Microtubule-membrane interactions in ctenophore swimming plate cilia

The cilia in ctenophore swimming plates are organized into long rows and the cilia within each of the rows are connected to one another by interciliary bridges. The interciliary bridges form a type of intracellular junction and are periodically spaced at 15 nm intervals along the long axis of a cilium. The bridges bind adjacent cilia together even after dissolution of the ciliary membrane by non-ionic detergent. Interciliary bridges are attached to the compartmenting lamellae, which are paracrystalline structures composed of spherical particles which are periodically attached to the outer doublet microtubules at the sites to which the microtubule-membrane bridges are bound. It is proposed that the compartmenting lamellae are modifications of the ciliary microtubule-membrane bridge found in other eukaryotic cilia and that it is associated with a junctional complex that binds adjacent cilia together in swimming plates.     

STUDIES ON CILIA : III. Further Studies on the Cilium Tip and a "Sliding Filament" Model of Ciliary Motility

This study confirms and extends previous work on the lateral cilia of the fresh-water mussel, Elliptio complanatus, in support of a "sliding filament" mechanism of ciliary motility wherein peripheral filaments (microtubules) do not change length during beat (see Satir, 1967). Short sequences of serial sections of tips are examined in control (nonbeating) and activated (metachronal wave) preparations. Several different tip types, functional rather than morphogenetic variants, are demonstrated, but similarly bent cilia have similar tips. The peripheral filaments are composed of two subfibers: a and b. The bent regions of cilia are in the form of circular arcs, and apparent differences in subfiber-b length at the tip are those predicted solely by geometry of the stroke without the necessity of assuming filament contraction. Various subfibers b apparently move with respect to one another during beat, since small systematic variations in relative position can be detected from cilium to cilium. While subfiber-b lengths are uniform throughout, subfiber-a lengths are morphologically different for each filament: 8 and 3 are about 0.8 µ longer than 1, 4 and 5, but each unique length is independent of stroke position or tip type. Subfiber-a does not contract, nor does it move, e.g. slide, with respect to subfiber-b of the same doublet. The central pair of filaments extends to the tip of the cilium where its members fuse. Subunit assembly in ciliary microtubules is evidently precise. This may be of importance in establishing the relationships needed for mechanochemical interactions that produce sliding and beat.     

STUDIES ON CILIA : The Fixation of the Metachronal Wave

Upon excision into spring water, the lateral cilia of the gill of the freshwater mussel Elliptio complanatus (Solander) stop beating, but 0.04 M potassium ion can activate the gill so that these cilia again beat with metachronal rhythm. One per cent osmium tetroxide quickly pipetted onto a fully activated gill fixes the lateral cilia in a pattern that preserves the form and arrangement of the metachronal wave, and permits the cilia to be studied with the electron microscope in all stages of their beat cycle. Changes are seen in the fixed active preparation that are not present in the inactive control, i.e., in the packing of the cilia, the position of the axis of the ciliary cross-section, and the diameter of the ring of peripheral filaments. Analysis of these parameters may lead to new correlations between ciliary fine structure and function.     

THE CILIARY NECKLACE : A Ciliary Membrane Specialization

Cilia, primarily of the lamellibranch gill (Elliptio and Mytilus), have been examined in freeze-etch replicas. Without etching, cross fractures rarely reveal the 9 + 2 pattern, although suggestions of ninefold symmetry are present. In etched preparations, longitudinal fractures through the matrix show a triplet spoke alignment corresponding to the spoke periodicity seen in thin sections. Dynein rows can be visualized along the peripheral microtubules in some preparations. Fracture faces of the ciliary membrane are smooth with few membrane particles, except in the regions adjacent to the basal plate. In the transition region below the plate, a unique particle arrangement, the ciliary necklace, is found. In the Elliptio gill, on fracture face A the necklace is comprised of three well-defined rows or strands of membrane particles that encircle the ciliary shaft. The rows are scalloped and each scallop corresponds to a peripheral doublet microtubule. In thin sections at the level of these particles, a series of champagne-glass structures link the microtubular doublets to the ciliary membrane. The ciliary necklace and this "membrane-microtubule" complex may be involved in energy transduction or the timing of ciliary beat. Comparative studies show that these features are present in all somatic cilia examined including those of the ameboflagellate Tetramitus, sea urchin embryos, rat trachea, and nonmotile cilia of cultured chick embryo fibroblasts. The number of necklace strands differs with each species. The necklace has not been found in rat or sea urchin sperm.     

