Distinct regulation of two flagella by calcium during chemotaxis of male gametes in the brown alga Mutimo cylindricus (Cutleriaceae,Tilopteridales)

Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high-speed recording system under different calcium conditions. The extracellular Ca²⁺ concentration at 10⁻³ M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca²⁺ concentrations below 10⁻² M. Real-time Ca²⁺ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca²⁺ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca²⁺-dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca²⁺ concentration.     

Digitized precision measurements of the movements of sea urchin sperm flagella.

High speed cinemicrographs were made of sea urchin sperm at temperatures varying from 22 to 6 degrees C. Apparatus, combining a television camera and a video digitizer, was constructed to scan individual flagellar images and to digitize the flagellar waveforms. With appropriate smoothing and averaging procedures, the rough data were condensed by a microcomputer into the coordinates of 20 points along a flagellum, spaced 2 microns apart. The curvature of the flagellum at these points was also computed. The coordinates of the flagellar positions were obtained to an accuracy of approximately +/- 0.1 micron, flagellar curvature to an accuracy of approximately +/- 50 cm-1. At all temperatures the amplitude of the flagella was found to vary with time in a purely sinusoidal fashion to within +/- 2%. The local curvature of the flagella had basically a purely sinusoidal time course to within +/- 50 cm-1, but a varying amount of asymmetry was present in the distal and the proximal ends of the flagella. This asymmetry in the curvature was related to the radius of the circular path of the sperm. The flagellar waveforms can probably be summarized in simple algebraic functions.     

Effect of Extracellular Ca2+ and Ca2+-Antagonists on the Movement and Chemoorientation of Male Gametes of Ectocarpus siliculosus (Phaeophyceae)

Chemoorientation in male gametes of Ectocarpus siliculosus in response to sexual pheromones is effected by two distinct mechanisms, chemokinesis and chemoklinotaxis. These are characterized by a strongly asymmetric bending pattern of the anteriorly-directed flagellum and transient unilateral bending of the hind flagellum, respectively. Removal of extracellular Ca²⁺ showed that normal flagellar movement and chemokinesis require millimolar concentrations of Ca²⁺ in the medium. The response to pheromones is strongly inhibited by La³⁺, whereas the Ca²⁺-channel drugs, verapamil and nifedipine, have only little effect. Nifedipine nethertheless effectively inhibited accumulation at pheromone sources. These results are interpreted as an indication for the involvement of two pharmacologically distinct Ca²⁺-channels in chemokinesis and chemoklinotaxis. The calmodulin-antagonist, trifluoperazine, induces, at low concentrations, the same flagellar response in chemokinesis as the pheromone, the mechanism of action remaining unknown.     

A FLAVIN-LIKE AUTOFLUORESCENT SUBSTANCE IN THE POSTERIOR FLAGELLUM OF GOLDEN AND BROWN ALGAE1

An autofluorescent substance occurs in the flagella of flagellate cells of the golden and brown algae. It is localized only in the posterior (short) flagellum and could not be detected in the anterior (long) one. It showed maximum fluorescence emission at 515–520 nm upon excitation of 440 nm; therefore, it is considered to be a flavin. This substance is distributed widely among flagellate cells of golden and brown algae irrespective of their nature (vegetative cells, zoospores, gametes, or sperm). It is absent, however, in some brown algal zoospores and sperm which lack an eyespot and flagellar swelling and are considered to lack phototaxis. Because the flagellar swelling in the posterior flagellum is a presumptive photoreceptor for phototaxis in these groups, it is suggested that the flavin located in the posterior flagellum acts as a photoreceptor pigment in phototaxis.     

Phototaxis and chemotaxis of brown algal swarmers

Brown algae exhibit three patterns of sexual reproduction: isogamy, anisogamy, and oogamy. Unicellular swarmers including gametes and zoospores bear two heterogenous flagella, an anterior flagellum with mastigonemes (fine tripartite hairs) and a posterior one. In seawater, these flagellates usually receive physico-chemical signals for finding partners and good habitats. It is well known that brown algal swarmers change their swimming direction depending on blue light (phototaxis), and male gametes do so, based on the sex pheromones from female gametes (chemotaxis). In recent years, the comparative analysis of chemotaxis in isogamy, anisogamy, and oogamy has been conducted. In this paper, we focused on the phototaxis and chemotaxis of brown algal gametes comparing the current knowledge with our recent studies.     

