Hierarchical Functional Construction of Cellular Motile Systems

我们专注于在生物系统的大范围层次组织中，从单个分子到马达组合，再到细胞，对力对分子马达活动的影响进行检查。主要研究成果如下：1)在ATP存在的情况下，对肌动蛋白V的单个分子施加不同角度的外力，使其在肌动蛋白丝上进行运动，以测试马达在不同负荷下运动的稳健性。此外，通过在不同方向施加负载并测量不同[ADP]下单个肌动蛋白-肌球蛋白V复合物的解结合力，揭示了在外部负载下确保肌动蛋白V运动高稳定性的机制（Oguchi et al., Nature Chem）。医学杂志。， 2010；关于肌球蛋白VI，见Oguchi et al.， PNAS, 2008)。对不同电机（包括肌凝蛋白V和VI，以及肌凝蛋白II和肌动蛋白）的解结合力测量的主要结果进行了总结。接口,2010)。2)在中等激活条件下，单个肌原纤维中单个肌节对外部机械刺激的反应揭示了两种类型的肌节共存：对施加负荷有反应的肌节和不施加负荷的肌节。这些观察证实了肌节间机械协调的存在，帮助我们建立了肌肉收缩和调节的特征性机制（Shimamoto等）。， pnas, 2009)。3)我们构建了明确描述横切肌收缩特征的理论模型，包括自振荡（SPOC） （Sato et al., 2010，提交）。4)我们基于对非洲爪蟾卵提取物中自组装的脊椎动物减数分裂纺锤体的研究发表了第一份报告，其中我们测试了它们的机械性能，如可变形性和刚度，以及对机械扰动的响应（Itabashi et al., Nature Methods, 2009）。这项工作报告，除其他外，不同尺寸的主轴的自发重新组装可以诱导施加大的控制外部负载。5)为了揭示减数分裂纺锤体特征“橄榄球”形状稳定性的机制，我们定量测试了它对极性（长）轴机械拉伸的响应，这是由一对微针的微操作产生的。这些测量清楚地表明，纺锤体对外部诱导的大变形有反应，能够通过调节微管的聚合/解聚以及运动蛋白和动力蛋白分子马达（MS in preparation）的活性来保持其形状。6)利用≧1秒热脉冲对HeLa细胞进行局部加热，使细胞内局部温度升高0.1~几摄氏度，引起细胞内振荡[Ca^<2+>]。此外，在37℃左右，细胞对~0.2℃的微小温度变化有反应，IP_3受体被证明参与其中（Suzuki et al., HFSP J., 2009）。少

We focused on the examination of the effect of force on the activity of the molecular motors in a wide range of the hierarchical organization of the biological systems, from single molecules to assemblies of the motors and further on to the cells. Main achievements are summarized as follows : 1) The external forces were applied at various angles to the individual molecules of myosin V, processively moving on actin filaments in the presence of ATP, to test the robustness of the motor's motility under various loads. Furthermore, the mechanism ensuring high stability of the myosin V's motility under external loads was revealed by applying loads in various directions and measuring the unbinding forces of individual actin-myosin V complexes at different [ADP] (Oguchi et al., Nature Chem. Biol., 2010 ; for myosin VI, see Oguchi et al., PNAS, 2008). The main results of the unbinding force measurements on different motors, including myosins V and VI, as well as myosin II and kinesin, were summ … More arized and published (Mikhailenko et al., J. Royal Soc. Interface, 2010). 2) The response of individual sarcomeres in a single myofibril to the external mechanical stimulation under intermediate activation conditions revealed the coexistence of two types of sarcomeres : those responding to the application of load, and those remaining indifferent. These observations confirmed the existence of a mechanical inter-sarcomere coordination, helping us to establish the characteristic mechanism of muscle contraction and regulation (Shimamoto et el., PNAS, 2009). 3) We constructed the theoretic model explicitly describing the characteristics of contraction, including the auto-oscillation (SPOC), in a striated muscle (Sato et al., 2010, submitted). 4) We published the first report based on our research on vertebrate meiotic spindles self-assembled in Xenopus egg extracts, in which we tested their mechanical properties, such as deformability and stiffness, and the response to mechanical perturbations (Itabashi et al., Nature Methods, 2009). This work reported, inter alia, that the spontaneous re-assembly of spindles of different sizes can be induced by applying large controlled external loads. 5) To reveal the mechanisms underlying the stability of a characteristic "rugby ball"-like shape of the meiotic spindle, we quantitatively tested its response to mechanical stretch along the polar (long) axis, produced by the micromanipulation with a pair of microneedles. These measurements clearly demonstrated that a spindle responds to the large externally induced deformations, being able to preserve its shape via the modulation of polymerization/depolymerization of microtubules and the activity of kinesin and dynein molecular motors (MS in preparation). 6) We found that the local heating of HeLa cells using the ≧1 sec heat pulses, which increase the local temperature within cells by 0.1～few degrees centigrade, induces the oscillation of intracellular [Ca^<2+>]. Moreover, around 37℃ the cell responds to small, ～0.2℃, temperature changes, in which the IP_3 receptors were shown to be involved (Suzuki et al., HFSP J., 2009). Less