and calculated L. Finally, we calculated the average distance between kinesins, dk, or between dyneins, dd, through the equation (1) with experimentally measured <S> and L. Figure 4: Sequential fluorescence images of a short MT making pivot turns. White arrow heads indicate the MT leading head. Red cross marks indicate the point of MT pivot at 20 s. MT glided rightward from 0 to 20 s. MT pivoted at 20 s, changed the gliding direction and kept gliding leftward from 30 to 40 s. Figure 5: Sequential fluorescence images of MTs gliding at the boundary between kinesin- and dynein-coated surface. Right sides in the images are the kinesin-coated surface, and left sides are the dynein-coated surface. Red arrow heads and white ones indicate MT’s plus end or minus end, respectively. a) MT gliding to the kinesin-coated surface from the boundary. b) MT gliding to the dynein-coated surface from the boundary. 1455 MT gliding at the boundary between kinesin and dynein: We injected MTs into the flow cell, where kinesin and dynein were selectively patterned in two regions. We focused on MTs which were pulled by kinesin and dynein from each region at the boundary. We observed their tug-of-war phenomena, and evaluated their position and velocity. Fig. 5 shows sequential fluorescence images of MT’s movements at the boundary between kinesin- and dynein-coated surfaces. RESULTS AND DISCUSSION To evaluate selective patterning of kinesin and dynein, MT’s polarity and velocity were measured on the kinesin- and dynein-coated surface. MTs were gliding with minus ends leading on the kinesin-coated surface (glass surface) (Fig. 3a) and with plus ends leading on the dynein-coated surface (Au-coated surface) (Fig. 3b). Also, considering their velocity on each surface, we conclude that the selective pattering of kinesin and dynein was successfully achieved. For the measurement of dk and dd, we evaluated the trajectory and curvature of 656 ± 35 nm and 526 ± 22 nm MTs gliding on kinesin- or dynein-coated surface, respectively. We calculated and concluded that S was 2.52 ±1.12 µm and 4.03 ± 2.34 µm, respectively. In conclusion, dk was 157 ± 38 nm and dd was 104 ± 25 nm through the equation (1). To evaluate MT’s movement at the boundary, we measured the time course of the ratio of MT’s length on each surface, and velocity. For example, MT in Fig. 5a moved longer distance from 6 to 9 minutes than from 0 to 3 minutes. It means the following two phenomena; The length of a MT on the kinesin- and dynein-coated surface increase and decrease, respectively, when the MT glides from the dynein- to kinesin-coated region. Then, velocity of the MT increased according to the increase of MT’s length in the kinesin-coated region. Similarly, in the case of MT gliding from the kinesin- to dynein-coated region, the length of MT on the dynein- and kinesin-coated surface increase and decrease, respectively. Then, velocity of the MT increased according to the increase of MT’s length in the dynein-coated region (Fig. 5b). We calculated the numbers of kinesin and dynein attached to a MT by dividing MT’s length on the kinesin- or dynein-coated surface by dk or dd.
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