Digital image analysis of the flagellar beat of activated and hyperactivated suncus spermatozoa

The flagellar beat of hyperactivated Suncus spermatozoa was analyzed by digital imaging and was compared to that of the nonhyperactivated (activated) spermatozoa in order to examine the function of the accessory fibers during the flagellar beat and the sliding filament mechanism inducing the motility of the hyperactivated spermatozoa. Unusual large and long characteristics of the accessory fibers were involved in generating the gently curved bends and a low beat frequency. Examination of the motility parameters of the flagellar beat of the activated and hyperactivated spermatozoa attached to a slide glass by their heads revealed that there were two beating modes: a frequency-curvature dependent mode in the activated flagellar beat and a nearly constant frequency mode in the hyperactivated flagellar beat. The hyperactivated flagellar beat was characterized by sharp bends in the proximal midpiece and a low beat frequency. The sharp bends in the proximal midpiece were induced by the increase in the total length of the microtubule sliding at the flagellar base. The rate of microtubule sliding (sliding velocity) in the axoneme remained almost constant in the flagellar beat of both the activated and hyperactivated spermatozoa. Comparison of the sliding velocity in Suncus, golden hamster, monkey, and sea urchin sperm flagella with their stiffness suggests that the sliding velocity is determined by the stiffness at the flagellar base and that the same sliding microtubule system functions in both mammalian and echinoderm spermatozoa.     

Evidence for a sliding-resistance at the tip of the trypanosome flagellum

Motility in trypanosomes is achieved through the undulating behaviour of a single "9 + 2" flagellum; normally the flagellar waves begin at the flagellar tip and propagate towards the base. For flagella in general, however, propagation is from base-to-tip and it is believed that bend formation, and sustained regular oscillation, depend upon a localised resistance to inter-doublet sliding - which is normally conferred by structures at the flagellar base, typically the basal body. We therefore predicted that in trypanosomes there must be a resistive structure at the flagellar tip. Electron micrographs of Crithidia deanei, Herpetomonas megaseliae, Trypanosoma brucei and Leishmania major have confirmed that such attachments are present. Thus, it can be assumed that in trypanosomes microtubule sliding at the flagellar tip is resisted sufficiently to permit bend formation.     

Flagellar oscillation: a commentary on proposed mechanisms

Eukaryotic flagella and cilia have a remarkably uniform internal ‘engine’ known as the ‘9+2’ axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive ‘switching’ event is assumed to occur; there are several proposals regarding the nature of the ‘switch’ and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The ‘provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force‐generating cycles and dynein‐to‐dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self‐triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem.     

Flagellar and ciliary beating in trypanosome motility

The single flagellum of Leishmania and Trypanosoma parasites is becoming an increasingly attractive model for the analysis of flagellar function-driven largely by the abundance of genomic and proteomic information available for the organelle, the genetic manipulability of the organisms and the importance of motility for the parasite lifecycle. However, as yet, there is a paucity of published data on the beating of any genetically malleable trypanosomatid species. Here we undertook an in-depth analysis using high-speed videomicroscopy of the beating of free-swimming Leishmania major cells in comparison to Crithidia species (for which there is some existing literature). In so doing, we describe a simple and generally-applicable technique to facilitate the quantitative analysis of free-swimming cells. Our analysis thoroughly defines the parameters of the expected tip-to-base symmetrical flagellar beat in these species. It also describes beat initiation from points other than the flagellum tip and a completely different, base-to-tip highly-asymmetric beat that represents a ciliary beat of trypanosomatid flagella. Moreover, detailed analysis of parameter interrelationships revealed an unexpected dependency of wavelength on oscillator length that may be the result of reversible constraint of doublet sliding at the tip or resonance of the flagellar beat.     

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.     

