Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia.

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.

Mechanical induction of oscillatory movement in demembranated, immotile flagella of sea urchin sperm at very low ATP concentrations.

Oscillation is a characteristic feature of eukaryotic flagellar movement. The mechanism involves the control of dynein-driven microtubule sliding under self-regulatory mechanical feedback within the axoneme. To define the essential factors determining the induction of oscillation, we developed a novel experiment by applying mechanical deformation of demembranated, immotile sea urchin sperm flagella at very low ATP concentrations, below the threshold of ATP required for spontaneous beating. Upon application of mechanical deformation at above 1.5 µmol l-1 ATP, a pair of bends could be induced and was accompanied by bend growth and propagation, followed by switching the bending direction. For an oscillatory, cyclical bending response to occur, the velocity of bend propagation towards the flagellar tip must be kept above certain levels. Continuous formation of new bends at the flagellar base was coupled with synchronized decay of the preceding paired bends. Induction of cyclical bends was initiated in a constant direction relative to the axis of the flagellar 9+2 structure, and resulted in the so-called principal bend. In addition, stoppage of the bending response occasionally occurred during development of a new principal bend, and in this situation, formation of a new reverse bend did not occur. This observation indicates that the reverse bend is always active, opposing the principal bend. The results show that mechanical strain of bending is a central component regulating the bend oscillation, and switching of the bend direction appears to be controlled, in part, by the velocity of wave propagation.

Effects of external strain on the regulation of microtubule sliding induced by outer arm dynein of sea urchin sperm flagella.

Oscillatory bending movement of eukaryotic flagella is powered by orchestrated activity of dynein motor proteins that hydrolyse ATP and produce microtubule sliding. Although the ATP concentration within a flagellum is kept uniform at a few millimoles per litre level, sliding activities of dyneins are dynamically coordinated along the flagellum in accordance with the phase of bending waves. Thus at the organellar level the dynein not only generates force for bending but also modulates its motile activity by responding to bending of the flagellum. Single molecule analyses have suggested that dynein at the molecular level, even if isolated from the axoneme, could alter the modes of motility in response to mechanical strain. However, it still remains unknown whether the coordinated activities of multiple dyneins can be modulated directly by mechanical signals. Here, we studied the effects of externally applied strain on the sliding movement of microtubules interacted with an ensemble of dynein molecules adsorbed on a glass surface. We found that by bending the microtubules with a glass microneedle, three modes of motility that have not been previously characterized without bending can be induced: stoppage, backward sliding and dissociation. Modification in sliding velocities was also induced by imposed bending. These results suggest that the activities of dyneins interacted with a microtubule can be modified and coordinated through external strain in a quite flexible manner, and that such a regulatory mechanism may be the basis of flagellar oscillation.

Versatile properties of dynein molecules underlying regulation in flagellar oscillation.

Dynein is a minus-end-directed motor that generates oscillatory motion in eukaryotic flagella. Cyclic beating, which is the most significant feature of a flagellum, occurs by sliding spatiotemporal regulation by dynein along microtubules. To elucidate oscillation generated by dynein in flagellar beating, we examined its mechanochemical properties under three different axonemal dissection stages. By starting from the intact 9 + 2 structure, we reduced the number of interacting doublets and determined three parameters, namely, the duty ratio, dwell time and step size, of the generated oscillatory forces at each stage. Intact dynein molecules in the axoneme, doublet bundle and single doublet were used to measure the force with optical tweezers. The mean forces per dynein determined under three axonemal conditions were smaller than the previously reported stall forces of axonemal dynein; this phenomenon suggests that the duty ratio is lower than previously thought. This possibility was further confirmed by an in vitro motility assay with purified dynein. The dwell time and step size estimated from the measured force were similar. The similarity in these parameters suggests that the essential properties of dynein oscillation are inherent to the molecule and independent of the axonemal architecture, composing the functional basis of flagellar beating.

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 regulating Ca2+-dependent mechanosensory behaviour in sea urchin spermatozoa.

