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.     

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.     

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin- section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.     

Tannic acid improves the visualization of the human erythrocyte membrane skeleton by freeze-etching

The effect of fixatives on the membrane skeleton underlying the human erythrocyte membrane was examined by freeze-etching. An anastomosing fibrillar network was readily observed on the protoplasmic surface of the erythrocyte membrane treated with tannic acid. Such structure was much less defined in unfixed membrane or membrane fixed with glutaraldehyde or glutaraldehyde followed by osmium tetraoxide. Tannic acid caused a marked increase in diameter of the fibrillar components of the membrane skeleton and of the protoplasmic surface particles of inside-out vesicles prepared by alkali treatment but did not affect the size of intramembranous particles seen on fracture faces nor the appearance of exoplasmic surfaces. The improved visualization of the membrane skeleton after treatment with tannic acid resulted from interactions between tannic acid and exposed membrane proteins.     

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.     

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.     

Fine structural localization of phosphatases in cilia and basal bodies of Tetrahymena pyriformis.

Cytochemical localization of ATPase activities in cilia and basal bodies of Tetrahymena pyriformis revealed a number of possible sites of ATPases. In basal bodies, reaction product was localized on the periphery of basal body microtubules, in the core of the B-microtubules, on the dense basal body core, and on the basal plate; some reaction product was associated with the postciliary and basal microtubules. In the cilium, reaction product was associated with the ciliary membrane, the basal granule, the periphery of the outer doublet microtubules, in the core of the B-microtubules, and on the arms and either the central microtubules or the radial spoke heads. Reaction product deposition required ATP and either Ca2+ or Mg2+ or ADP and Mg2+. When incubated in the presence of ATP and Na+, reaction product was only found at the base of the cilium in the region of the ciliary necklace. Implications of the various sites of activity are discussed with respect to possible mechanisms of ciliary motility.     

FEEDING APPARATUS OF THE COLORLESS FLAGELLATE KATABLEPHARIS (CRYPTOPHYCEAE)1

The feeding apparatus of Kalablepharis ovalis (isolated from a freshwater impoundment in Colorado) and Katablepharis clone G-2 (isolated from the littoral of the Black Sea near Yalta in the Crimea) consists of inner and outer oval-shaped arrays of microtubules that begin at the anterior end of the cell and pass into the posterior of the cell. Each array of microtubules contains groups of microtubules with two to eight microtubules per group depending on the position of the array in the cell. A specialized area of the plasma membrane, the mouth, occurs at the anterior end of the cell. The mouth is oval with the long axis oriented dorsoventrally and consists of a raised ridge surrounding a central depression. The anterior end of the microtubules of the inner and outer arrays supports the raised ridge of the mouth. In freeze-fracture replicas, the protoplasmic face of the plasma membrane contains intramembrane particles on the raised ridge of the mouth. Three small membrane-cisternae occur on the protoplasmic side of the plasma membrane in the area of the mouth. Katablepharis clone G-2 also has five or six additional large membrane-cisternae associated with the inner microtubular array in the anterior portion of the cell. These larger membrane-cisternae do not occur in K. ovalis. Vesicles with electron-dense contents occur in association with the microtubular arrays. Katablepharis ovalis has a second type of vesicle containing a single-membrane profile associated with the microtubule arrays. The structure of the microtubular arrays in Katablepharis is compared with similar structures in suctorian ciliates and dinoflagellates.     

The ultrastructure of the growing regions of aerial hyphae ofRhizopus sexualis (Smith) callen

Summary The fine structure of the growing region of the aerial hyphae ofRhizopus sexualis is described. The protoplasmic components are organised in zones. At the hyphal tip there are abundant cytoplasmic vesicles and few ribosomes. Mitochondria, ribosomes, and endoplasmic reticulum are present in the sub-apical region. Nuclei occur in the posterior subapical region, some have been observed containing microtubules and bearing long projections. Microtubules have been observed extending for some distance through the cytoplasm.     

