Effect of Extracellular pH on Motility and K+-Induced Ciliary Reversal in Paramecium caudatum1

ABSTRACT. Paramecium caudatum, reared on bacterized hay infusions at pH 6.5 to 6.9, were washed into various buffered solutions containing 0.016 mM CaCl₂ and a pH of 3.5 to 10.4. Solutions of pH 4.5 to 9.5 support strong swimming of the cells for at least 24 h. At pH values acid to the culture medium, cells show an increasing frequency of spontaneous ciliary reversal episodes (“avoiding reactions”). Uninterrupted forward swimming is usually observed over the pH range of 7.1 to 8.5, and above pH 8.5, forward motion is interrupted by circular swimming. For all pH values tested, transfer of cells to a more acidic test solution than the solutions into which they were washed (adaptation solution) usually induced short duration, periodic ciliary reversal behavior. With transfer to a more alkaline test solution than the adaptation solution, the cells shift from forward left spiralling motion to forward right spiralling motion. With decreasing pH, the cells show progressively less sensitivity to KC1 stimulation, and at pH values of less than 5.0, cells fail to show significant ciliary reversal response to any KC1 concentration tested (1 - 128 mM). At alkaline pH values and higher KC1 concentrations, the cells show very pronounced ciliary reversal behaviors but usually fail to regain forward swimming behavior.     

How cellular movement determines the collective force generated by the Dictyostelium discoideum slug

How the collective motion of cells in a biological tissue originates in the behavior of a collection of individuals, each of which responds to the chemical and mechanical signals it receives from neighbors, is still poorly understood. Here we study this question for a particular system, the slug stage of the cellular slime mold Dictyostelium discoideum (Dd). We investigate how cells in the interior of a migrating slug can effectively transmit stress to the substrate and thereby contribute to the overall motive force. Theoretical analysis suggests necessary conditions on the behavior of individual cells, and computational results shed light on experimental results concerning the total force exerted by a migrating slug. The model predicts that only cells in contact with the substrate contribute to the translational motion of the slug. Since the model is not based specifically on the mechanical properties of Dd cells, the results suggest that this behavior will be found in many developing systems.     

Expression of Na,K-ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells

We have analyzed the expression of Na,K-ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non-pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na,K-ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na,K-ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na,K-ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na,K-ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A x 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na,K-ATPase alpha subunit isoforms as compared to cultured cells.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Peter Satir -- investigating the structural basis for cell function

Peter Satir has devoted his research career to elucidating the structural basis for ciliary motility. His ingenious use of structural analysis, combined with identification of powerful model systems, provided a model for the sliding microtubule hypothesis of ciliary bending and led to the discovery that dynein is a 'minus-end'-directed motor whose regulated activity underpins the bending motion of cilia. Here, we focus on ciliary motility to illustrate Satir's pioneering contributions to cell biology.     

Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross- reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i- antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.     

Ciliary motion modeling, and dynamic multicilia interactions

This paper presents a rigorous and accurate modeling tool for ciliary motion. The hydrodynamics analysis, originally suggested by Lighthill (1975), has been modified to remove computational problems. This approach is incorporated into a moment-balance model of ciliary motion in place of the previously used hydrodynamic analyses, known as Resistive Force Theory. The method is also developed to include the effect of a plane surface at the base of the cilium, and the effect of the flow fields produced by neighboring cilia. These extensions were not possible with previous work using the Resistive Force Theory hydrodynamics. Performing reliable simulations of a single cilium as well as modeling multicilia interactions is now possible. The result is a general method which could now be used for detailed modeling of the mechanisms for generating ciliary beat patterns and patterns of metachronal interactions in arrays of cilia. A computer animation technique was designed and applied to display the results.     

Contractile events in the cilia of Paramecium, Opalina, Mytilus, and Phragmatopoma.

