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During muscle force generation, an elastic energy stored at the compliant region of myosin head is a source of mechanical work against the external environment. Thus, this elastic distortion of myosin molecule was modeled as an essential mechanical element of the force generation driven by the crossbridges (Huxley, 1957). In the crossbridge model, the central assumption of the constant stiffness implies that the negatively-strained myosins must be detached at much higher rate than the positively-strained myosins to avoid the significant drag effect. However, the molecular studies on processive motors, such as kinesin and unconventional myosins, show that the assemblies of these motors do not seem to impede the molecular interactions, although they have much higher duty ratio and thus, a higher chance to cause the molecular interference. Recently, we found that skeletal myosins have the non-linear elasticity, in which stiffness is much higher when they are positively-strained and lower when negatively-strained. Therefore, this non-linear elasticity seems to be the essential and complementary property of motors to avoid the drag force generation in the motor assembly. We have currently worked on the theoretical model to investigate the effect of non-linear elasticity on the force generation, particularly the force-velocity relationships and the T1-T2 curves obtained from the muscle fiber quick release experiment. Cytoplasmic dynein is a motor protein that exerts force on microtubules and in doing so, drives a myriad of intracellular activities from mitotic spindle positioning to chromosome movements in meiotic prophase. To exert force on microtubules, dynein needs anchorage, which is typically found at the cell cortex. The key question is how dynein finds the sites where a microtubule and an anchor protein are close enough for dynein to link them and subsequently pull on the microtubule. Here we directly observe single dyneins in fission yeast and show that they attach in two steps, first from the cytoplasm to a microtubule and then also to cortical anchors. Upon attachment to the microtubule, dynein moves in a diffusive manner along the microtubule. This is a surprising finding for a minus end directed motor and may help dynein to find cortical anchors. Our work demonstrates that dynein performs threedimensional diffusion in the cytoplasm and one-dimensional diffusion along the microtubule to find sites where it can exert pulling force on the microtubule.

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.