Particle Assemblies in the Plasma Membrane of Tetrahymena: Relationship to Cell Surface Topography and Cellular Morphogenesis1

During a freeze-fracture electron microscopical study of the plasma membrane of Tetrahymena, several different types of organized particle assemblies were observed. Three of these were found only on the protoplasmic face and were localized in the anterior-ventral region of the cell. These consisted of plate-like arrays composed of 4–25 triplet rows of small 3–4 nm particles; long, paired linear arrays localized at the tops of cortical ridges and composed of 7–8 nm particles; and elongated tetragonal arrays located in the grooves between ridges and composed of approximately 10 nm particles. The distribution of these arrays is consistent with roles in cellular morphogenesis, chemoreception, or cell-cell pairing during conjugation. In addition, a unique particle track associated with the cytoproct (anal pore) was observed in the external face of the plasma membrane. Furthermore, the protoplasmic face of the plasma membrane is characterized by a high density of particles organized into localized microarrays, consisting of small paracrystals or strings, which exhibit a loose higher-order patterning most evident toward the anterior end of the cell. Particle distributions on the protoplasmic face do not appear to be significantly altered by conditions that cause clumping of alveolar membrane particles. Taken together, these observations are consistent with the idea that the proteins of the plasma membrane are highly ordered and relatively immobile and that the structure of the plasma membrane is regionally differentiated.     

Fine structures of sponge cell membranes: comparative study with freeze-fracture and conventional thin section methods

Freeze-fracture replicas of sponge cell membranes revealed in general a low density of intramembranous particles, with the exceptions of the membrane (silicalemma) surrounding the siliceous spicules in Ephydatia and the membranes of spherulous cells in Chondrosia. In addition, several types of particle arrangements were observed. A classical necklace is present at the base of the choanocyte flagellum. Rosettes of particles are particularly obvious in the apical membranes of choanocytes, where they are associated with the fuzzy coat covering these cells. Parallel ridges of particles were observed along the microvilli of the choanocyte collar, at sites of insertion of connecting filaments. Rows of particles were observed in the plasma membrane of pinacocytes in Ephydatia where they are located on areas deformed by protruding fibrillar inclusions. Pinacocyte plasma membranes in this species also can contain accumulations of particles which are likely related to desmosomes. Single rows of aligned particles and double rows of staggered particles (sometimes organized in large plates) in addition to rhombic particle arrays were encountered on replicas of marine sponge cell membranes. No classical arrangements corresponding to gap junctions, tight junctions or septate desmosomes were observed. The significance of these data is analysed.     

Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method

Summary The fine structure of the glomerular basement membrane was re-evaluated by using a deep-etch replica method.The structure of the laminae rarae interna and externa of the rat glomerular basement membrane was basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. The size of the mesh was quite variable but most often ranged from 20 to 25 nm in width. In addition, a zipper-like substructure of the epithelial slit diaphragm was observed. By contrast, the lamina densa was composed of closely packed particles.After exposure of the bovine glomerular basement membrane to ultrasonic waves or trypsin, the particles of the lamina densa were effectively removed. The underlying structure showed the fibrillar network closely resembled that seen in the laminae rarae of the rat glomerular basement membrane.The glomerular basement membrane thus revealed was as principally composed of a fibrillar network, which might be regularly arranged units of type-IV collagen. Numerous fine particles, most likely proper components of the glomerular basement membrane, were attached onto this basic fibrillar structure, giving rise to a morphologic appearance different from that of the laminae rarae.     

