THE LOCOMOTOR BEHAVIOR OF LYGODIUM AND MARSILEA SPERM

Flagella of living sperm of the ferns, Lygodium japonicum (Thunb.) Sw. and Marsilea vestita Hook, and Grev., beat three dimensionally with a continuous traveling helical wave. The wave is propagated from base to tip of the flagellum. Flagella of Lygodium and Marsilea complete 65 and 30 beat cycles per sec, respectively. Each flagellum circumscribes an open conicoid oriented in a latero-posterior direction. Only dead sperm have anteriorly directed flagella as illustrated in plant morphology textbooks.     

Use of a Novel Cell Adhesion Method and Digital Measurement to Show Stimulus‐dependent Variation in Somatic and Oral Ciliary Beat Frequency in Paramecium

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid‐body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.     

THE RELEASE MECHANISM AND LOCOMOTOR BEHAVIOR OF EQUISETUM SPERM

When antheridia of gametophytes of Equisetum hyemale L. are placed in water, only spermatid cells are released. These spermatid cells have from 6–12 fibrils radiating from 2–4 loci on the cell wall. One sperm is released from each spermatid cell. With high speed cinemicrography, it can be shown that sperm flagella beat three dimensionally with a continuous, traveling helical wave. A flagellar beat cycle is completed every 0.03 sec. In water the sperm swim rapidly at a rate of 300 μ per sec and traverse a helical path of long wavelength and shallow amplitude. Calcium is essential for normal locomotor behavior. When calcium is chelated by ethylenediaminetetraacetate (EDTA), the sperm spin in place. All other monovalent and divalent ionic solutions tested cause the sperm to swim abnormally. Monovalent ionic solutions prevent the drastic change in sperm mobility patterns resulting from chelation by EDTA.     

Intraflagellar Transport 80 Is Required for Cilia Construction and Maintenance in Paramecium tetraurelia

Intraflagellar transport (IFT) represents a bidirectional dynamic process that carries cargo essential for cilia building and the maintenance of ciliary function, which is important for the locomotion of single cells, intracellular and intercellular signalling transduction. Accumulated evidence has revealed that defects in IFT cause several clinical disorders. Here, we determined the role of IFT80, an IFT‐B protein that is mutated in Jeune asphyxiating thoracic dystrophy. Using the RNAi method in the ciliate Paramecium as model, we found that loss of IFT80 prevents cilia biogenesis and causes strong cell lethality. A specific antibody against IFT80 was also prepared in our study, which labelled IFT80 in cilia of Paramecium. GFP fusion experiments were performed to illustrate the dynamic movement of IFT‐A and IFT‐B proteins in cilia of Paramecium; then, we found that the depletion of IFT80 in cells prevents IFT‐A and IFT‐B proteins from entering the cilia. Our results showed the distribution change of other IFT proteins in cells that were depleted of IFT80, and we discuss the possible roles of IFT80 in Paramecium.     

Estimation of effective concentrations of ATP-regenerating enzymes in cilia of Paramecium caudatum

The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP‐regenerating enzymes, we attempted to reconstitute certain ATP‐regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high‐energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 mg/ml creatine kinase and 0.4 mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP‐regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 μM based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 μM. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.     

Coordinated Flagellar and Ciliary Beating in the Protozoon Tritrichomonas foetus1

ABSTRACT. Tritrichomonas foetus is a flagellated protozoon found in urogenital tract of cattle. Its free movement in liquid medium is powered by the coordinated movement of three flagella projecting towards the anterior region of the cell, and one recurrent flagellum that forms a junction with the cell body and ends as a free projection in the posterior region of the cell. We have used video microscopy and digital image processing to analyze the relationships between the movements of these flagella. The anterior flagella beat in a ciliary type pattern displaying effective and recovery strokes, while the recurrent flagellum beats in a typical flagellar wave form. One of the three anterior flagella has a distinctive pattern of beating. It beats straight in its forward direction as opposed to the ample beats performed by the others. Frequency measurements obtained from cells swimming in a viscous medium shows that the beating frequency of the recurrent flagelium is approximate twice the frequency for the three anterior flagella. We also observed that the costa and the axostyle do not show any active motion. On the contrary, they form a cytoskeletal base for the anchoring and orientation of the flagella.     