Flagellar movement of the sea urchin sperm is regulated by intracellular Ca(2+). Flagellasialin, a polysialic acid-containing glycoprotein, as well as other membrane proteins seems responsible for the Ca(2+) control. To elucidate the mechanism of Ca(2+) dynamics underlying flagellar movement, we analysed the sperm's mechanosensory behavioural responses by using microtechniques. In sea water containing 10 mM Ca(2+), the sperm swim in circular paths. When a mechanical stimulus was applied to the sperm head with a glass microstylus, the sperm showed a series of flagellar responses, consisting of a stoppage of beating (quiescence) and a recovery of swimming in a straight path, followed by swimming in a circular path again; as the result the sperm avoided the obstacle. Ca(2+)-imaging with Fluo-4 showed that the intracellular Ca(2+) was high in the quiescence and gradually decreased after that. The effects of blockers and antibodies against candidate components revealed that the Ca(2+) influx was induced by Ca(2+) channels and the Ca(2+) efflux was induced by a flagellasialin-related Ca(2+)-efflux system, plasma membrane Ca(2+)-ATPases and the K(+)-dependent Na(+)/Ca(2+) exchanger. The results show that the Ca(2+)-dependent mechanosensory behaviour of the sea urchin sperm is regulated by organized functioning of the membrane environment including the plasma membrane proteins and flagellasialin.

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).

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.

Induction of beating by imposed bending or mechanical pulse in demembranated, motionless sea urchin sperm flagella at very low ATP concentrations.

A basic feature of the movement of eukaryotic flagella is oscillation. Although flagellar oscillation is thought to be regulated by a self-regulatory feedback system including the mechanical signal of bending itself, the mechanism regulating the dynein motile activity to produce oscillation is not well understood. To elucidate the mechanism, we developed a new experimental system which allowed us to analyze the conditions necessary for the induction of oscillation. When a mechanical signal of bending or a pulse was applied by micromanipulation to a demembranated motionless sea urchin sperm flagellar axoneme at very low ATP concentrations (1-3 microM), a localized pair of bends was induced. The bend formation was often followed by further responses including propagation of the distal bend of paired bends, growth and propagation of the paired bends, and cyclical beating. The beating was induced at 2.0 microM or higher concentrations of ATP, but appeared even at 1.5 microM ATP if a few muM of ADP was also present. When the proximal half of a flagellum was attached to a microneedle, beating could not be induced in the distal free region at 2 microM ATP. These results suggest that mechanical signal is involved in the mechanism regulating the motile activity of dynein to produce oscillation. Our results also showed that the presence of a small amount of ADP and the axial difference along the flagellum are factors essential for the induction of flagellar oscillation.

Effects of imposed bending on microtubule sliding in sperm flagella.

The movement of eukaryotic flagella is characterized by its oscillatory nature. In sea urchin sperm, for example, planar bends are formed in alternating directions at the base of the flagellum and travel toward the tip as continuous waves. The bending is caused by the orchestrated activity of dynein arms to induce patterned sliding between doublet microtubules of the flagellar axoneme. Although the mechanism regulating the dynein activity is unknown, previous studies have suggested that the flagellar bending itself is important in the feedback mechanism responsible for the oscillatory bending. If so, experimentally bending the microtubules would be expected to affect the sliding activity of dynein. Here we report on experiments with bundles of doublets obtained by inducing sliding in elastase-treated axonemes. Our results show that bending not only "switches" the dynein activity on and off but also affects the microtubule sliding velocity, thus supporting the idea that bending is involved in the self-regulatory mechanism underlying flagellar oscillation.

Dynein arms are strain-dependent direction-switching force generators.

Dynein is a minus-end-directed motor that can generate (forward) force to move along the microtubule toward its minus end. In addition, axonemal dyneins were reported to oscillate in the generation of forward force, and cytoplasmic dynein is observed to generate bidirectional forces in response to defined chemical states. Both dyneins can also respond to mechanically applied force. To test whether axonemal dynein can switch direction of force generation, we measured force using an optical trap and UV-photolysis of caged ATP. We observed that isolated dynein could repeatedly generate force in both directions along the microtubule. Bidirectional force was also observed for dynein arms that are still attached on the doublet microtubules. Axonemal dynein generated force to move backward (∼ 4 pN) as well as forward (5-6 pN) along microtubules. Furthermore, backward force could be stimulated by plus-end directed external force applied to axonemal dynein before ATP application. The results show that axonemal dynein is unique exhibiting multiple modes of force generation including backward and forward force, oscillatory force and slow, repetitive bidirectional force. The results also demonstrate that mechanical strain is important for switching the directionality of force generation in axonemal dyneins.