FIBRILLAR DIFFERENTIATION IN A MICROPLASMODIUM OF THE SLIME MOLD PHYSARUM POLYCEPHALUM

The motility of Physarum polycephalum microplasmodia depends upon the conditions under which they are cultured. To investigate the relation between protoplasmic streaming and filamentous structures observed in the cytoplasm, microplasmodia were collected from shaken cultures, agar plates and shaken cultures of the organism which had previously been plate-cultured.
No sign of streaming could be found in materials in shaken culture, even in those which were shaken after they had once been motile on an agar plate. The immotile microplasmodia in both cases failed to contain any filamentous structures.
Microplasmodia on agar plates were motile, showing vigorous peripheral movements (projection of pseudopods) and inner protoplasmic streaming. In the motile organisms two types of filamentous structures were observed: loose networks just inside the plasma membrane of rounded pseudopods with smooth surfaces; and compact, straight bundles beneath the pseudopods or in much deeper locations.     

Studies on the olfactory epithelium of the frog and the toad with the aid of light and electron microscopy

Summary Epithelial structures in the olfactory region of the frog and the toad have been examined with the aid of light and electronmicroscopy. The olfactory cells send out 6–12 extremely long cilia with a central fibrillar structure and a peripheral sheath. They are motile but only in a small degree as their peripheral ends are weaved together in a network which covers the epithelial surface. The sustentacular cells show a great number of small protoplasmic protrusions on their surface. Their importance is discussed.The cells of the glands of Bowman are also provided with small protoplasmic protrusions but they are smaller than those of the sustentacular cells and do not show their inner structures. In all three celltypes mitochondria were found containing systems of double-membranes and in the supporting cells and Bowman's cells intracellular cytoplasmic membranes were observed.     

The restructuring of the flagellar base and the flagellar necklace during spermatogenesis of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Transmission electron microscopy was used to study the development of the flagellar base and the flagellar necklace during spermatogenesis in a moth (Ephestia kuehniella Z.). Until mid-pachytene, two basal body pairs without flagella occur per cell. The basal bodies, which contain a cartwheel complex, give rise to four flagella in late prophase I. The cartwheel complex appears to be involved in the nucleation of the central pair of axonemal microtubules. In spermatids, there is one basal body; this is attached to a flagellum. At this stage, the nine microtubular triplets of the basal body do not terminate at the same proximal level. The juxtanuclear triplets are shifted distally relative to the triplets distant from the nuclear envelope. Transition fibrils and a flagellar necklace are formed at the onset of axoneme elongation. The flagellar necklace includes Y-shaped elements that connect the flagellar membrane and the axonemal doublets. In spindle-containing spermatocytes, the flagellar necklace is no longer detectable. During spermatid differentiation, the transition fibrils move distally along the axoneme and a prominent middle piece appears. Our observations and those in the literature indicate certain trends in sperm structure. In sperms with a short middle piece, we expect the presence of a flagellar necklace. The distal movement of the transition fibrils or equivalent structures is prevented by the presence of radial linkers between the flagellar membrane and the axonemal doublets. On the other hand, the absence of a flagellar necklace at the initiation of spermiogenesis enables the formation of a long middle piece. Thus, in spermatozoa possessing an extended middle piece, a flagellar necklace may be missing.     

Localization of Possible Calcium-Binding Sites in the Cilia of Paramecium caudatum

SYNOPSIS. Electron-dense deposits indicating possible Ca-binding sites were found at the ciliary base of Paramecium caudatum fixed in a glutaraldehyde solution containing 5 mM CaCl₂. The deposits appeared mainly at the inner surface of the ciliary membrane above the "ciliary necklace" region, although they could also occur in the space between the outer and the central microtubules. In some cases a ring of exactly 9 deposits was found in a ciliary cross section of a cilium.     

Spermatogenesis of a marine snail,Littorina sitkana

Summary The fine structure of the spermatogonium, spermatocyte and spermatid of a marine snail, Littorina sitkana is described. The ring centriole (annulus) is formed from the distal centriole and it migrates to the base of the mitochondrial region where it lies in a joint-like structure which is formed by an area of invaginated plasma membrane. The distal and proximal centrioles are at first perpendicular to each other but the proximal centriole rotates to a position coaxial with the distal centriole and fuses with it. The peripheral doublet fibers are continuous between the two centrioles but the central fibers originate only in the distal centriole. The acrosome differentiates from the proacrosomal granule which is derived from a Golgi body. Microtubules, present at this stage, may assist acrosomal formation. Chromatin condensation begins with the formation of fibrous strands, then to lamellar plates which become folded and later twisted around the flagellar shaft. In the final stages the lamellae appear in cross section as concentric rings which eventually fuse to form a homogeneously dense nuclear tube.     

