Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement

Phagosome fusion vesicles (PFVs), a new population of relatively large granules in Paramecium caudatum which fuse with the first stage of digestive vacuoles (DV-I) shortly after these vacuoles are released from the cytopharynx (their site of formation), have been studied by using the freeze-fracture technique. Identification of PFVs is possible in the resulting replicas at all sites where they are commonly found in thin sections, at the cytopharynx, bound but not fused with nascent digestive vacuoles and fused with released vacuoles in the cell's posterior end. These PFVs have membranes which do not resemble the membranes of the forming digestive vacuole membrane or the discoidal vesicle membranes from which vacuole membrane is derived. Their smooth E-fracture face with only 50 to 100 intramembrane particles (IMPs) per micrometers 2 and particulate P-face (approximately 2500 IMPs/micrometers) do resemble the second vacuole stage (DV-II) which is characterized by a smaller diameter and acid pH. Evidence is presented for PFV fusion with the DV-I and for membrane replacement, at least in part, as the DV-I becomes a DV-II. Membrane replacement entails first adding PFVs to the DV-I and then removing the original discoidal vesicle-derived membrane as tubules as the vacuole condenses. Implications of the possible role of PFVs in forming intravacuolar symbiotic relationships are also discussed.     

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.     

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.     

CILIARY MEMBRANE DIFFERENTIATIONS IN TETRAHYMENA PYRIFORMIS : Tetrahymena Has Four Types of Cilia

We have examined thin sections and replicas of freeze-fractured cilia of Tetrahymena pyriformis. The ciliary necklace located at the base of all freeze-fractured oral and somatic cilia has been studied in thin sections. Since electron-dense linkers have been found to connect both microtubule doublets and triplets to the ciliary membrane at the level of the necklace, the linkers and the associated necklace seem to be related to the transition region between the doublets and triplets of a cilium. Plaque structures, consisting of small rectangular patches of particles located distal to the ciliary necklace, are found in strain GL, but are absent in other strains examined in this study. In freeze-cleaved material, additional structural differentiations are observed in the distal region of the ciliary membranes of somatic and oral cilia. Somatic cilia contain many randomly distributed particles within their membrane. Oral cilia can be divided into three categories on the basis of the morphology of their freeze-fractured membranes: (a) undifferentiated cilia with very few randomly distributed particles: (b) cilia with particles arranged in parallel longitudinal rows spaced at intervals of 810–1080 Å that are located on one side of the cilium; and (c) cilia with patches of particles arranged in short rows oriented obliquely to the main axis of the cilium. The latter particles, found on one side of the cilium, seem to serve as attachment sites for bristles 375–750 Å long and 100 Å wide which extend into the surrounding medium. The particles with bristles are located at the tips of cilia in the outermost membranelle and may be used to detect food particles and/or to modify currents in the oral region so that food particles are propelled more efficiently into the buccal cavity. Examination of thin-sectioned material indicates that the particles in oral cilia which form the longitudinal rows could be linked to microtubule doublets. Linkage between microtubule doublets and adjacent membrane areas on one side of the cilium could modify the form of ciliary beat by restricting the sliding of the microtubules. It is suggested that membrane-microtubule interactions may form the basis for the various forms of ciliary beat observed in different organisms.     

Ciliary beat co-ordination by calcium

Motile cilia in the airway epithelium are the engine for mucociliary clearance, the mechanism responsible for cleaning the airways from inhaled particles. Human airway epithelial cilia appear to have a slow constitutive rate of beating, driven by inherent and spontaneous dynein ATPase activity. Additionally, cilia can increase their beating frequency by activation of several different control mechanisms. One of these controllers is calcium. Its intracellular concentration is regulated by purinergic and acetylcholine receptors. Besides the rate regulatory effect of calcium on ciliary beat, calcium is also involved in synchronizing the beat among cilia of one single cell as well as between cilia on different cells. This article gives an overview of the complex effects of calcium on the beating of motile cilia in the airways.     

Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium

An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation.     

Versatile ciliary behaviour in capture of particles by the bryozoan cyphonautes larva

Cyphonautes larvae of a bryozoan, Membranipora membranacea, used several ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding current. (1) Lateral cilia changed beat and a backcurrent occurred at the time and place that particles were retained. (2) Algal cells were sieved and held stationary at the upstream (frontal) side of a row of laterofrontal cilia that were not beating. (3) Localized extension of cilia toward the inhalant chamber, coincident with particle captures, indicated that laterofrontal cilia flick toward the inhalant chamber. These flicks may aid transport of captured particles toward the mouth. Thus my earlier report that larvae only sieve, in contrast to the adults (which have an active ciliary response) was in error. The similar ciliary bands in adult and larval bryozoans and in other lophophorates (brachiopods, and phoronids) suggest that these animals share a core repertoire of ciliary behaviours in the capture and concentration of suspended food particles.     