Chemotaxis in Laminaria digitata (Phaeophyceae): I. ANALYSIS OF SPERMATOZOID MOVEMENT

The chemotactic behaviour of Laminaria digitata spermatozoidsupon stimulation with sex pheromone was studied using videomicroscopyand high speed cinematography. The cells show phobic (‘shock-’)reorientation reactions when perceiving decreasing stimulusconcentrations in a pheromone gradient and are able to adaptto constant concentrations. The phobic responses are executedby regulation of flagellar activity: drastic changes in thedirection of cell movement are induced by rapid deflexions ofthe posterior flagellum. Key words: Laminaria, chemotaxis, pheromone, flagellar movement.     

Behavior of flagella and flagellar root systems in the planozygotes and settled zygotes of the green alga Bryopsis maxima Okamura (Ulvophyceae,Chlorophyta) with reference to spatial arrangement of eyespot and cell fusion site

Behaviors of male and female gametes, planozygotes and their microtubular cytoskeletons of a marine green alga Bryopsis maxima Okamura were studied using field emission scanning electron microscopy, high‐speed video microscopy, and anti‐tubulin immunofluorescence microscopy. After fusion of the biflagellate male and female gametes, two sets of basal bodies lay side by side in the planozygote. Four long female microtubular roots extended from the basal bodies to the cell posterior. Four short male roots extended to nearly half the distance to the posterior end. Two flagella, one each from the male and female gametes, become a pair. Specifically, the no. 2 flagellum of the female gamete and one male flagellum point to the right side of the eyespot of the female gamete, which is located at the cell posterior and which is associated with 2s and 2d roots of the female gamete. This spatial relationship of the flagella, microtubular roots, and the eyespot in the planozygote is retained until settlement. During forward swimming, the planozygote swings the flagella backward and moves by flagellar beating. The male and female flagella in the pair usually beat synchronously. The cell withdraws the flagella and becomes round when the planozygote settles to the substratum 20 min after mixing. The axoneme and microtubular roots depolymerize, except for the proximal part and the basal bodies. Subsequently, distinct arrays of cortical microtubules develop in zygotes until 30 min after mixing. These results are discussed with respect to the functional significance of the spatial relationships of flagellar apparatus‐eyespot‐cell fusion sites in the mating gametes and planozygote of green algae.     

Locomotion of free-swimming ghost knifefish: anal fin kinematics during four behaviors

The maneuverability demonstrated by the weakly electric ghost knifefish (Apteronotus albifrons) is a result of its highly flexible ribbon-like anal fin, which extends nearly three-quarters the length of its body and is composed of approximately 150 individual fin rays. To understand how movement of the anal fin controls locomotion we examined kinematics of the whole fin, as well as selected individual fin rays, during four locomotor behaviors executed by free-swimming ghost knifefish: forward swimming, backward swimming, heave (vertical) motion, and hovering. We used high-speed video (1000 fps) to examine the motion of the entire anal fin and we measured the three-dimensional curvature of four adjacent fin rays in the middle of the fin during each behavior to determine how individual fin rays bend along their length during swimming. Canonical discriminant analysis separated all four behaviors on anal fin kinematic variables and showed that forward and backward swimming behaviors contrasted the most: forward behaviors exhibited a large anterior wavelength and posterior amplitude while during backward locomotion the anal fin exhibited both a large posterior wavelength and anterior amplitude. Heave and hover behaviors were defined by similar kinematic variables; however, for each variable, the mean values for heave motions were generally greater than for hovering. Individual fin rays in the middle of the anal fin curved substantially along their length during swimming, and the magnitude of this curvature was nearly twice the previously measured maximum curvature for ray-finned fish fin rays during locomotion. Fin rays were often curved into the direction of motion, indicating active control of fin ray curvature, and not just passive bending in response to fluid loading.     

Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.

Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C. A roughly linear relation between swimming speed and flagellar rotation rate was observed. The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation. At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate. That is, the cell with a faster-rotating flagellum did not always swim faster. To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered. The model could be analytically solved, and it qualitatively explained the experimental results. The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20%. The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force.     

A theory of piezoelectric activity and ion-movements in the relation of flagellar structures and their movements to the phototaxis of Euglena

A review of the literature on the flagellar undulations and phototactic movements of Euglena indicates that the flagellum functions as an ATP-using motor, triggered and mediated by cations, especially H₃O⁺, K^+, Mg²⁺ and Ca²⁺, and driven by energy from ATP. The undulatory waves are assumed to be started by means of repetitive pulses due to a redox reaction at the base of the flagellum. It is also assumed that the axoneme and paraflagellar rod are composed of asymmetrically-crystalline proteinaceous fibrils which are piezoelectric, i.e. they bend when energy passes through or along them, thus acting as a motor, and when bending they deliver a current, thus acting as a generator. This piezoelectric activity displaces cations and drives them ahead of it, triggering sequential bending and straightening of segments of the flagellum from base to tip. The paraflagellar swelling (“photoreceptor”) is also assumed to be piezoelectric, reactive to light, acting as a capacitor. It discharges as the intensity of light striking it is changed by the alternative shading effect of the stigma (“eyespot”) and exposure to light as the Euglena gyrates in swimming. The charge delivered by the photoreceptor augments the effects of ion-movements along the flagellum, also augmenting the amplitude and force of the flagellar undulations and altering the position of the flagellum relative to the body and the direction of swimming. The body is tipped away from the original path and swims either toward or away from the light, depending on the ultimate alteration of the path of swimming.     

