Cyclical interactions between two outer doublet microtubules in split flagellar axonemes

Abstract

The beating of cilia and flagella is based on the localized sliding between adjacent outer doublet microtubules; however, the mechanism that produces oscillatory bending is unclear. To elucidate this mechanism, we examined the behavior of frayed axonemes of Chlamydomonas by using high-speed video recording. A pair of doublet microtubules frequently displayed association and dissociation cycles in the presence of ATP. In many instances, the dissociation of two microtubules was not accompanied by noticeable bending, suggesting that the dynein-microtubule interaction is not necessarily regulated by the microtubule curvature. On rare occasions, association and dissociation occurred simultaneously in the same interacting pair, resulting in a tip-directed movement of a stretch of gap between the pair. Based on these observations, we propose a model for cyclical bend propagation in the axoneme.

FIGURE 1

Split axoneme obtained by protease/ATP treatment. Video-recorded dark-field micrograph. The axonemes tend to split into single outer doublets and bundles, while their proximal ends remain connected. Bar, 5 μm.

FIGURE 2

Cyclical interaction between a pair of outer doublet microtubules. A series of video frames taken every 10 ms (see supplementary movie 1 (fourfold slowed down)). The number in each frame shows the time in milliseconds. This pair repeats an association/dissociation cycle at ∼9 Hz. Note that no significant bending occurs in the associated portion. ATP concentration, 500 μM. Bar, 5 μm.

FIGURE 3

Doublet pair showing a bending movement. Video frames taken every 30 ms. This type of bending movement was frequently observed at lower ATP concentrations. ATP concentration, 5 μM. Bar, 5 μm.

FIGURE 4

Change in the positions at which association (○; curve 1) and dissociation (•; curve 2) occurred between two doublet microtubules shown in Fig. 2. The doublet pair repeats association and dissociation at an almost constant frequency.

FIGURE 5

Maximal velocities of the association/dissociation front progression at different ATP concentrations. (○) The association front. (•) The dissociation front. (Solid line) Data for the dissociation front fitted to Michaelis-Menten kinetics with the following parameters: maximal velocity = 240 μm/s; Km = 107 μM. For each data point, the average and standard deviation (error bars) measured in eight samples are shown.

FIGURE 6

Pair of doublets displaying a distally traveling gap. Video frames taken every 5 ms (see supplementary movie 2 (fourfold slowed down)). ATP concentration, 100 μM. Bar, 5 μm.

FIGURE 7

Bend propagation in a microtubule bundle in a split axoneme. One of the two microtubule bundles in a split axoneme (left photo) is propagating a bend, as seen in the sequential photos shown right. Video frames taken every 5 ms. ATP 100 μM. Bar, 5 μm.

FIGURE 8

Model of the association/dissociation movement displayed by a doublet pair. (a) When two outer doublet microtubules are associated for a certain length, dynein (depicted by small projections on the right microtubule) generates sliding force. (b) The large bending moment at the base results in dissociation of the microtubules. (c and d) The dissociated portion spreads as the dissociation front moves toward the tip. (e) After complete dissociation, the two outer doublets start to reassociate at the base. The associated portion spreads as the association front moves toward the tip.

FIGURE 9

Model of wave propagation based on a “gap” movement. (A) Model for a traveling “gap” similar to that shown in Fig. 6. Simultaneous movements of the dissociation and association fronts (arrowheads) produce a distally traveling gap. (B) Model for bend propagation. If we assume that a gap is formed as in A but that the two outer doublets are connected by some loose structures, then interdoublet sliding will produce a bend (asterisk) that advances distally.