Oral Monokinetids in the Free-Living Haptorid Ciliate Enchelydium polynucleatum (Ciliophora,Enchelyidae): Ultrastructural Evidence and Phylogenetic Implications1

Special ultrastructural characteristics of the haptorid soil ciliate Enchelydium polynucleatum Foissner, 1984 are the restriction of the parasomal sacs to the area of the “brush” and finger-like projections of the food vacuole membrane into the lumen of the vacuole. The general organization of the infraciliature is similar to that of Spathidium and some buetschliids because the anterior ends of the somatic kineties are condensed and obliquely bent. Enchelydium is similar to haptorids and buetschliids in possessing monokinetid somatic fibrillar structures with the classical fibrillar associates: 1) a short kinetodesmal fiber; 2) two transverse microtubular ribbons; 3) a long postciliary microtubular ribbon; and 4) a system of overlapping subkinetal microtubules, which seems to be absent in the buetschliids. Unlike Spathidium and all other haptorids so far investigated ultrastructurally, serial sections show that there are no oral dikinetids, as in the endocommensal buetschliids and balantidiids. Instead, three to six anterior kinetids in each ciliary row have nematodesmal bundles extending into the cytoplasm and surrounding the cytopharynx. These kinetids lack cilia and all fibrillar associates except enlarged transverse ribbons, which extend anteriorly and inwards to support the cytopharynx. Other similarities between the buetschliids and Enchelydium are the conspicuous rough endoplasmic reticulum and abundant sausage-like vesicles in the oral region. As in other haptorids, Enchelydium has two types of toxicysts and one type of mucocyst. These observations strongly suggest that Enchelydium belongs to the ancestral stock of both the Haptorida and the Archistomatida. The similarities in the somatic and oral infraciliature and ultrastructure of the Haptorida and the Archistomatida suggest that they belong to the same subclass, Haptoria Corliss, 1974.     

Digestive system membranes: freeze-fracture evidence for differentiation and flow in Paramecium

Freeze-fractured membranes of digestive vacuoles of randomly feeding Paramecium caudatum exhibit dramatic differences in intramembrane particle (IMP) number and distribution on both E- and P-fracture faces. By pulse-feeding latex spheres to cells we have demonstrated that these differences are related to the age of the digestive vacuoles, and that the membranes of such vacuoles undergo a specific sequence of changes during the digestive cycle. Young digestive vacuoles (DV-I; less than or equal to 6 min), nascent vacuoles still connected to the cytopharynx, and discoidal vesicles, from which vacuole membrane is derived, all have a highly particulate E face and a less particulate P face. As early as 3 min after feeding, a second category of digestive vacuoles (DV-II) can be recognized, which are both considerably smaller in diameter and lack particles on their E face. These findings suggest that the endocytic removal of DV-I membrane material associated with the formation of DV-II vacuoles involves a concomitant and selective removal of E-face particles, as essentially no changes are seen in the density of P-face particles on the two types of vacuoles. Beginning at 10 min the first DV-III vacuoles are encountered. These are both larger than the DV-II vacuoles and possess very prominent E-face particles, which resemble those on the E face of the numerous lysosomes bordering the digestive vacuoles. DV-III vacuoles also exhibit a substantial increase in P-face particles. These membrane changes closely parallel, and are probably correlated with, the physiological events occurring within the vacuole lumen: concentration of food, killing of prey, and digestion. Calculations of the amount of membrane removed from DV-I to form DV-II and of the increase in membrane surface area during the transition from DV-II to DV-III indicate that as much as 90% of the initial phagosome (DV-I) membrane can be removed before digestion begins. The enlargment of DV-II must be caused by fusion with adjacent lysosomes which also contribute the new populations of IMPs to the DV- III membrane. The appearance of numerous endocytic structures on older DV-III vacuoles suggests that membrane is retrieved from DV-III before defecation.     

Digestion in the peritrich ciliateOphrydium versatile

Summary Digestion in the peritrich ciliateOphrydium versatile O.F.M. involves a complex sequence of intracytotic and exocytotic membrane fusion and recycling events. Food particulates are concentrated in the lower cytopharynx which forms a fusiform-shaped food vacuole. Upon release from the cytopharynx, this food vacuole begins to condense, concentrating the food particulates. Excess membrane is removed intracytotically. These released membranes pieces form discoidal vesicles which are recycled to the base of the cytopharynx, thus providing additional membrane for subsequent food vacuole formation. In the condensed food vacuole, digestion proceeds; hydrolytic enzymes are delivered to the food vacuole via rough endoplasmic reticulum and/or by the cup-shaped coated vesicles (CSCV). As these vesicles fuse with the food vacuole, the food vacuole enlarges, digestion proceeds and an electron-dense membrane coat appears along the luminal surface of the food vacuole. Prior to defecation, the food vacuole undergoes a final condensation; irregularly-shaped, electron dense, single-membrane bound vesicles are cut-off intracytotically from the old food vacuole. These vesicles undergo condensation and invagination to form the cup-shaped coated vesicles (CSCV) which fuse with younger food vacuoles.     

