Reconstitution of Eukaryotic Flagellar Axonemes by the Bottom-Up Strategy

The complexity of the eukaryotic flagellar axoneme is derived from 200-600 types of modular building blocks assembled hierarchically. Nine doublet microtubules surround a pair of singlet microtubules. On each of nine doublet microtubules, dyneins are aligned in two rows, outer- and inner-arm dyneins. In Chlamydomonas, the model organism for flagellar motility, several subspecies of dyneins have been identified; one outer-arm dynein with three different heavy chains, one heterodimeric inner-arm dynein and six inner-arm dyneins. Each of the heavy chains is reported to have different mechanical properties. They are precisely arranged along doublet microtubules and regulated in a coordinated fashion to produce periodic flagellar beating. The coordination among these building block under strict spatio-temporal regulation makes flagella beat in organized manner. To reveal the mechanism of coordination and regulation, we have carried out in vitro reconstitution of axonemal structures in bottom-up manner and has compared mechanical properties of the reconstituted axonemes with those of intact ones. Tubulins were polymerized into microtubules from fragmented axonemes working as seeds. To these microtubule bundles with the same polarity, we added crude outer-arm dynein extract from Chlamydomonas axonemes. The dynein formed regular arrays (24nm-repeat) on the microtubules in self-organized manner and made stiff microtubules bundles. In addition of ATP, a pair of microtubules occasionally displayed association and dissociation cycles. When both ends of a microtubule bundle were clamped, shear between microtubules at the middle part elicited looping out of microtubules, forming characteristic S-shaped bending and then recoiling. Force generated at the middle part was calculated using Euler formula to be ca. 1 pN per dynein arm. These cyclic interaction between dynein and microtubules regulated by microtubule bending could be essence of the beating mechanism working in an axoneme. (This work is supported by Takeda Science Foundation and Grant-in-Aid for Scientific Research (C) 26440089).