Measuring the regulation of dynein activity during flagellar motility.

Flagellar and ciliary motility are driven by the activity of dynein, which produces microtubule sliding within the axonemes. Our goal is to understand how dynein motile activity is regulated to produce the characteristic oscillatory movement of flagella. Analysis of various parameters, such as frequency and shear angle in beating flagella, is important for understanding the time-dependent changes of microtubule sliding amounts along the flagellum. Demembranated flagella can be reactivated in a wide range of ATP concentrations (from 2 µM to several mM) and the beat frequency increases with an increase in ATP. By imposed vibration of a micropipette that caught a sperm head by suction, however, the oscillatory motion can be modulated so as to synchronize to the vibration frequency over a range of 20-70Hz at 2mM ATP. The time-averaged sliding velocity calculated as a product of shear angle and vibration frequency decreases when the imposed frequency is below the undriven flagellar beat frequency, but at higher imposed frequencies, it remains constant. In addition to the role of ATP, the mechanical force of bending is involved in the activation of dynein. In elastase-treated axonemes, bending-dependent regulation of microtubule sliding is achieved. This chapter provides an overview of several approaches, using sea urchin sperm flagella, to studying the measurements in the regulation of dynein activity with or without mechanical force.

Effects of iodide on the coupling between ATP hydrolysis and motile activity in axonemal dynein.

Dynein transduces the chemical energy of ATP hydrolysis into mechanical work through conformational changes. To identify the factors governing the coupling between the ATPase activity and the motile activity of the dynein molecule, we examined the effects of potassium iodide, which can unfold protein tertiary structures, on dynein activity in reactivated sea urchin sperm flagella. The presence of low concentrations of KI (0.05-0.1 M) in the reactivating solution did not influence the stable beating of demembranated flagella at 0.02-1 mM ATP, when the total concentration of potassium was kept at 0.15 M by adding K-acetate. However, double-reciprocal plots of ATP concentration and beat frequency showed a mixed type of inhibition by KI, indicating the possibility that KI inhibits the ATP hydrolysis and decreases the maximum sliding velocity. The ATPase activity of 21S dynein with or without microtubules did not decrease with the KI concentration. In the elastase-treated axonemes, KI decreased the velocity of sliding disintegration, while it increased the frequency of occurrence of axonemes showing no sliding. This may be related to some defect in the coordination of dynein activities. On 21S dynein adsorbed on a glass surface, however, the velocity of microtubule sliding was increased by KI, while KI lowered the dynein-microtubule affinity. The velocity further increased under lower salt conditions enhancing the dynein-microtubule interactions. The results suggest the importance of organized regulation of the dynamic states of dynein-microtubule interactions through the stalk for the coupling between the ATPase activity and the motile activity of dynein in beating flagella.

Analysis of the role of nucleotides in axonemal dynein function.

Axonemal dynein in flagella and cilia is a motor molecule that produces microtubule sliding, powered by the energy of ATP hydrolysis. Our goal is to understand how dynein motile activity is controlled to produce the characteristic oscillatory movement of flagella. ATP, the energy source for dynein, is also important as a regulator of dynein activity. Among the four nucleotide-binding sites of a dynein heavy chain, one is the primary ATP hydrolyzing site while the others are noncatalytic sites and thought to perform regulatory functions. Stable binding of both ATP and ADP to these regulatory sites is probably essential for the chemomechanical energy transduction in dynein. Although the ATP concentration in beating flagella is physiologically high and constant, at any moment in the oscillatory cycle some dynein molecules are active while others are not, and the motile activity of dynein oscillates temporally and spatially in the axoneme. It is likely that the basic mechanism underlying the highly dynamic control of dynein activity involves the ATP-dependent inhibition and ADP-dependent activation (or release of inhibition) of dynein. How the inhibition and activation can be induced in beating flagella is still unknown. It seems, however, that the mechanical force of bending is involved in the activation of dynein, probably through the control of noncatalytic nucleotide binding to dynein. This chapter provides an overview of several approaches, using sea urchin sperm flagella, to studying the roles of ATP and ADP in the regulation of dynein activity with or without the mechanical signal of bending.

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.

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 Ca(2+)-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.

The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella.