Discocilia—A new type of kinocilia in the larvae of Lanice conchilega (Polychaeta,Terebellomorpha)

A modified type of kinocilia has been found in the Aulophora-Iarva of the sedentarian polychaete Lanice conchilega. For this newly described cilium type the term "discocilium" is proposed. The only structural difference from usual locomotory cilia is the tip, which possesses a discoidal head. The head is formed from the terminal part of the cilium shaft, which is bent to give rise to a loop-like ring covered by the ciliary membrane. Three types of discocilia can be distinguished: a) discocilia having swollen, bulblike heads with a central straight axoneme; b) discocilia having heads with a curved lateral axoneme and c) discocilia in which the axoneme forms a loop. The internal structure shows the usual 9 + 2 arrangement of the filaments. The head shows no sign of secretion; it appears structureless in electron microscopical examination. There are two kinds of discocilia arrangements: 1) isolated bunches of cilia especially at the tentacles and in the frontal region, and 2) segmental dorsal rows of cilia. The possible formation of discocilia is described.     

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.     

Adaptations of ciliary systems for the propulsion of water and mucus

1. The characteristics of ciliary systems are determined by the dominance of viscous effects over inertial effects. 2. The velocity of water propulsion depends on ciliary length, beat frequency, pattern of beating, the arrangement of the cilia and their co-ordination. Beating cilia influence a layer of water only two or three cilium lengths deep, with maximal velocity near the ciliary tip. 3. Mucus is propelled by the tips of short cilia that penetrate the mucus; these cilia are closely spaced on epithelia, and achieve slow propulsion that is relatively independent of load and does not require strong ciliary co-ordination.     

FINE STRUCTURE OF THE CILIA OF ROTIFERS

The fine structure of the coronal cilia of the rotifer Philodina citrina has been studied in detail. Specimens were fixed with OsO₄ and embedded in butyl—methyl methacrylate, Epon 812, or Vestopal and sectioned with a Porter-Blum microtome. The details of structure of the rootlets, basal bodies, basal plates, and free cilia are described. The general structure of the rotifer ciliary apparatus conforms well to that established for other species. One of the main observations is the difference in structure of the peripheral filaments in the opposing halves of a cross-section of the free cilium. Also, in longitudinal sections evidence is offered for the existence of a helical structure in the peripheral filaments.     