The rate of change in Ca(2+) concentration controls sperm chemotaxis

During chemotaxis and phototaxis, sperm, algae, marine zooplankton, and other microswimmers move on helical paths or drifting circles by rhythmically bending cell protrusions called motile cilia or flagella. Sperm of marine invertebrates navigate in a chemoattractant gradient by adjusting the flagellar waveform and, thereby, the swimming path. The waveform is periodically modulated by Ca(2+) oscillations. How Ca(2+) signals elicit steering responses and shape the path is unknown. We unveil the signal transfer between the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and path curvature (κ). We show that κ is modulated by the time derivative d[Ca(2+)](i)/dt rather than the absolute [Ca(2+)](i). Furthermore, simulation of swimming paths using various Ca(2+) waveforms reproduces the wealth of swimming paths observed for sperm of marine invertebrates. We propose a cellular mechanism for a chemical differentiator that computes a time derivative. The cytoskeleton of cilia, the axoneme, is highly conserved. Thus, motile ciliated cells in general might use a similar cellular computation to translate changes of [Ca(2+)](i) into motion.     

Flagellum-mediated motility in Pelotomaculum thermopropionicum SI

The basic functions of a propionate-oxidizing bacterium Pelotomaculum thermopropionicum flagellum, such as motility and chemotaxis, have not been studied. To investigate its motility, we compared with that of Syntrophobacter fumaroxidans, an aflagellar propionate-oxidizing bacterium, in soft agar medium. P. thermopropionicum cells spread, while S. fumaroxidans cells moved downward slightly, indicating flagellum-dependent motility in P. thermopropionicum SI. The motility of P. thermopropionicum was inhibited by the addition of carbonyl cyanide m-chlorophenyl hydrazone, a proton uncoupler, which is consistent with the fact that stator protein, MotB of P. thermopropionicum, shared sequence homology with proton-type stators. In addition, 5-N-ethyl-N-isopropyl amiloride, an Na⁺ channel blocker, showed no inhibitory effect on the motility. Furthermore, motAB of P. thermopropionicum complemented the defective swimming ability of Escherichia coli ?motAB. These results suggest that the motility of P. thermopropionicum SI depends on the proton-type flagellar motor.     

Unidirectional, intermittent rotation of the flagellum of Rhodobacter sphaeroides.

The single flagellum of the photosynthetic bacterium Rhodobacter sphaeroides was found to be medially located on the cell body. Observation of free-swimming bacteria, and bacteria tethered by their flagellar filaments, revealed that the flagellum could only rotate in the clockwise direction; switching of the direction of rotation was never observed. Flagellar rotation stopped periodically, typically several times a minute for up to several seconds each. Reorientation of swimming cells appeared to be the result of Brownian rotation during the stop periods. The flagellar filament displayed polymorphism; detached and nonrotating filaments were usually seen as large-amplitude helices of such short wavelength that they appeared as flat coils or circles, whereas the filaments on swimming cells showed a normal (small-amplitude, long-wavelength) helical form. With attached filaments, the transition from the normal to the coiled form occurred when the flagellar motor stopped rotating, proceeding from the distal end towards the cell body. It is possible that both the relaxation process and the smaller frictional resistance after relaxation may act to enhance the rate of reorientation of the cell. The transition from the coiled to the normal form occurred when the motor restarted, proceeding from the proximal end outwards, which might further contribute to the reorientation of the cell before it reaches a stable swimming geometry.     

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s^-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s^-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.     