Electron tomographic analysis of post-meiotic cytokinesis during pollen development in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The mechanism of cell wall formation after male meiosis was studied in microsporocytes of Arabidopsis thaliana (L.) Heynh. by means of thin-section and immuno-electron microscopy and dual-axis electron tomography of high-pressure-frozen/freeze-substituted cells. The cellularization of four-nucleate microsporocytes involves a novel type of cell plate, called a post-meiotic-type cell plate. As in the syncytial endosperm, the microsporocyte cell plates assemble in association with mini-phragmoplasts. However, in contrast to the endosperm cell plates, post-meiotic type cell plates arise simultaneously across the entire division plane. Vesicles are transported along mini-phragmoplast microtubules by putative kinesin proteins and, prior to fusion, they become connected together by 24-nm-long linkers that resemble exocyst complexes. These vesicles fuse with each other to form wide tubules and wide tubular networks. In contrast to endosperm cell plates, the wide tubular networks in microsporocytes completely lack callose and do not appear to be constricted by dynamin rings. The most peripheral wide tubular networks begin to fuse with the plasma membrane before the more central cell plate assembly sites become integrated into a coherent cell plate. Fusion with the parental plasma membrane triggers callose synthesis and the wide tubular domains are converted into convoluted sheets. As the peripheral convoluted sheets accumulate callose and arabinogalactan proteins, they are converted into stub-like projections, which grow centripetally, i.e. toward the interior of the syncytium, fusing with the wide tubular networks already assembled in the division plane. We also demonstrate that the ribosome-excluding cell plate assembly matrix is delivered to the mini-phragmoplast with the first vesicles, and encompasses all the linked vesicles and intermediate stages in cell plate formation.Abbreviations AGP Arabinogalactan protein - MT Microtubule     

Tonoplast fine structure and osmotic regulation in Porphyra umbilicalis

In Porphyra, an intertidal red alga, the fine structure of the tonoplast was studied by freeze-fracture electron microscopy. It was shown that density and size of intramembraneous particles on the protoplasmic fracture face vary with external osmotic potential. The frequency of particles grouped in size classes (calculated per cell) increases with increasing osmotic stress and shows a maximum in 3 to 4 x artificial seawater medium ASP₁₂. It is concluded that the intensity of tonoplast transport, which probably is enhanced with increasing osmotic stress from 1 to 4 x media, is most likely correlated with a change in membrane fine structure of the tonoplast.     

Periplast structure of the cryptomonad flagellateHemiselmis brunnescens

Summary The periplast ofHemiselmis brunnescens Butcher is a complex cell covering comprised of the plasma membrane (PM) sandwiched between a surface periplast component (SPC) and an inner periplast component (IPC). The SPC is revealed by deep-etching, and consists of hexagonal plates composed of tripartite subunits that appear to self-assemble into a crystalline layer with a hexagonal symmetry. Small scales (termed fibrillar scales) accumulate on the crystalline plates during cell growth, eventually forming a carpet that itself may appear crystalline when fully formed. Heptagonal rosette scales are occasionally observed on the surface as well. The position of the crystalline plates is precisely mirrored by both the E and P fracture faces of the PM. The plate proper is underlain by membrane with a high concentration of intramembrane particles (IMPs) while the bands of membrane underlying the plate borders lack IMPs. Access of subunits and fibrillar scales to the cell surface following initial plate formation appears to be at the plate boundaries. This study suggests that cryptomonad flagellates may provide model systems for studying the self-assembly of cell surface components, and for relating membrane structure to function, as evidence suggests a major role for the PM in mediating periplast assembly and development.