High-Resolution Structural Aanlyases of Dynein Molecules During Sliding By Quick-Freeze Deep-Etoh Replica Electron Microscopy

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This project originally aimed to determine three-dimensional (3-D) structure of cytoplasmic dyncin/microtubule complex and its change upon sliding movement. Because of difficulty in obtaining very fresh materials with high activity, we switched the material to sea-urchin sperm flagella and studied the structure/function relationship of axonemal dyncin subspecies. We also investigated the 3-D structure of sliding actomyosin complex, using a reconstruction method which we recently developed according to the concept of "single molecule physiology". With the results of such studies as a basis, we discussed the molecular mechanism of processive motor activity which often seems to play crucial roles in dynein/microotuble sliding systems.Many members of unconventional myosins evoke processive movement of actin filament. Myosin-V is among the most intriguing for its long lever-arm moiety and its "walking" movement with 36 nm steps, along actin filament. Such characteristic behavior of myosin-V … More has been attributed as one of the strongest evidences to support the validity of "tilting lever-arm mechanisms". We examined the behavior of myosin-V mutant with truncated lever-arm and myosin-VI which naturally has short lever-arm moiety. Interestingly, both of them showed processive movement of actin with 〜36 nm step size that is identical to original myosin-V with long lever-arm. We checked the mode of binding of those myosin heads together with dyctyostelium myosin mutant whose ADP-Pi state is extraordinarily stabilized, to actin filament by electron microscopy. Surprisingly, we found that all of those myosin heads seem to bind to actin with 〜36 nm regular intervals, and only to one side of the filament. All of these phenomena are quite difficult to explain by "tilting lever-arm hypothesis" and might suggest the possibility that the binding of one energized myosin head to actin might evoke the special high affinity site(s) (hot-spot(s)) 〜36 nm away from the original site.According to our replica images, the structure of functioning myosin heads was roughly classified into three categories,' those bound to actin through upper 50kD domain, those bound through lower 50kD domain and those bound through both 50kD domains. If the particles with different structures are postulated to represent the time-course of myosin's structural change, we might assume that myosin head proceeds actin filament involving certain "rocking motion".The B-band component of sea urchin axoneme turned out to be a dyncin subspecies. Less