3P167 Kinetic characterization of rice plant specific kinesin E11 using fluorescent ATP analogue(Molecular motor,Poster,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))
3P167 蛍光標識ATP アナログを用いたイネ特有のキネシンE11 の速度論的解析(分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

Taniguchi Hironobu, Miyabe Kouichi, Umezu-Furutani Nozomi, Maruta Shinsaku
2014 Seibutsu Butsuri  
Centralspindlin, kinesin-6 (motor)/CYK-4(Rho-GAP) complex is essential for assembly of the central spindle. While the motility of kinesin-6 bound to CYK-4 was examined in a microtubule sliding assay, it is not clear how binding of CYK-4 to kinesin-6 regulates the motor activity. In this work, using 3-D tracking microscopy, three dimensional movements of both kinesin-6 and centralspindlin along a suspended microtubule were quantified. We found that both kinesin-6 and centralspindlin displayed a
more » ... eft-handed spiraling motion around the microtubule. The velocity of centralspindlin was slower than that of kinesin-6 and the rotational pitch of centralspindlin was longer than that of kinesin-6, indicating that CYK-4 modulates the motor activity of kinesin-6. Cytoplasmic dynein is a microtubule-based motor responsible for diverse intracellular movements. Here, we show single dynein molecules are in an autoinhibited state, in which the two motor heads are stacked together. In this state, dynein moves diffusively along a microtubule with a small bias toward the minus end of the microtubule. When the two heads were physically separated, the movement of dynein molecules became directed and processive. Furthermore, assembling of multiple dynein molecules on a single cargo enabled directed movement and cooperative force production. We propose a mechanism of autonomous on-off switching of cargo transport, in which single dynein molecules in the cell is autoinhibited and become active when assembled as a team on a cargo. 3P165 ミオシンの協調的首振りとアクチン滑り運動のゆらぎ Cooperative lever-arm swings of myosins and fluctuation of actin sliding Yota Kondo, Kazuo Sasaki (Dept. Appl. Phys., Sch. Eng., Tohoku Univ.) Myosin II is a motor protein which swings a lever arm and generates in a group the sliding movement of an actin filament. It is customarily thought that the actions of individual myosin motors are independent when an actin filament slides. If this is the case, the effective diffusion coefficient of actin will be inversely proportional to the number of myosins (K. Sekimoto and K. Tawada, Biophys. Chem., 2001). However, a measurement of actin sliding in vitro showed that the effective diffusion coefficient is almost independent of the number of myosins (N. Noda et al., Biophysics, 2005), which implies that lever-arm swings of myosins are not independent. We construct a theoretical model that can explain the experiment and analyze cooperative lever-arm swings of myosins. To elucidate domain motions of the catalytic β subunits of a molecular motor F 1 -ATPase at the single molecular level, we have developed TIRFM under polarization modulation. A pair of images under sand p-polarized illuminations is continuously captured by fast switching between two beams. Single emitting images of spots were rotated at the camera plate synchronously with the rotation of the polarization orientation under spolarized light, and resultant circular images were fitted with an approximated equation. Feasibility of the analyses was validated by reconstructed images to estimate the changes in tilting and azimuthal angles of the C-terminal helix of β. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures. In this study, we focused on rice kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed in E.coli BL21(DE3) and purified by Co-chelate column in order to characterize biochemical and ATPase kinetic properties. We employed two fluorescent ATP analogues, Mant-ATP and NBD-ATP for the kinetic characterization. we have successfully observed significant FRET between Mant-ATP and intrinsic tryptophane (Trp23) in E11. The rates of initial binding of Mant-ATP and release of Mant-ADP from E11 were analyzed by monitoring the FRET using stopped flow apparatus. 3P168 金ナノロッドを用いた高速配向イメージングシステムの開発 と F1-ATPase の構造変化検出への応用 Development of high-speed orientation imaging system for gold nanorod and application to detection of conformational change of F1-ATPase
doi:10.2142/biophys.54.s276_5 fatcat:dt6tkctwwfhybhgl7d7bhk6qry