The velocity of microtubule sliding: its stability and load dependency

It is now well understood that ATP-driven active sliding between the doublet microtubules in the sperm axoneme generates flagellar movement. However, much remains to be learned about how this movement is controlled. Detailed analyses of the flagellar beating of the mammalian spermatozoa revealed that there were two beating modes at a constant rate of microtubule sliding: that is, a nearly constant-curvature beating in nonhyperactivated spermatozoa and a nearly constant-frequency beating in hyperactivated spermatozoa. The constant rate of microtubule sliding suggests that the beat frequency and waveform of the flagellar beating are dependently regulated. Comparison of the sliding velocity of several mammalian and sea urchin sperm flagella with their mechanical property clarified that the sliding velocity of the microtubule was determined by the stiffness of the flagellum at its base, and that its relationship was expressed by a logarithmic equation that is similar to the classical force-velocity equation of the muscle contraction. Data from sea urchin spermatozoa also satisfied the equation, suggesting that the same microtubule sliding system functions in both the mammalian and echinoderm spermatozoa.     

Basal sliding and the mechanics of oscillation in a mammalian sperm flagellum

The mechanism of oscillation in cilia and flagella has been a long-standing mystery. This article raises the possibility of a mechanical explanation based on new findings relating to where in the flagellum microtubule sliding can occur--and where it cannot occur. All theoretical analyses of flagellar bending have until now made the assumption that sliding displacements at the base of the flagellum cannot occur. One consequence of this has been the need to accept that sliding must be transmitted through propagating bends, an idea that has been tolerated even though it becomes paradoxical if bends are the result of resistance to sliding. Our observations, of spermatozoa from the chinchilla, have led us to a contradictory view. We have shown directly, by light microscopy and by two methods of electron microscopy, that basal sliding does occur. Also, evidence from video microscopy indicates that a propagating bend cannot transmit sliding through it. We have analyzed a movement pattern in which the beat frequency increases fourfold in a phasic manner. Our analysis of this suggests that new bends terminate when no further sliding is possible. At this point the bend direction immediately reverses. That is, the flagellar beat frequency increases when there is a limitation to sliding. One can see directly the alternation in basal sliding direction under these circumstances. This suggests a mechanism for the initiation of a new bend in the opposite direction to the bend just completed: we propose that the initiating trigger is the reversal of elastic deformations at the base, which reverses the direction of interdoublet sliding.     

Evidence for axonemal distortion during the flagellar beat of Chlamydomonas

In order to understand the working mechanism that governs the flagellar beat it is essential to know if the axoneme undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell was able to preserve the waveform of Chlamydomonas flagella much as it appears during normal flagellar beating [Mitchell, Cell Motil Cytoskeleton 2003;56:120-129]. This conservation of the waveform suggests that the stress responsible for the production of bending is also trapped by the fixation procedure. Longitudinal sections of these well-preserved flagella were used to document variations in the relative axonemal diameter. Sections aligned to the plane of bending, showing both the central pair microtubules and outer doublets, were examined for this purpose. Micrographs were selected that continuously showed both the outer doublets and the central pair from a straight region to a curved region of the flagellum. Axoneme diameters measured from these select micrographs showed an increase in relative diameter that averaged 39 nm greater at the crest of the bent region. This constituted a 24% increase in the axoneme diameter in the bends. The transverse stress acting across the axoneme during bending was calculated from the Geometric Clutch computer model for a simulated Chlamydomonas-like flagellar beat. If we assume that this is representative of the transverse stress acting in a real flagellum, then the Young's modulus of the intact axoneme is approximately 0.02 MPa. The possibility that the distortion of the axoneme during the beat could play a significant role in regulating dynein function is discussed.     

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved ‘9+2’ axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26–57%) and beat frequencies (by 8–16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.     

