Beat Direction Research Articles

The ultrastructure of the Chlamydomonas reinhardtii basal apparatus: identification of an early marker of radial asymmetry inherent in the basal body.

The biflagellate unicellular green alga Chlamydomonas reinhardtii is a classic model organism for the analysis of flagella and their organizers, the basal bodies. In this cell, the two flagella-bearing basal bodies, along with two probasal bodies and an array of fibers and microtubules, form a complex organelle called the basal apparatus. The ultrastructure of the basal apparatus was analysed in detail by serial thin-section electron microscopy of isolated cytoskeletons and several newly discovered features are described, including a marker for the rotational asymmetry inherent in the basal bodies and probasal bodies. In addition, the complex three-dimensional basal apparatus ultrastructure is resolved and illustrated, including the attachment sites of all basal apparatus elements to specific microtubular triplets of the basal bodies and probasal bodies. These data will facilitate both the localization of novel basal apparatus proteins and the analysis of mutants and RNA interference cells with only subtle defects in basal apparatus ultrastructure. The early harbinger of radial asymmetry described here could play a crucial role during basal body maturation by orienting the asymmetric attachment of the various associated fibers and therefore might define the orientation of the basal bodies and, ultimately, the direction of flagellar beating.

Time-frequency analysis of electronystagmogram signals in patients with congenital nystagmus.

To evaluate the usefulness of the short-time Fourier transform (STFT) in the analysis of temporal congenital nystagmus characteristics. We carried out an observational case series in patients with congenital nystagmus. An electronystagmogram (ENG) recording was performed for 300 s in 13 consecutive subjects with congenital nystagmus (age: 7-68 years). Digitized ENG signals (12 bits, 100 Hz) were divided into 2-s segments with a Hanning window, and a Fourier transform was carried out for each segment. The power spectrum obtained for each segment was graphically reconstructed as a function of time. The results were compared among the patients. Clear periodicity in the time-frequency distribution was observed in 3 of the 13 patients (23%). One of the three patients was diagnosed with periodic alternating nystagmus, but the other two patients showed periodicity without alternation of beat direction. No significant difference was observed in clinical characteristics, except for sex, between patients who showed periodicity and those who did not. Some patients with congenital nystagmus showed clear periodicity in the nystagmus waveform without alternation of beat direction. The STFT is a useful tool to evaluate the temporal characteristics of congenital nystagmus in clinics.

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.

Swimming behavior regulation by GABA B receptors in Paramecium

In Paramecium, internal Ca 2+ concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca 2+ influx, and the addition of drugs that block the Ca 2+ current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABA B receptors in P. primaurelia swimming control. The presence of GABA B immunoanalogue in Paramecium was evidenced using SDS-PAGE, Western blotting, and confocal laser scanning microscopy. By applying the specific GABA B receptor agonist baclofen, a dose-dependent inhibition of the membrane depolarization-induced CR duration was observed. This inhibition was antagonized by phaclofen, persisted when K + channel blockers were applied, and disappeared after treatment with nifedipine and verapamil. Moreover, the action of baclofen on depolarization-induced CR was suppressed by treatment with pertussis toxin. Therefore, these experiments suggest that baclofen modulates CR by a G protein (G 0 or G 1) mediated inhibition of dihydropyridine-sensible calcium channels. Finally, synthesis and release of GABA in the environment by Paramecium have been demonstrated by HPLC. Possible correlations between GABA B receptor activation and the regulation of intracellular Ca 2+ levels are discussed.

Photolysis of caged calcium in cilia induces ciliary reversal in Paramecium caudatum.

Intracellular Ca(2+) concentration controls both the pattern and frequency of ciliary and flagellar beating in eukaryotes. In Paramecium, it is widely accepted that the reversal of the direction of ciliary beating (ciliary reversal) is induced by an increase in intra-ciliary Ca(2+) levels. Despite this, the Ca(2+)-sensitive region of the cilium that initiates ciliary reversal has not been clearly identified. We injected caged calcium into living P. caudatum cells and applied ultraviolet (UV) light to portions of the injected cells to raise artificially the intracellular Ca(2+) level ([Ca(2+)](i)). UV application to the upper ciliary region above the basal body induced ciliary reversal in injected cells. Furthermore, UV application to the tips of cilia induced weak ciliary reversal. Larger areas of photolysis in the cilium gave rise to greater angles of ciliary reversal. These results strongly suggest that the Ca(2+)-sensitive region for ciliary reversal is distributed all over the cilium, above the basal body.

Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum.

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.

Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila.

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.

Spontaneous reversal of nystagmus in the dark

AIMTo report five children with horizontal jerk nystagmus in whom eye movement recordings in the dark revealed a spontaneous reversal in the direction of the nystagmus beat. Three patients were...

Bistable membrane potential of the ciliate Coleps hirtus.