Evidence for Two Extremes of Ciliary Motor Response in a Single Swimming Microorganism

Because arrays of motile cilia drive fluids for a range of processes, the versatile mechano-chemical mechanism coordinating them has been under scrutiny. The protist Paramecium presents opportunities to compare how groups of cilia perform two distinct functions, swimming propulsion and nutrient uptake. We present how the body cilia responsible for propulsion and the oral-groove cilia responsible for nutrient uptake respond to changes in their mechanical environment accomplished by varying the fluid viscosity over a factor of 7. Analysis with a phenomenological model of trajectories of swimmers made neutrally buoyant with magnetic forces combined with high-speed imaging of ciliary beating reveal that the body cilia exert a nearly constant propulsive force primarily by reducing their beat frequency as viscosity increases. By contrast, the oral-groove cilia beat at a nearly constant frequency. The existence of two extremes of motor response in a unicellular organism prompts unique investigations of factors controlling ciliary beating.     

Evidence for Two Extremes of Ciliary Motor Response in a Single Swimming Microorganism

Because arrays of motile cilia drive fluids for a range of processes, the versatile mechano-chemical mechanism coordinating them has been under scrutiny. The protist Paramecium presents opportunities to compare how groups of cilia perform two distinct functions, swimming propulsion and nutrient uptake. We present how the body cilia responsible for propulsion and the oral-groove cilia responsible for nutrient uptake respond to changes in their mechanical environment accomplished by varying the fluid viscosity over a factor of 7. Analysis with a phenomenological model of trajectories of swimmers made neutrally buoyant with magnetic forces combined with high-speed imaging of ciliary beating reveal that the body cilia exert a nearly constant propulsive force primarily by reducing their beat frequency as viscosity increases. By contrast, the oral-groove cilia beat at a nearly constant frequency. The existence of two extremes of motor response in a unicellular organism prompts unique investigations of factors controlling ciliary beating.     

Roles of Adenylate Cyclases in Ciliary Responses of Paramecium to Mechanical Stimulation

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K⁺‐outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K⁺ channels in the membrane. Using gene‐specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus‐induced hyperpolarization‐coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)‐gene and 2 (ac2)‐gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8‐Br‐cAMP and dilution of extracellular [K⁺] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization‐activated inward K⁺ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus‐induced acceleration of ciliary beat in Paramecium.     

High-speed video observation of swimming behavior and flagellar motility ofProrocentrum minimum (Dinophyceae)

Summary To understand the functions of the longitudinal and transverse flagella of dinoflagellates, the flagellar waveform and frequency of each flagellum were observed by high-speed video-recording. The longitudinal flagellum emerged from the anterior end of the cell and beat with a planar undulating wave whose plane was perpendicular to the valval sutural plane. The transverse flagellum curved around the anterior end of the cell and beat with a helical wave, with different alternating half pitches. The half pitch corresponding to the parts farther from the cellular antero-posterior axis was shorter than that of the parts closer to the axis. This pattern is described by the ratio of the outer-parts half pitch to the pitch of the whole period of the helix and seems to be characteristic of the dinoflagellates' transverse flagellum.Abbreviations p _in half pitch corresponding to the inner parts of the transverse flagellum - p _out half pitch corresponding to the outer parts of the transverse flagellum - P _p pitch of helical swimming trajectory - R _p radius of helical swimming trajectory - _c rotational frequency of the cell     

Ciliary sieving and active ciliary response in capture of particles by suspension-feeding brachiopod larvae

Larvae of a brachiopod, Glottidia pyramidata, used at least two ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding/swimming current. (1) Filtration: the larvae retained algal cells on the upstream (frontal) side of a sieve composed of a row of stationary laterofrontal cilia. Movement of the laterofrontal cilia could not be observed during capture or rejection of particles, but the laterofrontal cilia can bend toward the beating lateral cilia, a possible mechanism for releasing rejected particles from the ciliary sieve. (2) Localized changes of ciliary beat: the larvae may also concentrate particles by a local change in beat of lateral cilia in response to particles. The evidence is that the beat of lateral cilia changed coincident with captures of algal cells and that captured particles moved on paths consistent with a current redirected toward the frontal side of the tentacle by an induced local reversal of the lateral cilia. The change of beat of lateral cilia could have been an arrest rather than a reversal of ciliary beat, however. The similar ciliary bands in adult and larval lophophorates (brachiopods, phoronids, and bryozoans) suggest that these animals share a range of ciliary behaviours. The divergent accounts of ciliary feeding of lophophorates could be mostly the result of different authors observing different aspects of ciliary feeding.     

Computation of the internal forces in cilia: application to ciliary motion, the effects of viscosity, and cilia interactions.