The motion of the abnormal cilia of Opalina and Mytilus can be described by the recently developed model for ciliary motion, provided the activation of the contractility during the effective stroke is reduced by three- to fivefold compared with that in the recovery stroke. The stiffness of the Mytilus cilium during the effective stroke is found several hundred times larger than that predicted by the model, however. The stiffness of the cilia of Paramecium, Opalina, Phragmatopoma, and of Mytilus in the recovery phase, is predicted approximately correctly by the model. The activation of contractility in Mytilus and Phragmatopoma cilia increases with the viscosity of the medium, as the velocity of the ciliary motion slows down. This leads to the equivalent of a force-velocity relation. The velocity of propagation of the bend in the cilia during the recovery stroke is shown to be dependent only on the elastic properties of the ciliary shaft, and to be independent of the contractile activiey.     

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. Effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

How Does Cilium Length Affect Beating?

The effects of cilium length on the dynamics of cilia motion were investigated by high-speed video microscopy of uniciliated mutants of the swimming alga, Chlamydomonas reinhardtii. Cells with short cilia were obtained by deciliating cells via pH shock and allowing cilia to reassemble for limited times. The frequency of cilia beating was estimated from the motion of the cell body and of the cilium. Key features of the ciliary waveform were quantified from polynomial curves fitted to the cilium in each image frame. Most notably, periodic beating did not emerge until the cilium reached a critical length between 2 and 4 μm. Surprisingly, in cells that exhibited periodic beating, the frequency of beating was similar for all lengths with only a slight decrease in frequency as length increased from 4 μm to the normal length of 10–12 μm. The waveform average curvature (rad/μm) was also conserved as the cilium grew. The mechanical metrics of ciliary propulsion (force, torque, and power) all increased in proportion to length. The mechanical efficiency of beating appeared to be maximal at the normal wild-type length of 10–12 μm. These quantitative features of ciliary behavior illuminate the biophysics of cilia motion and, in future studies, may help distinguish competing hypotheses of the underlying mechanism of oscillation.     

'Dicty dynamics': Dictyostelium motility as persistent random motion

We model the motility of Dictyostelium cells in a systematic data-driven manner. We deduce a minimal dynamical model that reproduces the statistical features of experimental trajectories. These are trajectories of the centroid of the cell perimeter, which is more sensitive to pseudopod activity than the usual tracking by centroid or nucleus. Our data account for cell individuality and dictate a model that extends the cell-type specific models recently derived for mammalian cells. Two generalized Langevin equations model stochastic periodic pseudopod motion parallel and orthogonal to the amoeba's direction of motion. This motion propels the amoeba with a random periodic left-right waddle in a direction that has a long persistence time. The model fully accounts for the statistics of the experimental trajectories, including velocity power spectra and auto-correlations, non-Gaussian velocity distributions, and multiplicative noise. Thus, we find neither need nor place in our data for an interpretation in terms of anomalous diffusion. The model faithfully captures cell individuality as different parameter values in the model, and serves as a basis for integrating the local mechanics of cell motion with our observed long-term behavior.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Distinct protein domains regulate ciliary targeting and function of C. elegans PKD-2

TRPP2 (transient receptor potential polycystin-2) channels function in a range of cells where they are localized to specific subcellular regions including the endoplasmic reticulum (ER) and primary cilium. In humans, TRPP2/PC-2 mutations severely compromise kidney function and cause autosomal dominant polycystic kidney disease (ADPKD). The Caenorhabditis elegans TRPP2 homolog, PKD-2, is restricted to the somatodendritic (cell body and dendrite) and ciliary compartments of male specific sensory neurons. Within these neurons PKD-2 function is required for sensation. To understand the mechanisms regulating TRPP2 subcellular distribution and activity, we performed in vivo structure-function-localization studies using C. elegans as a model system. Our data demonstrate that somatodendritic and ciliary targeting requires the transmembrane (TM) region of PKD-2 and that the PKD-2 cytosolic termini regulate subcellular distribution and function. Within neuronal cell bodies, PKD-2 colocalizes with the OSM-9 TRP vanilloid (TRPV) channel, suggesting that these TRPP and TRPV channels may function in a common process. When human TRPP2/PC-2 is heterologously expressed in transgenic C. elegans animals, PC-2 does not visibly localize to cilia but does partially rescue pkd-2 null mutant defects, suggesting that human PC-2 and PKD-2 are functional homologs.     