In normal recording solution, the swimming pattern of the freshwater ciliate Coleps hirtus, belonging to the class Prostomatea, consists of alternating periods of nearly linear forward swimming and circular swimming within a small area. Current-clamp recordings were performed to elucidate the mechanism for this behaviour. No members of this class have previously been studied using electrophysiological techniques. The ciliates were maintained in culture and fed on the planctonic alga Rhodomonas minuta. The membrane potential showed spontaneous shifts between a more negative (deep) level of approximately -50 mV and a less negative (shallow) level of approximately -30 mV. The input resistance and capacitance at the more negative level were approximately 400 M capomega and 120 pF respectively. C. hirtus displayed a pronounced inward rectification, which was virtually insensitive to 1 mmol l(-1) Cs(+) and almost completely blocked by 1 mmol l(-1) Ba(2+). Depolarising current injections failed to evoke graded, regenerative Ca(2+) spikes. However, current-induced depolarisations from the more negative potential level (-50 mV) showed a pronounced shoulder during the repolarising phase. Increased current injections prolonged the shoulder, which occasionally stabilised at the shallow membrane potential (-30 mV). The membrane potential could be shifted to the deep level by brief hyperpolarising current injections. Similar biphasic membrane properties have not been reported previously in any ciliate. The bistability of the membrane potential was abolished in Ca(2+)-free solution containing Co(2+) or Mg(2+). In Ca(2+)-free solution containing 1 mmol l(-1) Ba(2+), brief depolarising current injections at the deep potential level evoked all-or-nothing action potentials with a prolonged plateau coinciding with the shallow potential. We conclude that the deep membrane potential in C. hirtus corresponds to the traditional resting potential, whereas the shallow level is a Ca(2+)-dependent plateau potential. In normal solution, the direction of the ciliary beat was backwards at the deep potential level and forwards at the shallow membrane potential, probably reflecting the two main phases of the swimming pattern.

Directional spectrum of ocean waves from array measurements using phase/time/path difference methods

Wave direction has for the first time been consistently, accurately and unambiguously evaluated from array measurements using the phase/time/path difference (PTPD) methods of Esteva in case of polygonal arrays and Borgman in case of linear arrays. We have used time series measurements of water surface elevation at a 15-gauge polygonal array, in ≅8 m water depth, operational at the CERC's Field Research Facility at Duck, North Carolina, USA. Two modifications have been made in the methodology. One modification is that we use the true phase instead of the apparent phase, the other modification being that estimates of wave direction are registered only if the relevant gauges in the array are coherent at 0.01 significance level. PTPD methods assume that in a spectral frequency band the waves approach from a single direction, and are simple, expedient and provide redundant estimates of wave direction. Using Esteva's method with the above modifications, we found that at Duck: (i) the directions of swell and surf beat, when energetic swell is present, conform to the schematic diagram of surf beat generation given by Herbers et al., (ii) surf beat of remote origin occurs when the significant wave height, H mo, falls below 0.41 m, (iii) the surf beat of remote origin is not normally incident at the shore contrary to Herbers et al. In fact we found that the surf beat of remote origin is incident at angles in excess of 45° with respect to the shore normal, and (iv) the surf beat of remote origin is largely trans-oceanic in origin.

A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video‐microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step‐up and step‐down stimuli. After a step‐up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step‐down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.

Long-term follow-up of the clinical relevance of short outer dynein arms in human nasal cilia.

Clinical significance of short outer dynein arms was examined in a long-term follow-up study of 76 patients with various respiratory symptoms. Clinical evaluations, nasal mucociliary transport rate (NMTR) measurement and transmission electron microscopy were performed. Follow-up examinations took place 5-11 years later. In the initial examination four patients and on follow-up seven patients were found to have short outer dynein arms in their nasal mucosal biopsies. Short dynein arms were associated with a slow NMTR, poorly coordinated ciliary beat direction, and clinical symptoms of perennial rhinitis and recurrent sinusitis. Short dynein arms were found to be a significant histologic finding and probably represent a variant of primary ciliary dyskinesia. In some cases short outer dynein arms may be a reversible finding with improving clinical symptoms. It is obvious that at least part of dynein defects are acquired.

Effect of Colcemid on Distribution and Beat Direction of Cilia from Ciliated Cell of Newt Lung

Effect of Colcemid on Distribution and Beat Direction of Cilia from Ciliated Cell of Newt Lung

Motility in the colonial and multicellular Volvocales: structure, function, and evolution

The colonial Volvocales are often said to be composed of Chlamydomonas-like cells, but there are substantial differences in motility and flagellar apparatus construction between the unicellular forms and the individual members of a colony or spheroid. These changes appear to be required for effective organismal motion and might possibly limit the rate at which new colonial forms evolve from unicellular ones. The flagellar-beat envelopes in colonial members are modified such that they beat in the same direction and in parallel planes with their effective strokes at right angles to the cellular anterior-posterior axis. These changes result from a series of developmental events of the flagellar apparatus of the colonial forms while the colony is still an embryo. Differences in the flagellar-apparatus structure in the members of the Goniaceae and Volvocaceae are not obviously correlated with the traditional placement of these algae in a simple volvocine lineage. Effective colonial motion clearly requires precise positioning and rotational orientation of the cells within the colony. Almost any arrangement where the cells are placed with rotational symmetry within the colony results in colonial progression with rotation. Such rotational symmetry is present from the time of embryogenesis. The mechanism that leads to organismal steering in behavioral responses (e.g., phototaxis) must likewise differ between colonial and unicellular forms. In at least some cases, this appears to result from changes in beat frequency in some parts of the spheroid, but changes in beat direction cannot be ruled out for all forms.