This paper presents a simple and reasonable method for generating a phenomenological model of the internal mechanism of cilia. The model uses a relatively small number of parameters whose values can be obtained by fitting to ciliary beat shapes. Here, we use beat patterns observed in Paramecium. The forces that generate these beats are computed and fit to a simple functional form called the "engine." This engine is incorporated into a recently developed hydrodynamic model that accounts for interactions between neighboring cilia and between the cilia and the surface from which they emerge. The model results are compared to data on ciliary beat patterns of Paramecium obtained under conditions where the beats are two-dimensional. Many essential features of the motion, including several properties that are not built in explicitly, are shown to be captured. In particular, the model displays a realistic change in beat pattern and frequency in response to increased viscosity and to the presence of neighboring cilia in configurations such as rows of cilia and two-dimensional arrays of cilia. We found that when two adjacent model cilia start beating at different phases they become synchronized within several beat periods, as observed in experiments where two flagella are brought into close proximity. Furthermore, examination of various multiciliary configurations shows that an approximately antiplectic wave pattern evolves autonomously. This modeling evidence supports earlier conjectures that metachronism may occur, at least partially, as a self-organized phenomenon due to hydrodynamic interactions between neighboring cilia.     

Relations Between Photobehavior and Ionically Stimulated Swimming Behavior in Paramecium bursaria1

ABSTRACT. Cells of Paramecium bursaria, which harbor a symbiotic alga of the genus Chlorella, reverse the effective beat of their cilia and swim backward when stimulated in either of two ways. Ionic stimulation is introduction of cells into a solution high in K⁺ while step-down photostimulation is a sharp reduction in the intensity of light falling on the culture. Much is known about the mechanisms of ionic stimulation of ciliary reversal, but little is known about step-down photobehavior. Inhibitors of ionically stimulated ciliary reversal were applied to cells undergoing step-down photobehavior; Ca-channel inhibitors, neomycin and W-7, inhibit both behaviors. Activation of Ca-channels in the ciliary membrane is involved in step-down photobehavior, suggesting that the algae may alter the Paramecium membrane to make it more excitable.     

Evolutionary implications of localization of the signaling scaffold protein Parafusin to both cilia and the nucleus

Parafusin (PFUS), a 63 kDa protein first discovered in the eukaryote Paramecium and known for its role in apicomplexan exocytosis, provides a model for the common origin of cellular systems employing scaffold proteins for targeting and signaling. PFUS is closely related to eubacterial rather than archeal phosphoglucomutases (PGM) – as we proved by comparison of their 88 sequences – but has no PGM activity. Immunofluorescence microscopy analysis with a PFUS‐specific peptide antibody showed presence of this protein around the base region of primary cilia in a variety of mammalian cell types, including mouse embryonic (MEFs) and human foreskin fibroblasts (hFFs), human carcinoma stem cells (NT‐2 cells), and human retinal pigment epithelial (RPE) cells. Further, PFUS localized to the nucleus of fibroblasts, and prominently to nucleoli of MEFs. Localization studies were confirmed by Western blot analysis, showing that the PFUS antibody specifically recognizes a single protein of ca. 63 kDa in both cytoplasmic and nuclear fractions. Finally, immunofluorescence microscopy analysis showed that PFUS localized to nuclei and cilia in Paramecium. These results support the suggestion that PFUS plays a role in signaling between nucleus and cilia, and that the cilium and the nucleus both evolved around the time of eukaryotic emergence. We hypothesize that near the beginnings of eukaryotic cell evolution, scaffold proteins such as PFUS arose as peripheral membrane protein identifiers for cytoplasmic membrane trafficking and were employed similarly during the subsequent evolution of exocytic, nuclear transport, and ciliogenic mechanisms.     

FLAGELLAR MOTION AND FINE STRUCTURE OF THE FLAGELLAR APPARATUS IN CHLAMYDOMONAS

The biflagellate alga Chlamydomonas reinhardi was studied with the light and electron microscopes to determine the behavior of flagella in the living cell and the structure of the basal apparatus of the flagella. During normal forward swimming the flagella beat synchronously in the same plane, as in the human swimmer's breast stroke. The form of beat is like that of cilia. Occasionally cells swim backward with the flagella undulating and trailing the cell. Thus the same flagellar apparatus produces two types of motion. The central pair of fibers of both flagella appear to lie in the same plane, which coincides with the plane of beat. The two basal bodies lie in a V configuration and are joined at the top by a striated fiber and at the bottom by two smaller fibers. From the area between the basal bodies four bands of microtubules, each containing four tubules, radiate in an X-shaped pattern, diverge, and pass under the cell membrane. Details of the complex arrangement of tubules near the basal bodies are described. It seems probable that the connecting fibers and the microtubules play structural roles and thereby maintain the alignment of the flagellar apparatus. The relation of striated fibers and microtubules to cilia and flagella is reviewed, particularly in phytoflagellates and protozoa. Structures observed in the transitional region between the basal body and flagellar shaft are described and their occurrence is reviewed. Details of structure of the flagellar shaft and flagellar tip are described, and the latter is reviewed in detail.     