Distinct axonemal processes underlie spontaneous and stimulated airway ciliary activity

Cilia are small organelles protruding from the cell surface that beat synchronously, producing biological transport. Despite intense research for over a century, the mechanisms underlying ciliary beating are still not well understood. Even the nature of the cytosolic molecules required for spontaneous and stimulated beating is debatable. In an effort to resolve fundamental questions related to cilia beating, we developed a method that integrates the whole-cell mode of the patch-clamp technique with ciliary beat frequency measurements on a single cell. This method enables to control the composition of the intracellular solution while the cilia remain intact, thus providing a unique tool to simultaneously investigate the biochemical and physiological mechanism of ciliary beating. Thus far, we investigated whether the spontaneous and stimulated states of cilia beating are controlled by the same intracellular molecular mechanisms. It was found that: (a) MgATP was sufficient to support spontaneous beating. (b) Ca(2+) alone or Ca(2+)-calmodulin at concentrations as high as 1 microM could not alter ciliary beating. (c) In the absence of Ca(2+), cyclic nucleotides produced a moderate rise in ciliary beating while in the presence of Ca(2+) robust enhancement was observed. These results suggest that the axonemal machinery can function in at least two different modes.     

An electro-optic monitor of the behavior of Chlamydomonas reinhardtii cilia

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.     

Scoring a backstage pass: mechanisms of ciliogenesis and ciliary access

Cilia are conserved, microtubule-based cell surface projections that emanate from basal bodies, membrane-docked centrioles. The beating of motile cilia and flagella enables cells to swim and epithelia to displace fluids. In contrast, most primary cilia do not beat but instead detect environmental or intercellular stimuli. Inborn defects in both kinds of cilia cause human ciliopathies, diseases with diverse manifestations such as heterotaxia and kidney cysts. These diseases are caused by defects in ciliogenesis or ciliary function. The signaling functions of cilia require regulation of ciliary composition, which depends on the control of protein traffic into and out of cilia.     

Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis

The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.     

The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia

The connecting cilium of rat retinal rods was studied by freeze-fracture and thin-sectioning techniques. Transverse strands of intramembranous particles could be observed on fracture face B on the ciliary plasma membrane. The strands were essentially similar to those found at the transitional zone of motile cilia ("ciliary necklace"). The larger number of intramembranous particles obscured the pattern on fracture face A of the membrane. On longitudinal sections of the cilia, beads showing a periodicity similar to the necklace strands were observed. Each bead consisted of two structures apposed to both sides of the plasma membrane. Transverse sections of the cilia revealed radial Y-shaped structures that connected each ciliary doublet with the plasma membrane. Axial tubules, central sheath, radial spokes and dynein arms were missing in the connecting cilium. Comparing the fine structure of the retinal cilia with that of motile cilia it becomes evident that the connecting cilium is analogous in structure with the transitional zone of motile cilia. The present observations suggest that periodic membrane beads along the plasma membrane on thin sections correspond to strands of necklace particles as observed on freeze-fractured membranes. The arrangement of the particles in transverse strands is probably ensured by the radial connecting structures.     

Automated measurement of ciliary beat frequency

Measurements of ciliary beat frequency using video images are dependent on observer interpretation. To obtain objective estimates of ciliary beat frequency from video-image sequences, a computer-based method was developed. Regions of interest of video-image sequences were selected and digitized. Variations in numerical values representing light intensity resulting from cilia beating were extracted and analyzed using autocorrelation techniques. The ciliary beat frequencies obtained for 14 in vitro experiments on ciliated cells or epithelium from the frog palate (Rana catesbeiana) over the range of frequencies 2-25 Hz correlated well with independent observer measurements (r = 0.979). The addition of such computer-based methods to video observer-based systems allows more objective and efficient determinations of ciliary beat frequency.     

A numerical study of muco-ciliary transport under the condition of diseased cilia

Structural and functional disorders of pulmonary cilia may result from genetic disorders and acquired insults. A two-dimensional numerical model based on the immersed boundary method coupled with the projection method is used to study the flow physics of muco-ciliary transport of the human respiratory tract under various abnormalities of cilia. The effects of the cilia beat pattern (CBP), ciliary length, immotile cilia, beating amplitude and uncoordinated beating of cilia are investigated. As expected, the mucus velocity decreases as the beating amplitude reduces. The windscreen wiper motion and rigid planar motion, which are two abnormal CBPs owing to genetic disorders, greatly reduce or almost stop the mucus transport. If the ciliary length varies from its standard length, the mucus velocity would decrease. The mucus velocity decreases rather linearly if the number of uniformly distributed immotile cilia increases. The numerical results show that the mucus velocity would be further reduced marginally when the uniformly distributed immotile cilia are rearranged as a cluster of immotile cilia. Furthermore, if half of the cilia are immotile and uniformly distributed and motile cilia beat at reduced amplitude, the incoordination between the active motile cilia would not significantly affect the mucus velocity.     

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.     

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.