THE FINE STRUCTURE OF CORTICAL COMPONENTS OF PARAMECIUM MULTIMICRONUCLEATUM

The electron microscope was used to study the structure and three dimensional relationships of the components of the body cortex in thin sections of Paramecium multimicronucleatum. Micrographs of sections show that the cortex is covered externally by two closely apposed membranes (together ~250 A thick) constituting the pellicle. Beneath the pellicle the surface of the animal is molded into ridges that form a polygonal ridgework with depressed centers. It is these ridges that give the surface of the organism its characteristic configuration and correspond to the outer fibrillar system of the light microscope image. The outer ends of the trichocysts with their hood-shaped caps are located in the centers of the anterior and posterior ridges of each polygon. The cilia extend singly from the depressed centers of the surface polygons. Each cilium shows two axial filaments with 9 peripheral and parallel filaments embedded in a matrix and the whole surrouned by a thin ciliary membrane. The 9 peripheral filaments are double and these are evenly spaced in a circle around the central pair. The ciliary membrane is continuous with the outer member of the pellicular membrane, whereas the plasma membrane is continuous with the inner member of the pellicular membrane. At the level of the plasma membrane the proximal end of the cilium is continuous with its tube-shaped basal body or kinetosome. The peripheral filaments of the cilium, together with the material of cortical matrix which tends to condense around them, form the sheath of the basal body. The kinetodesma connecting the ciliary kinetosomes (inner fibrillar system of the light microscopist) is composed of a number of discrete fibrils which overlap in a shingle-like fashion. Each striated kinetosomal fibril originates from a ciliary kinetosome and runs parallel to other kinetosomal fibrils arising from posterior kinetosomes of a particular meridional array. Sections at the level of the ciliary kinetosomes reveal an additional fiber system, the infraciliary lattice system, which is separate and distinct from the kinetodesmal system. This system consists of a fibrous network of irregular polygons and runs roughly parallel to the surface of the animal. Mitochondria have a fine structure similar in general features to that described for a number of mammalian cell types, but different in certain details. The structures corresponding to cristae mitochondriales appear as finger-like projections or microvilli extending into the matrix of the organelle from the inner membrane of the paired mitochondrial membrane. The cortical cytoplasm contains also a particulate component and a system of vesicles respectively comparable to the nucleoprotein particles and to the endoplasmic reticulum described in various metazoan cell types. An accessory kinetosome has been observed in oblique sections of a number of non-dividing specimens slightly removed from the ciliary kinetosome and on the same meridional line as the cilia and trichocysts. Its position corresponds to the location of the kinetosome of the newly formed cilium in animals selected as being in the approaching fission stage of the life cycle.     

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.     

Ultrastructural evidence for the occurrence of three types of mechanosensitive cells in the tentacles of the cubozoan polypCarybdea marsupialis

Summary The ectodermal cell layer in the tentacles of the cubozoan polypCarybdea marsupialis contains four types of cells (types 1–4) bearing specialized cilia. Epitheliomuscular cells (type 1) are characterized by motile cilia with dynein-decorated axonemes. 200 nm long extramembranous filaments of unknown function are restricted to a belt-like region distal to the transition zone. Up to 40 rn long rigid cilia formed by a slender epithelial cell type (type 2) are surrounded by rings of short microvilli. The axonemes of these cilia are composed of incomplete microtubules and lack dynein. Microvilli and cilia are linked by intermembrane connectors. Microtubuledoublets and ciliary membrane are interconnected by microtubule-associated cross-bridges only within this contact region. At the tip of each tentacle a single nematocyte (type 3) is surrounded by 7–10 accessory cells (type 4). These both cell types are equipped with similar cilium-stereovilli-complexes consisting of a cone-like arrangement of stereovilli and a modified cilium. The axonemal modifications of the cilium, its interconnections with the surrounding stereovilli and the linkages between ciliary axoneme and ciliary membrane are similar to those known from the cnidocil-complexes of hydrozoons and other epithelial mechanosensitive cells of the collar-receptor type. Our data indicate that besides the nematocyte two other types of mechanosensory cells (types 2 and 4) are integrated in the ectodermal cell layer ofCarybdea which possibly affect the triggering mechanism of nematocyst discharge.     

A comparative study of ciliary differentiations in the branchial stigmata of ascidians

In a correlated thin sectioning and freeze-fracturing study, we have examined species belonging to the orders of the ascidian class: Stolidobranchiata (Botryllus schlosseri, Botrylloides leachi, Molgula socialis, Styela plicata), Phlebobranchiata (Ascidiella aspersa, Phallusia ingeria, Ciona intestinalis) and Aplousobranchiata (Clavelina lepadiformis). Though the branchial basket varies in the complexity and filtration efficiency in the three orders, the ciliated epithelia aroand the stigmata contain a common pattern of organization; seven rows of flattened cells, each bearing a single row of long cilia flanked by a single row of microvilli. All the species examined possess ciliary specializations represented by: (a) bridges connecting doublets number 5 and 6 as well as 9.1 and 2; (b) dense material lying between the above mentioned axonemal doublets (5-6 and 1-2) and the ciliary membrane, sometimes in the shape of longitudinal strands or often as lines of dots; (c) a fuzzy coat protruding from the ciliary membrane, consisting of tufts or scattered filaments; (d) intramembrane particles (IMPs) associated with the P-face of the membrane, often arranged in clusters and orderly alignments related to the anderlying axonemal doublets; these IMPs decorate the opposite sides of each cilium facing the adjacent cilia forming the ciliary rows of adjacent cells and are absent on the lateral sides. The stigmatal cilia propel water through the stigmata and their effective strokes follow a line at right angles to the row of cilia in each cell. The usual direction of the effective stroke is toward doublets 5-6. It is possible, therefore, to refer to structure in relation to the ciliary beat cycle. The importance of these specializations is unknown, but the structures appear to vary in the different species. A correlation between the richness of the specializations and the complexity of the branchial basket was not evidenced. It was suggested that the ciliary specializations relate to the peculiar organization of the stigmatal margin and that all are involved in the regulation of the ciliary activity.     