THE METACHRONAL WAVE OF LATERAL CILIA OF MYTILUS EDULIS

The form of beat of cilia and the structure of the metachronal wave on the lateral gill epithelium of Mytulus edulis have been studied on living material by interference-contrast microscopy and stroboscopic illumination, and compared with the same features in rapid-fixed preparations studied by light microscopy and with the scanning electron microscope. The most striking finding is that the beat of the cilia is not planar, as previously assumed, but involves a sideways movement in the recovery stroke Previous reports on nonplanar ciliary beating from protozoan examples describe a planar effective stroke and a counterclockwise rotation in the recovery stroke; in this molluscan example there is a clockwise rotation in the recovery stroke The lateral inclination of the cilia in the recovery stroke is in the same direction as the propagation of the waves, and the orientation of cilia in the recovery stroke is thought to determine whether the waves move to the left or right of the direction of the effective stroke     

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a "high-load environment," we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.     

Passive Electrical Properties of Paramecium and Problems of Ciliary Coordination

Potential recordings made simultaneously from opposite ends of the cell indicate that the cytoplasmic compartment of P. caudatum is nearly isopotential. Measured decrements of the spread of steady-state potentials are in essential agreement with calculated decrements for a short cable model of similar dimensions and electrical constants. Action potentials and passively conducted pulses spread at rates of over 100 µm per msec. In contrast, metachronal waves of ciliary beat progress over the cell with velocities below 1 µm per msec. Thus, electrical activity conducted by the plasma membrane cannot account for the metachronism of ciliary beat. The electrical properties of Paramecium are responsible, however, for coordinating the reorientation of cilia (either beating or paralyzed by NiCl₂) which occurs over the entire cell in response to current passed across the plasma membrane. In response to a depolarization the cilia assume an anteriorly directed orientation ("ciliary reversal" for backward locomotion). The cilia over the anterior half of the organism reverse more strongly and with shorter latency than the cilia of the posterior half. This was true regardless of the location of the polarizing electrode. Since the membrane potential was shown to be essentially uniform between both ends of the cell, the cilia of the anterior and posterior must possess different sensitivities to membrane potential.     

FLAGELLAR APPARATUS,EYESPOT AND BEHAVIOR OF MICROTHAMNION KUETZINGIANUM (CHLOROPHYCEAE) ZOOSPORES1,2,3

The flagellar apparatus of Microthamnion kuet-zingianum Naegeli differs from, that of Chlamydomonas reinhardtii Dangeard in that the zoospores can autonomously orient their basal bodies for different types of swimming behavior, including forward, and backward progression with, stationary intervals. Reorientation of the basal regions of the flagella and of the basal bodies were documented by cinefilms and by stroboscopic and electron micrographs. Even when the flagella. were sheared off, the remaining stubs (containing the basal bodies) were capable of being reoriented, by the organism. Thus the mechanism of basal body reorientation cannot reside in the 9 + 2 flagellar shaft. Rather, the reorienting process involves a shortening or lengthening of the distal fiber and of the plasma membrane region overlying an anterior papilla. In their helical and spiral motions, the zoospores trace complicated, but surprisingly regular curves. Such motion might result from the inherent 3-dimensional structure and beat of the flagella. The eyespot has an invariable, highly asymmetric location within the cell in direct proximity with a specific microtubular band (MT_E), but nevertheless may occur in either the anterior or posterior region of the chloroplast. Further, multiple eyespots may occur along the same side of MT_E. This observation is consistent with the discovery (in Fucus sperm) that microtubules serve to align individual eyespot granules in eyespot-ontogeny. By this means the position of the eyespot within a cell could well be determined.     

Growth of Paramecium tetraurelia in Bacterized,Monoxenic Cultures1

Wild type and mutant Paramecium tetraurelia were grown in monoxenic cultures by first growing Enterobacter aerogenes on a defined medium and then adding the Paramecium to the stationary phase bacterial culture. The bacterial growth was proportional to the concentration of the carbon source (citrate), and the Paramecium growth was dependent upon both the bacterial density and the starting density of Paramecium. The behavior, electrophysiological properties, ciliary lipid composition, and growth characteristics were similar to the commonly used bacterized medium (Cerophyl) except that 5–10 times greater Paramecium yields were reliably obtained.