Chikako Shingyoji

Dynein arms are oscillating force generators

Eukaryotic flagella beat rhythmically. Dynein is a protein that powers flagellar motion, and oscillation may be inherent to this protein. Here we determine whether oscillation is a property of dynein arms themselves or whether oscillation requires an intact axoneme, which is the central core of the flagellum and consists ofa regular array of microtubules. Using optical trapping nanometry,, we measured the force generated by a few dynein arms on an isolated doublet microtubule. When the dynein arms on the doublet microtubule contact a singlet microtubule and are activated by photolysis of caged ATP, they generate a peak force of ∼6 pN and move the singlet microtubule over the doublet microtubule in a processive manner. The force and displacement oscillate with a peak-to-peak force and amplitude of ∼2 pN and ∼30 nm, respectively. The geometry of the interaction indicates that very few (possibly one) dynein arms are needed to generate the oscillation. The maximum frequency of the oscillation at 0.75 mM ATP is ∼70 Hz; this frequency decreases as the ATP concentration decreases. A similar oscillatory force is also generated by inner dynein arms alone on doublet microtubules that are depleted of outer dynein arms. The oscillation of the dynein arm may be a basic mechanism underlying flagellar beating.

Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes

The movement of eukaryotic flagella and cilia is regulated by intracellular calcium. We have tested a model in which the central pair of microtubules mediate the effect of Ca2+ to modify the dynein activity. We used a novel microtubule sliding assay that allowed us to test the effect of Ca2+ in the presence or absence of the central-pair microtubules. When flagellar axonemes of sea-urchin sperm were exposed to ATP in the presence of elastase, they showed different types of sliding disintegration depending on the ATP concentration: at low concentrations of ATP (≤50μ M), all the axonemes were disintegrated into individual doublets by microtubule sliding; by contrast, at high ATP concentrations (≥100 μM), a large proportion of the axonemes showed limited sliding and split lengthwise into a pair of two microtubule bundles, one of which was thicker than the other. The sliding behaviour of the axonemes was also influenced by Ca2+. Thus, at 1 mM ATP, the proportion of axonemes that split into two bundles increased from 25% at <10–9 M Ca2+ to 60% at 10–4 M Ca2+, whereas the sliding velocity of doublets during the splitting did not change. Electron microscopy of split bundles showed that the thicker bundles contained five or six doublets and the central pair, whereas the thinner bundles contained three or four doublets but not the central pair. Closer examinations revealed that the thicker bundles were dominated by four patterns of doublet combinations: doublets 8-9-1-2-3-4, 8-9-1-2-3, 4-5-6-7-8 and 3-4-5-6-7-8. This indicates that the sliding occurred preferentially at one or two fixed interdoublet sites on either side of the central-pair microtubules, whereas the sliding at the remaining interdoublet sites was inhibited under these conditions. Ca2+ reduced the appearance of the 4-5-6-7-8 and 3-4-5-6-7-8 patterns and increased the 8-9-1-2-3-4 and 8-9-1-2-3 patterns. The splitting patterns are possibly related to the switching mechanism of the dynein activity underlying the cyclical flagellar bending. To investigate the role of the central pair in the regulation of the dynein activity by Ca2+, we studied the behaviour of singlet microtubules applied to the dynein arms exposed on the doublets of the split bundles that were either associated with the central pair or not. Microtubules moved along both the thicker and the thinner bundles but the frequency of microtubule sliding on the thinner (i.e. the central-pair-less) bundles was three to four times (at≤ 10–5 M Ca2+) and ten times (at 10–4 M Ca2+) as large as that on the thicker, central-pair-associated bundles. Furthermore, the velocity of microtubule sliding at 1 mM ATP on the thicker bundles were significantly reduced by 10–7-10–4 M Ca2+, whereas that on the thinner bundles was not changed by the concentration of Ca2+. These results indicate that Ca2+ inhibits the activity of dynein arms on the doublets through a regulatory mechanism that involves the central pair and the radial spoke complex. This mechanism might control the switching of the dynein activity within the axoneme to induce the oscillatory bending movement of the flagellum.