Analysis of gamete and zygote motility in Allomyces

To study the mechanisms of chemotaxis in eukaryotes, the motility patterns of the gametes and zygotes and the chemotactic responses of the male gametes of the lower eukaryote Allomyces macrogynus were examined. Dark-field microscopy of the male gametes showed a smooth swimming pattern interrupted by very brief ‘jerks’ of the cell body that caused a change in swimming direction. Female gametes had a slower swimming velocity than the males and underwent more jerks or turns which accounted for their sluggish motility. The zygotes swam with the fastest velocity and were observed to have a helical swimming pattern involving a continuous turning of the cell body, a behavior absent from the gametes. Introduction of female gametes that produce the chemoattractant sirenin brought about an immediate change in the behavior of the male gametes. They moved in spirals (or helices) towards the source of the chemoattractant (the female gametes), undergoing only a few jerks to reorient the male cells. When very near the female cells, or in high concentrations of added sirenin, many very short motility tracks were observed that finally resulted in contact between the two gamete types. The results indicate that the poor swimming ability of the female gametes facilitates gamete contact, resulting in as many as 30–40 male gametes clustered on a single female cell. Further, male gamete orientation to the sirenin gradient is caused by the chemoattractant suppressing the jerk motion.     

Mannose-Binding Lectin Inhibits the Motility of Pathogenic Salmonella by Affecting the Driving Forces of Motility and the Chemotactic Response

Mannose-binding lectin (MBL) is a key pattern recognition molecule in the lectin pathway of the complement system, an important component of innate immunity. MBL functions as an opsonin which enhances the sequential immune process such as phagocytosis. We here report an inhibitory effect of MBL on the motility of pathogenic bacteria, which occurs by affecting the energy source required for motility and the signaling pathway of chemotaxis. When Salmonella cells were treated with a physiological concentration of MBL, their motile fraction and free-swimming speed decreased. Rotation assays of a single flagellum showed that the flagellar rotation rate was significantly reduced by the addition of MBL. Measurements of the intracellular pH and membrane potential revealed that MBL affected a driving force for the Salmonella flagellum, the electrochemical potential difference of protons. We also found that MBL treatment increased the reversal frequency of Salmonella flagellar rotation, which interfered with the relative positive chemotaxis toward an attractive substrate. We thus propose that the motility inhibition effect of MBL may be secondarily involved in the attack against pathogens, potentially facilitating the primary role of MBL in the complement system.     

Domain structure of flagellin

The chemotaxis of bacteria such as Salmonella and Escherichia coli involves smooth swimming punctuated by periods of tumbling. In smooth swimming the flagellar filaments are left-handed, in tumbling they are right-handed with a different wavelength. The filaments are constructed from a globular protein, flagellin, by a process of self-assembly. The existing models assume that the flagellin molecule is bistable and longitudinal rows of subunits take one of the two possible conformations. Such a model explains the observed different morphology of the flagellum. We have studied Salmonella and E. coli flagellins in polymeric and monomeric forms by scanning microcalorimetry and circular dichroism. We have inferred that a flagellin molecule consists of several domains, two of which are structured at physiological temperatures and are in the monomeric form, while the others acquire a regular form only in the process of polymerization. This phenomenon may be the basis of a process during which the flagellin molecule, fitting into the flagellum, acquires a conformation analogous to that of the neighbouring molecule in the longitudinal row.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.     

Three-dimensional motion of avian spermatozoa

Observations have been made on spermatozoa from the domestic fowl, quail and pigeon (non-passerine birds) and also from the starling and zebra finch (passerine birds). In free motion, all these spermatozoa roll (spin) continuously about the progression axis, whether or not they are close to a plane surface. Furthermore, the direction of roll is consistently clockwise (as seen from ahead). The flagellar wave has been shown to be helical and dextral (as predicted) for domestic fowl sperm when they swim rapidly in low viscosity salines. Calculations have shown that their forward velocity is consistent with their induced angular velocity but that the size of the sperm head is suboptimal for progression speed under these conditions. Dextrally helical waves also occur on the distal flagellum of fowl, quail and pigeon sperm in high viscosity solutions. But in other cases, the mechanism of torque-generation is more problematical. The problem is most profound for passerine sperm, in that typically these cells spin rapidly while seeming to remain virtually straight. Because there is no evidence for a helical wave on these flagella, we have considered other possible means whereby rotation about the local flagellar axis (self-spin) might be achieved. Sometimes, passerine sperm, while maintaining their spinning motion, adopt a fixed curvature; this must be an instance of bend-transfer circumferentially around the axonemal cylinder-though the mechanism is obscure. It is suggested that the self-spin phenomenon may be occurring in non-passerine sperm that in some circumstances spin persistently, yet without expressing regular helical waves. More complex waves are apparent in non-passerine sperm swimming in high viscosity solutions: added to the small scale bends is a large scale, sinistrally helical curvature of the flagellum. It is argued that the flagellum follows this sinistrally helical path (i.e. "screws" though the fluid) because of the shape of the sperm head and the angle at which the flagellum is inserted into it. These conclusions concerning avian sperm motility are thought to have relevance to other animal groups. Also reported are relevant aspects of flagellar ultrastructure for pigeon and starling sperm.