A Computational Model of Dynein Activation Patterns that Can Explain Nodal Cilia Rotation

Normal left-right patterning in vertebrates depends on the rotational movement of nodal cilia. In order to produce this ciliary motion, the activity of axonemal dyneins must be tightly regulated in a temporal and spatial manner; the specific activation pattern of the dynein motors in the nodal cilia has not been reported. Contemporary imaging techniques cannot directly assess dynein activity in a living cilium. In this study, we establish a three-dimensional model to mimic the ciliary ultrastructure and assume that the activation of dynein proteins is related to the interdoublet distance. By employing finite-element analysis and grid deformation techniques, we simulate the mechanical function of dyneins by pairs of point loads, investigate the time-variant interdoublet distance, and simulate the dynein-triggered ciliary motion. The computational results indicate that, to produce the rotational movement of nodal cilia, the dynein activity is transferred clockwise (looking from the tip) between the nine doublet microtubules, and along each microtubule, the dynein activation should occur faster at the basal region and slower when it is close to the ciliary tip. Moreover, the time cost by all the dyneins along one microtubule to be activated can be used to deduce the dynein activation pattern; it implies that, as an alternative method, measuring this time can indirectly reveal the dynein activity. The proposed protein-structure model can simulate the ciliary motion triggered by various dynein activation patterns explicitly and may contribute to furthering the studies on axonemal dynein activity.     

Biophysical effects of the natural product euplotin C on the <Emphasis Type="Italic">Paramecium</Emphasis> membrane

The effect of euplotin C—a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus—on the voltage-dependent Ca²⁺ channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca²⁺ concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca²⁺ influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca²⁺ channel blockers, we demonstrate that euplotin C modulates Ca²⁺ channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca²⁺ channel functions through the reduction of cell membrane fluidity.     

Modelling of primary ciliary dyskinesia using patient‐derived airway organoids

Patient‐derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long‐term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient‐specific differences in ciliary beating are observed and are in agreement with the patients'' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient‐specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.     

Nematode Chemosensilla: Form and Function

As an introduction to a symposium of nematode chemoreception, the anatomy of nematode chemosensilla, their distribution on plant parasitic nematodes, and their possible functional roles is briefly reviewed. Comparison of nematode chemosensilla with those of other animals shows their greater resemblance to olfactory primary sense cells of vertebrates. Although the sensory process is obviously derived from a cilium, the absence of many ciliary features is noted. Retention of the ciliary necklace may be important functionally. A simple model is proposed, wherein binding of stimulant molecules to receptors in the membrane of the cilium-derived process results in entry of Na⁺ and Ca⁺⁺ (the latter via the ciliary necklace) to produce a receptor potential that spreads along the dendrite to the cell body where action potentials continue along the short axon to synapses.     

Growth factors induce neurogenesis in the ciliary body

The ciliary body of the eye is a nonneural tissue that is derived from the anterior rim of the optic cup, an extension of the neural tube. This tissue normally does not contain neurons and functions to produce components of the aqueous humor. We found that intraocular injections of insulin, EGF, or FGF2 stimulate NPE cells to proliferate and differentiate into neurons. These growth factors had region-specific effects along the radial axis of the ciliary body, with insulin and EGF stimulating proliferation of NPE cells close to the retina, while FGF2 stimulated the proliferation of NPE cells further toward the lens. Similar region-specific effects were observed for accumulations of neurons in the NPE in response to injections of different growth factors. The neurons derived from NPE cells express neurofilament, beta3 tubulin, RA4, calretinin, Islet1, or Hu, and a few produced long axonal projections, several millimeters in length that extend across the ciliary body. Our results suggest that the ciliary body has the capacity to generate retinal neurons, but normally neurogenesis is actively inhibited.