Hierarchical Functional Construction of Cellular Motile Systems

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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