ASPECTS OF CILIARY FINE STRUCTURE IN EUPLOTES PATELLA

1. The functional unity of cirri and membranelles can result structurally only from extensions of the ciliary membrane. 2. The pellicle is composed of an outer pellicular membrane and an inner cytoplasmic membrane. 3. The ciliary rootlets are composed of numerous filaments 120 A in diameter with central areas of low density. They have no periodic structure. 4. The ciliary membrane is a double-layered structure continuous with the pellicular membrane. The cilia show the typical arrangement of nine double, peripheral and two single, central fibrils. All fibrils pass into the basal region, the peripheral ones joining with the rootlet filaments, while the central fibrils from the extreme proximal position of the basal region turn back toward the pellicle and appear to unite just beneath the cytoplasmic membrane. 5. The cilia (300 mµ diameter) taper at their tips to a diameter at least as small as 50 mµ. At a diameter of about 150 mµ, the fibrils begin to show a reduction in number. 6. The central ciliary fibrils may determine the possible directions of ciliary beat. These fibrils show an intrafibrillar structure in their basal portion, which involves regularly spaced 40 A granules. 7. These observations on Euplotes, together with the other evidence cited, are consistent with the hypothesis that ciliary motion is produced by the contraction of the peripheral fibrils, while the central fibrils perhaps determine the plane in which the cilia can bend.     

FINE STRUCTURAL CHANGES IN THE FLAGELLUM OF THE SPERMATID IN EXPERIMENTAL CRYPTORCHIDISM OF THE RAT

Rat testes were confined to the abdominal cavity by operation. After 1 to 26 days they were excised, fixed with osmium tetroxide, sectioned, and examined with the electron microscope. Changes in the axial filament complex of the spermatid flagellum appeared 2 days after operation, and the arrangement of filaments in the middle- and main pieces of some spermatid tails was disordered as compared to the 9 + 2 filament arrangement in the tails of the control spermatids and in other flagella and cilia. In cross-sections, the filaments in the experimental material were nine or less in number, and each of them was single and dense. Occasionally some were double, and in those instances one filament was dense and the other was light and tubular. The central filaments were obscure. In longitudinal sections,the filaments were not parallel to the main axis of the flagella or to each other. It was assumed that the central filaments were more sensitive to the experimental conditions than the peripheral pairs of filaments. Furthermore, the light filaments of the peripheral pairs were more sensitive than the dense filaments. Besides the axial filament complex, the fibrous sheath which surrounds it in the main piece was also changed. The plasma membrane of the changed flagella disappeared or became fragmented.     

Distinctive features of cilia in metazoans and their significance for systematics.

A comparative study of epidermal cilia in the Turbellaria and Nemertea has revealed features in these organelles that are specific to certain taxonomic groups. Turbellarians of the order Acoela, in particular, have a characteristic pattern of axonemal filament termination in the distal tips of their cilia and a characteristic ciliary rootlet system that is not seen in other turbellarian orders nor in other metazoans. Each epidermal cilium in acoels has a typical 9 + 2 axonemal pattern through the main part of its length, but near its distal tip there is an abrupt shelf-life narrowing at which filaments 4-7 terminate; filaments 1, 2, 8 and 9 continue into the thinner distal-most part of the shaft along with singlet microtubules from the axonemal center. The rootlet system in acoel cilia involves an interconnecting pattern with lateral connectives. The unique structure of these cilia has systematic and phylogenetic significance for the Acoela, and it is argued that ultrastructural characters in general, including characters of organelles, can be validly applied to the phylogeny and systematics of the Metazoa.     