The regulation of dynein activity to produce microtubule sliding in flagella has not been well understood. To gain more insight into the roles of ATP and ADP in the regulation, we examined the effects of fluorescent ATP analogues and fluorescent ADP analogues on the ATPase activity and motile activity of dynein. 21S dynein purified from the outer arms of sea urchin sperm flagella hydrolyzed BODIPY(R) FL ATP (FL-ATP) at 78% of the rate for ATP hydrolysis. FL-ATP at 0.1-1 mM, however, induced neither microtubule translocation on a dynein-coated glass surface nor sliding disintegration of elastase-treated axonemes. Direct observation of single molecules of the fluorescent analogues showed that both the ATP and ADP analogues were stably bound to dynein over several minutes (dissociation rates = 0.0038-0.0082/s). When microtubule translocation on 21S dynein was induced by ATP, the initial increase of the mean velocity was accelerated by preincubation of the dynein with ADP. Similar increase was also induced by the preincubation with the ADP analogues. Even after preincubation with ADP, FL-ATP did not induce sliding disintegration of elastase-treated axonemes. After preincubation with a nonhydrolyzable ATP analogue, AMPPNP (adenosine 5'-(beta:gamma-imido)triphosphate), however, FL-ATP induced sliding disintegration in approximately 10% of the axonemes. These results indicate that both noncatalytic ATP binding and stable ADP binding, in addition to ATP hydrolysis, are involved in the regulation of the chemo-mechanical transduction in axonemal dynein.

Inhibition by ATP and activation by ADP in the regulation of flagellar movement in sea urchin sperm.

ATP and ADP are known to play inhibitory and activating roles, respectively, in the regulation of dynein motile activity of flagella. To elucidate how these nucleotide functions are related to the regulation of normal flagellar beating, we examined their effects on the motility of reactivated sea urchin sperm flagella at low pH. At pH 7.0-7.2 which is lower than the physiological pH of 8, about 90% of reactivated flagella were motionless at 1 mM ATP, while about 60% were motile at 0.02 mM ATP. The motionless flagella at 1 mM ATP maintained a single large bend or an S-shaped bend, indicating formation of dynein crossbridges in the axoneme. The ATP-dependent inhibition of flagellar movement was released by ADP, and was absent in outer arm-depleted flagella. Similar inhibition was also observed at 0.02 mM ATP when demembranated flagella were reactivated in the presence of Li+ or pretreated with protein phosphatase 1 (PP1). ADP also released this type of ATP-inhibition. In PP1-pretreated axonemes the binding of a fluorescent analogue of ADP to dynein decreased. Under elastase-treatment at pH 8.0, the beating of demembranated flagella at 1 mM ATP and 0.02 mM ATP lasted for approximately 100 and 45 s, respectively. The duration of beating at 0.02 mM ATP was prolonged by Li+, and that at 1 mM ATP was shortened by removal of outer arms. These results indicate that the regulation of on/off switching of dynein motile activity of flagella involves ATP-induced inhibition and ADP-induced activation, probably through phosphorylation/dephosphorylation of outer arm-linked protein(s).

Effects of trypsin-digested outer-arm dynein fragments on the velocity of microtubule sliding in elastase-digested flagellar axonemes.

Flagellar movement is caused by the coordinated activity of outer and inner dynein arms, which induces sliding between doublet microtubules. In trypsin-treated flagellar axonemes, microtubule sliding induced by ATP is faster in the presence than in the absence of the outer arms. To elucidate the mechanism by which the outer arms regulate microtubule sliding, we studied the effect of trypsin-digested outer-arm fragments on the velocity of microtubule sliding in elastase-treated axonemes of sea urchin sperm flagella. We found that microtubule sliding was significantly slower in elastase-treated axonemes than in trypsin-treated axonemes, and that this difference disappeared after the complete removal of the outer arms. After about 95% of the outer arms were removed, however, the velocity of sliding induced by elastase and ATP increased significantly by adding outer arms that had been treated with trypsin in the presence of ATP. The increase in sliding velocity did not occur in the elastase-treated axonemes from which the outer arms had been completely removed. Among the outer arm fragments obtained by trypsin treatment, a polypeptide of about 350 kDa was found to be possibly involved in the regulation of sliding velocity. These results suggest that the velocity of sliding in the axonemes with only inner arms is similar to that in the axonemes with both inner and outer arms, and that the 350 kDa fragment, probably of the alpha heavy chains, increases the sliding activity of the intact outer and inner arms on the doublet microtubules.