Electric control of ciliary beat direction and curvature of gliding in Loxodes (ciliata)

Summary The gliding locomotion of Loxodes on solid surfaces is commonly curved in the counterclockwise direction. Experiments using K+-dependent modulation of the membrane potential showed that depolarization induced gliding tracks of clockwise curvature, whereas hyperpolarization induced an increase of track curvature in the counterclockwise direction. Galvanotaxis of Loxodes in a linear DC-field is unorthodox in that cells become oriented parallel to the isopotentials with their oral sides facing the cathode. The experimental data were combined with a novel geometric model which predicts locomotion from differential ciliary activity. The model accounts for the body shape, distribution and density of ciliation, and beat direction of the cilia. Ciliary beat direction shifts in the counterclockwise direction (cilia seen tip-to-base) from posteriad to anteriad upon membrane depolarization. During hyperpolarization, beat direction is reoriented in the clockwise direction toward the posterior cell end. The characteristics of electromotor coupling with respect to the beat direction are in accordance with those which were previously documented in Paramecium.

Modification of ciliary beating in sea urchin larvae induced by neurotransmitters: beat-plane rotation and control of frequency fluctuation

The modification of ciliary beating by neurotransmitters in sea urchin larvae at the four-armed pluteus stage was analyzed in terms of the direction of beating and fluctuation in the beat period. Application of dopamine to Pseudocentrotus depressus causes the cilia to turn their beat plane but retain its characteristic planar feature up to the complete 'reversal' of the beat direction. This new type of response was termed the 'beat-plane turning response'. It was also found that neurotransmitters, especially dopamine and serotonin, can modify the length of the beating cycle in P. depressus and Hemicentrotus pulcherrimus. Dopamine decreased and serotonin increased the beat frequency averaged over the ciliated epithelium with the standard deviation from the mean increasing in the presence of dopamine and decreasing with serotonin. The beat-period fluctuation and its modification suggested by this observation was confirmed from measurements of the beating of individual cilia in the presence or absence of these neurotransmitters. Further analysis of the correlation between angular velocity and beat period indicates that variation in the beat period is not controlled by the same processes as those that modulate angular velocity. These findings in sea urchin larvae suggest that both the stability and the direction of ciliary beating is under nervous control.

Effect of Colcemid on Frequency and Direction of Ciliary Beat in Newt Lung

Effect of Colcemid on Frequency and Direction of Ciliary Beat in Newt Lung

Ciliary Currents of Non-Feeding Veligers in Putative Basal Clades of Gastropods

Ciliary Currents of Non-Feeding Veligers in Putative Basal Clades of Gastropods

Clinical Relevance of Tubulus Anomalies and Compound Cilia

Transmission electron microscopy (TEM) from nasal mucosa and nasal mucociliary transport rate (MTR) with a radio-isotopic method were examined in 144 patients with various respiratory symptoms. Examinations were not performed during acute infections. In cases of increased amount of tubulus anomalies: nasal MTR was not significantly slower than in other patients, the disorientation of ciliary beat direction was significantly larger, there was no connection with increased amount of compound cilia and there was no correlation to any specific symptom or respiratory disease. In cases of increased amount of compound cilia: nasal MTR did not differ from other patients and the disorientation of ciliary beat direction did not differ from other patients. In a follow- up study of 76 patients 5–11 years (mean 9.3 years) later the amount of tubulus anomalies and compound cilia had changed in most patients. In patients with less symptoms at the follow-up than primarily, there was a non-significant tendency to have less tubulus anomalies, too, but no change in the amount of compound cilia. The amount of tubulus anomalies and compound cilia is not stable. Their number can either increase or decrease during a follow-up. Tubulus anomalies are non- specific and probably secondary changes of minor clinical importance and compound cilia would seem to be quite irrelevant findings in the ultrastructural evaluation of respiratory cilia.

Scanning electron microscopy of the equine oviduct and observations on ciliary currents in vitro at day 2 after ovulation

There are considerable differences between mammalian species in the distribution and activity of ciliated cells within the oviduct, and limited information is available concerning either the distribution or activity of cilia within the equine oviduct. Patterns of ciliary activity were characterized in the ampulla and isthmus of oviducts recovered at 2 d after ovulation from 10 mares, and scanning electron microscopy was used to examine regional differences in the distribution of cilia in oviducts from 3 of these mares. Based upon the motility of 15-μm latex microspheres, ciliary activity was significantly (P < 0.001) greater in the ampullar oviduct compared with that of the isthmic oviduct. The direction of ciliary beat was consistently toward the uterus in all regions of the oviduct. Scanning electron microscopy revealed ciliated and secretory cells in both regions of the oviduct at 2 d after ovulation, with no apparent differences in the proportion of ciliated versus secretory cells.