A selective effect of Ni2+ on wave initiation in bull sperm flagella

Bull sperm that are extracted with 0.1% Triton X-100 and restored to motility with Mg2+-ATP lose coordination and stop swimming in the presence of 0.5 mM NiSO4. Although spontaneous coordination of flagellar waves is lost after exposure to Ni2+, other functions of the flagellum remain intact. The capacity for wave propagation along the flagellum is maintained together with the capacity for microtubular sliding. Wave motility can be restored to Ni2+-inhibited sperm by inducing a permanent bend onto the flagellum by micromanipulation. In the absence of such intervention, the loss of wave coordination is complete and irreversible. Ni2+-inhibited demembranated cells that are kept active by maintaining a bend in the flagellum exhibit a normal beat frequency. Both intact and demembranated sperm can retain spontaneous wave production at considerably slower rates of motion than Ni2+-inhibited cells. Short segments from the distal tip of the flagellum contain only the 9 + 2 microtubular axoneme. These short segments are able to propagate imposed bends even in the presence of Ni2+. In addition to wave propagation Ni2+-treated sperm can be shown to exhibit a normal sliding tubule phenomenon by direct assay. Although Ni2+-treated cells have a functional sliding tubule mechanism, and consequently the axoneme can propagate bends, it appears that these retained functions are not sufficient to cause spontaneous bend initiation. Our findings show that bend initiation is inhibited by Ni2+, and therefore is an independent process separate from the sliding tubule mechanism responsible for wave propagation.     

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.     

Bending motion of Chlamydomonas axonemes after extrusion of central- pair microtubules

Relatively little is known about the functions of central-pair microtubules (Tamm, S. L., and G. A. Horridge, 1970, Proc. Roy. Soc. Lond. B, 175: 219-233; Omoto, C. K., and C. Kung, 1979, Nature (Lond.). 279:532-534) and radial spokes (Warner, F. D., and P. Satir, 1974, J. Cell Biol., 63:35-63), although a sliding microtubule mechanism has been established for the flagellar movement (Summers, K. E., and I. R. Gibbons, 1971, Proc. Natl. Acad. Sci. USA., 68:3092-3096). In the present report, an attempt was made to determine the functions of central-pair microtubules in flagellar motility. Central-pair microtubules were found to extrude from the tips of elastase-digested axonemes of demembranated Chlamydomonas flagella after the addition of ATP. The length of the extruded central-pair microtubules was approximately 70-100% that of the axoneme. After extrusion, axonemes continued to swim slowly backwards in the reactivation medium, with a trailing central pair attached like a tail to the flagellar tip. During bending movement of the axonemes, partially extruded central pairs rotated counterclockwise about the axoneme axis, as viewed from the distal end (Kamiya, R., 1982, Cell Motil. [Suppl.]:169-173). Axonemes swam backwards with a symmetric waveform and a beat frequency of approximately 10 Hz in the reactivation medium containing 10(-9)-10(-4) M Ca ions. Even at a lower Ca++ concentration, no ciliary-type swimming was noted on the axonemes.     

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.     

The Spermatozoon of Brachionus plicatilis(Rotifera,Monogononta) With Some Notes on Sperm Ultrastructure in Rotifera

The spermatozoon of B. plicatilisis a thread–like cell with an anterior flagellar portion and a posterior cell body. The flagellum has a lateral ‘undulating membrane’, containing a folded longitudinal cisterna and an axoneme. The basal body of the axoneme is at the anterior tip. The axoneme lacks outer dynein arms and extends through the entire flagellar region and most of the cell body. The main portion of the flagellum and of the cell body contains a series of vesicles with tightly packed tubules that may serve as a cytoskeleton. The cell body contains a partly condensed nucleus, several mitochondria and some cytoplasm. Some elongated mitochondria are arranged in the postnuclear region. When the spermatozoon moves, the undulations propagate from the basal body at the flagellar tip. Late spermatids can be recognized by the nucleus and the flagellum being coiled and enclosed within a common cell membrane. As in other rotifers, there are cigar–like cell products (‘rods’) in the testes. The general organization of the cell, including the absence of an evident acrosome, resembles that of the other known monogonont sperm types.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".     