Dynein pulls microtubules without rotating its stalk

Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, “cryo-positive stain” electron microscopy. The dynein molecules in the complex were shown to be active in in vitro motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP·vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).

INDUCTION OF TEMPORARY BEATING IN PARALYZED FLAGELLA OF CHLAMYDOMONAS MUTANTS BY APPLICATION OF EXTERNAL FORCE

To help understand the mechanism by which the sliding movement of outer-doublet microtubules in cilia and flagella is converted into bending waves, we examined the effect of mechanical force imposed on the flagella of Chlamydomonas mutants lacking the central pair or multiple dyneins. These mutants were almost completely nonmotile under normal conditions. A bend was produced in a flagellum either by holding a cell with a micropipette and quickly moving it with a piezoelectric actuator; or by pushing a flagellum with a microneedle. After removal of the external force, mutants lacking the central pair (pf18 and pf19) displayed beating at irregular intervals of > 1 second for one to several cycles. Similarly, a double mutant (ida2ida4) lacking four species of inner-arm dynein displayed beating at intervals of > 0.1 second for up to 80 cycles. However, paralyzed flagella of double mutants that lack the outer dynein arm in addition to the central pair or the inner dynein arm did not show cyclical movements upon application of external force. These results indicate that the central pair and the inner dynein arm are important for both stable bend formation at the base and efficient bend propagation along the flagellar length. They also suggest that the outer dynein arm, and not the inner dynein arm, enables the flagellar axoneme to propagate bends independently of the central pair. We propose that the axoneme is equipped with two independent motor systems for oscillatory movements: an outer-arm system controlled by the axonemal mechanical state independently of the central pair/radial spoke system, and an inner-arm system controlled by both the axonemal mechanical state and the central pair/radial spokes.

Comparative structural analysis of eukaryotic flagella and cilia from Chlamydomonas, Tetrahymena, and sea urchins.

Although eukaryotic flagella and cilia all share the basic 9+2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions.

Mechanism of flagellar oscillation–bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm

Oscillatory movement of eukaryotic flagella is caused by dynein-driven microtubule sliding in the axoneme. The mechanical feedback from the bending itself is involved in the regulation of dynein activity, the main mechanism of which is thought to be switching of the activity of dynein between the two sides of the central pair microtubules. To test this, we developed an experimental system using elastase-treated axonemes of sperm flagella, which have a large Ca2+-induced principal bend (P-bend) at the base. On photoreleasing ATP from caged ATP, they slid apart into two bundles of doublets. When the distal overlap region of the slid bundles was bent in the direction opposite to the basal P-bend, backward sliding of the thinner bundle was induced along the flagellum including the bent region. The velocity of the backward sliding was significantly lower than that of the forward sliding, supporting the idea that the dynein activity alternated between the two sides of the central pair on bending. Our results show that the combination of the direction of bending and the conformational state of dynein-microtubule interaction induce the switching of the dynein activity in flagella, thus providing the basis for flagellar oscillation.

Conversion of beating mode in Chlamydomonas flagella induced by electric stimulation

Electric stimulation of a single Chlamydomonas cell by means of a suction electrode induced a temporary conversion of flagellar waveform from an asymmetric forward mode to a symmetric reverse mode. The reverse mode continued for about 0.5 seconds, after which the forward mode was resumed. Anodic stimulation (current passing outward through the membrane outside the suction pipette) was more effective in inducing the flagellar response than cathodic stimulation. No flagellar response was induced in the absence of free Ca2+ or in the presence of calcium channel inhibitors, pimozide (5 microM) and diltyazem (0.3 mM). These findings indicate that the flagellar response by membrane depolarization followed by a Ca2+ influx through voltage-dependent calcium channels. This experimental system allowed us to quantitatively analyze the behavior of flagella during the waveform conversion. The flagellar bending pattern quickly changed from the forward mode to the reverse mode and, thereafter, gradually resumed the forward mode through two discrete phases: changes during reverse mode beating (phase I) and a distinct transitional phase (phase II). Recovery in curvature and sliding velocity of principal bends occurred mostly in phase I. Almost all of the recovery of reverse bends, returning the curvature to the low values characteristic of asymmetric forward mode beating, occurred in phase II. Beat frequency recovered through both phases. Phase II was often interrupted by a temporary stoppage of beating. These findings indicate that the bending pattern is converted through multiple steps that are controlled by Ca2+.

Bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm—Roles of Ca2+ and ADP

Flagellar beating is caused by microtubule sliding, driven by the activity of dynein, between adjacent two of the nine doublet microtubules. An essential process in the regulation of dynein is to alternate its activity (switching) between the two sides of the central pair microtubules. The switching of dynein activity can be detected, in an in vitro system using elastase-treated axonemes of sea urchin sperm flagella, as a reversal of the relative direction of ATP-induced sliding between the two bundles of doublets at high Ca(2+) (10(-4) M) at pH 7.8-8.0. The reversal is triggered by externally applied bending of the doublet bundle. However, the mechanism of this bending-induced reversal (or backward sliding) remains unclear. To understand how the switching of dynein activity in flagella can be induced by bending, we studied the roles of ADP, which is an important factor for the dynein motile activity, and of Ca(2+) in the bending-induced reversal of microtubule sliding between two bundles of doublets at pH 7.5 and 7.2. We found that the reversal of sliding direction was induced regardless of the concentrations of Ca(2+) at low pH, but occurred more frequently at low Ca(2+) (<10(-9) M) than at high Ca(2+). At pH 7.5, an application of ADP increased the frequency of occurrence of backward sliding at high as well as low concentrations of Ca(2+). The results indicate that ADP-dependent activation of dynein, probably resulting from ADP-binding to dynein, is involved in the regulation of the bending-induced switching of dynein activity in flagella.

Cyclical training enhances the melanophore responses of zebrafish to background colours.

Zebrafish respond to visual stimuli to adapt their body colour to the background. If, rather than being a simple on/off reaction to visual stimulation, the colour change involves cognitive and memory‐related processes, training fish with cyclical changes of the background would be expected to increase its ability to change colour. To test this, we developed a standardized procedure for quantifying the responses of melanophores to background changes in living adult specimens of leopard, a zebrafish mutant with spotted stripes. After training with 2‐day cyclical alternation of white and black backgrounds for over 20 days, the proportion of the melanosome‐filled area of dorsal melanophores, which was 20% on the black background before the training, increased up to 97%. In these trained fish, a rapid melanosome aggregation occurred within 10 s of the background change from black to white. The results indicate that the zebrafish melanophore responses can be modulated by learning, in which areal and speed control of melanosome movement are important for dispersion and aggregation, respectively.

The phase of sperm flagellar beating is not conserved over a brief imposed interruption.

We have studied the phase component of flagellar beating by holding the head of a sea urchin sperm in the tip of a sinusoidally vibrating micropipet and then abruptly displacing the pipet laterally at a speed of 2.5 microns/ms for various durations. This rapid displacement of the pipet delayed the initiation of the next bend for as long as the displacement continued, up to a duration of 1 beat cycle, corresponding to a delay of 0.5 beat cycle. At the end of this displacement, the movement of the pipet was stopped completely without resumption of the initial vibration. Analysis of the flagellar waveform showed that immediately when the pipet was stopped, the flagellum started to beat by spontaneously initiating the bend that had been delayed. The flagellum then continued steady-state beating, with normal waveform and a new phase that was independent of the original phase of beating. These data suggest that the information on the phase of beating is located only at the basal end of the flagellum, and not in oscillators distributed along the axoneme. After this information has been lost, the flagellum can resume beating at any arbitrary phase relative to its original phase.