Control of the ciliary beat by cyclic nucleotides in intact cortical sheets from Paramecium

The locomotor behavior of Paramecium depends on the ciliary beat direction and beat frequency. Changes in the ciliary beat are controlled by a signal transduction mechanism that follows changes in the membrane potential. These events take place in cilia covered with a ciliary membrane. To determine the effects of second messengers in the cilia, cortical sheets were used with intact ciliary membrane as a half-closed system in which each cilium is covered with a ciliary membrane with an opening to the cell body. Cyclic nucleotides and their derivatives applied from an opening to the cell body affected the ciliary beat. cAMP and 8-Br-cAMP increased the beat frequency and the efficiency of propulsion and acted antagonistically to the action of Ca(2+). cGMP and 8-Br-cGMP increased the efficiency of propulsion accompanying clear metachronal waves but decreased the beat frequency. These results indicate that the cyclic nucleotides affect target proteins in the ciliary axonemes surrounded by the ciliary membrane without a membrane potential and increase the efficiency of propulsion of the ciliary beat. In vitro phosphorylation of isolated ciliary axonemes in the presence of cyclic nucleotides and their derivatives revealed that the action of cAMP was correlated with the phosphorylation of 29-kDa and 65-kDa proteins and that the action of cGMP was correlated with the phosphorylation of a 42-kDa protein.     

The role of cortical orientation in the control of the direction of ciliary beat in Paramecium

The swimming behavior of many ciliate protozoans depends on graded changes in the direction of the ciliary effective stroke in response to depolarizing stimuli (i.e., the avoiding reaction of Paramecium). We investigated the problem of whether the directional response of cilia with a variable plane of beat is related to the polarity of the cell as a whole or to the orientation of the cortical structures themselves. To do this, we used a stock of Paramecium aurelia with part of the cortex reversed 180 degrees. We determined the relation of the orientation of the kineties (ciliary rows) to the direction of beat in these mosaic paramecia by cinemicrography of particle movements near living cells and by scanning electron microscopy of instantaneously fixed material. We found that the cilia of the inverted rows always beat in the direction opposite to that of normally oriented cilia during both forward and backward swimming. In addition, metachronal waves of ciliary coordination were present on the inverted patch, travelling in the direction opposite to those on the normal cortex. The reference point for the directional response of Paramecium cilia to stimuli thus resides within the cilia or their immediate cortical surroundings.     

Ciliary behavior of a negatively phototactic Chlamydomonas reinhardtii

With an instrument that can record the motion of both cilia of the unicellular alga Chlamydomonas reinhardtii for many hours, the behavioral differences of its two cilia have been studied to determine their specific role in phototaxis. The organism was held on a fixed micropipette with the plane of ciliary beating rotated into the imaging plane of a quadrant photodetector. The responses to square-wave light patterns of a wide range of temporal frequencies were used to characterize the responses of each cilium. Eighty-one cells were examined showing an unexpectedly diverse range of responses. Plausible common signals for the linear and nonlinear signals from the cell body are suggested. Three independent ciliary measures--the beat frequency, stroke velocity, and phasing of the two cilia--have been identified. The cell body communicates to the cilia the direction of phototaxis the cell desires to go, the absolute light intensity, and the appropriate graded transient response for tracking the light source. The complexity revealed by each measure of the ciliary response indicates many independent variables are involved in the net phototactic response. In spite of their morphological similarity, the two cilia of Chlamydomonas respond uniquely. Probably the signals from the cell body fan out to independent pathways in the cilia. Each cilium modifies the input in its own way. The change in the pattern of the effective and recovery strokes of each cilium associated with negative phototaxis has been demonstrated and its involvement in phototactic turning is described.