Comparative analysis of movement characteristics of lancelet and fish spermatozoa having different morphologies

The movement characteristics of the sperm and their flagella obtained from a lancelet and 35 species from almost all orders of fishes were examined using high-speed video microscopy. The aim was to clarify the relationship between the motility parameters of the spermatozoa having different morphologies and how these motility parameters affect the swimming speed of the spermatozoa. The motility parameters representing the flagellar waveform, the wavelength, and the amplitude were neither very different between the spermatozoa of the different species nor related to the swimming speed. In contrast, the beat frequency was remarkably changed in the different spermatozoa and was proportional to the swimming speed. The maximum shear angle of the flagellar wave, which is directly related to the maximum sliding displacement between the doublet microtubules, remained nearly constant while the beat frequency varied widely; therefore, the spermatozoa beat in the constant sliding displacement mode. An analysis of the relationship between swimming speed and flagellar length revealed that short flagella were at a disadvantage in developing swimming speed; however, so were extra-long flagella. The ratio of the swimming speed to the wave velocity calculated from the wavelength and the beat frequency depended on the distance from the glass surface. The swimming speeds calculated using the original resistive-force theory were greater than the measured values. To rationalize the measured values, the ratio between the normal and tangential drag coefficient in the resistive-force theory was corrected; namely, 1.99 at 1 μm and 1.63 at 3 μm from the glass surface.     

Motility of triton-demembranated sea urchin sperm flagella during digestion by trypsin

The survival curves for a population of reactivated spermatozoa exposed to digestion by trypsin indicate that a large number of trypsin-sensitive targets must be digested before the flagellum disintegrates. Changes in flagellar movement during trypsin digestion can be very small, especially when the spermatozoa are reactivated at 0.25 M KCl. They are not the changes which would be expected if elastic resistance of the trypsin-sensitive structures responsible for maintaining the integrity of the axoneme is a significant determinant of flagellar bend amplitude. By carrying out trypsin digestion under a variety of conditions, at least six distinct effects of trypsin digestion on parameters of flagellar movement have been detected. These include a gradual increase in the rate of sliding between tubules, gradual and abrupt changes in beat frequency accompanied by reciprocal decreases in bend angle, changes in the symmetry and planarity of bending, and selective interference with mechanisms for bend initiation and bend propagation.     

Hyperactivation is the mode conversion from constant-curvature beating to constant-frequency beating under a constant rate of microtubule sliding

Flagellar beating of hyperactivated golden hamster spermatozoa was analyzed in detail using digital image analysis and was compared to that of nonhyperactivated (activated) spermatozoa in order to understand the change in flagellar beating during hyperactivation and the active microtubule sliding that brought about the change in flagellar beating. Hyperactivated flagellar beating, which was characterized by a sharp bend in the proximal midpiece and low beat frequency, was able to alter the waveform with little change in beat frequency (constant-frequency beating), whereas activated flagellar beating, which was characterized by a slight bend in the proximal midpiece and high beat frequency, was able to alter beat frequency with little change in the waveform (constant-curvature beating). These results demonstrate that flagellar beating of hyperactivated and activated spermatozoa were essentially different modes and that hyperactivation was the mode conversion from constant-curvature beating to constant-frequency beating. Detailed analysis of flagellar bends revealed that the increase in curvature in the proximal midpiece during hyperactivation was due to the increase in total length of microtubule sliding in a nearly straight region between bends, while the rate of microtubule sliding remained almost constant.     

Rib72, a conserved protein associated with the ribbon compartment of flagellar A-microtubules and potentially involved in the linkage between outer doublet microtubules

Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized "ribbon" of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an approximately 96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility.     

Microtubule termination patterns in mammalian sperm flagella

Detailed reconstructions of the flagellar tip (end-piece) in rodent spermatozoa have shown patterns of displacement between the termination points of the axonemal doublets (judging the terminations by the loss of electron density from the A-tubule). The patterns are in good agreement with those derived from sliding microtubule theory. In the hamster at least, the axis of major displacement passes approximately through doublet 1 and between doublets 5 and 6, though there may be some skewness in the clockwise direction. Microtubules derived from the plane at right angles to this (the central pair and presumably one or both of doublets 3 and 8) continue beyond the rest to the extreme tip, where they appear to be linked together at the cell membrane. This arrangement suggests that the tapering form of the end-piece, and of flagellar terminal filaments and ciliary tips in general, may be an adaptation to contain the sliding microtubules and prevent them impinging on the membrane overlying the tip.