Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Stages of ciliogenesis and regulation of ciliary length

Cilia and flagella are highly conserved eukaryotic microtubule-based organelles that protrude from the surface of most mammalian cells. These structures require large protein complexes and motors for distal addition of tubulin and extension of the ciliary membrane. In order for ciliogenesis to occur, coordination of many processes must take place. An intricate concert of cell cycle regulation, vesicular trafficking, and ciliary extension must all play out with accurate timing to produce a cilium. Here, we review the stages of ciliogenesis as well as regulation of the length of the assembled cilium. Regulation of ciliogenesis during cell cycle progression centers on centrioles, from which cilia extend upon maturation into basal bodies. Centriole maturation involves a shift from roles in cell division to cilium nucleation via migration to the cell surface and docking at the plasma membrane. Docking is dependent on a variety of proteinaceous structures, termed distal appendages, acquired by the mother centriole. Ciliary elongation by the process of intraflagellar transport (IFT) ensues. Direct modification of ciliary structures, as well as modulation of signal transduction pathways, play a role in maintenance of the cilium. All of these stages are tightly regulated to produce a cilium of the right size at the right time. Finally, we discuss the implications of abnormal ciliogenesis and ciliary length control in human disease as well as some open questions.     

An electromechanical hypothesis for ciliary coordination in ciliated protozoa

A new electrochemical hypothesis is proposed to explain ciliary coordination. The hypothesis states that, whenever a cilium beats, the membrane immediately surrounding the cilium is strained. The strain allows a local increase in ionic permeability so ions may flow down an electrochemical gradient and trigger activity in nearby cilia not in a refractory state.     

High voltage electron stereomicroscopy of the cilium-stereociliary complex of perioral sensory cells in Hydra

The cilium-stereociliary complex in perioral neurons of Hydra was examined by electron microscopy, with emphasis on stereomicrographs of serial, 0.5 micron thick, longitudinal and transverse sections. Longitudinal sections revealed (1) flat-topped cones in which the cilium was bent and the ciliary chamber appeared heart-shaped, and (2) pointed cones in which the cilium was straight and the ciliary chamber appeared triangular. Transverse sections revealed 10-12 stereocilia forming a cone over a central cilium with nine peripheral doublets of microtubules but with often more than two central microtubules. The ciliary membrane was fluted; fine filaments connected the outfoldings of membrane with the center of the microtubule doublets. Thin sections revealed 7 nm microfilaments in the stereocilia cores which branched basally into thick and thin roots; the thick roots surrounded the base of the central cilium. The cilium-stereociliary complex was enveloped by an epitheliomuscular cell sheath with a free margin distally and a septate junction proximally. In flat-topped cones the free margin of the enveloping epitheliomuscular cell was closely applied to the top of the cilium-stereociliary complex, whereas in pointed cones the cilium-stereociliary complex projected above the free margin of the sheath. Thus, the 7 nm actin-like filaments in the stereocilia might function to contract and open the complex in response to favorable stimuli so that the cilium is in contact with the aqueous environment.     

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.     

Ultrastructure and membrane topography of special ciliary organelles in the ciliate Eufolliculina uhligi (Protozoa)

Summary In Eufolliculina uhligi and other folliculinid ciliates, a territory has been identified that differs ultrastructurally from other areas of the cell, and that is especially sensitive to mechanical stimuli. This territory is located around the anterior oral apparatus of the loricate trophont and posterior to the membranellar spiral of the swarmer. Each cilium in this territory is closely apposed to a small membrane-covered pin that is supported by transverse microtubules of the cilium. In front of the pin, the base of the cilium bulges out; the ciliary membrane is interconnected with the axoneme by filamentous material. Freeze-fractured cilia show a large rectangular particle array at the site of the basal swelling. Only scattered particles have been observed in the pin membrane. It is suggested that the cilium and the pin act as a unit, which has therefore been named the ciliumpin-complex. Comparison with ciliary organelles of unicellular and multicellular organisms indicates that, because of their polar organization, the complexes are involved in the transduction of oriented, presumably mechanical, stimuli.     

Cytoskeletal modifications of the sensorimotor interneurons ofHydra vulgaris (Cnidaria,Hydrozoa), indicating a sensory function similar to chordotonal receptors of insects

Summary The battery mother cell complexes in the tentacles ofHydra vulgaris contain a neuronal cell known as sensorimotor interneuron that is characterized by a modified cilium lying parallel to the mesoglea. The cilium is surrounded by up to three rings of microvilli. Microvilli and cilium arise in an unusual antiparallel orientation from the opposite poles of a central cellular cavity. The lumen of this cavity communicates with the extracellular environment by way of a straight channel-like opening that is encircled by the microvillar rings. The modified cilium extends into the channel and terminates outside in the intercellular space. The wall of the cavity and the channel are stabilized by bundles of microtubules. A prominent glycocalyx interconnects all microvilli and links the innermost microvillar ring to the cilium. Within this contact region approximately 0.7 m in length the ciliary axoneme is specifically modified: all nine microtubule doublets and up to six additional microtubules are embedded in electron-dense material. The microtubule doublets are connected to the ciliary membrane by ledges of Y-shaped cross-bridging elements. These axonemal modifications resemble those known from the hydrozoan cnidocil complex or from the outer segments of insect mechanoreceptor cells. Distribution and orientation of the sensorimotor interneuron within the tentacles indicate a mechanosensory function of the cell similar to that of chordotonal receptors of insects.     

Cell differentiation during the larval development of the ophiuroid <Emphasis Type="Italic">Amphipholis kochii</Emphasis> Lütken, 1872 (Echinodermata: Ophiuroidea)

The differentiation of the ectodermal, entodermal, and mesodermal cell lines in developing plutei of the ophiuroid Amphipholis kochii was examined using electron microscopy and the immunochemical staining technique. The ectodermal cells form the pseudostratified epithelium of the ciliary band, the flattened epithelium of the body wall, and the esophageal epithelium. The epithelium of the ciliary band consists of ciliated and mucous cells; at its base is an axonal tract formed of the processes of neurons. The serotoninergic neurons form two lateral ganglia located along the paraoral ciliary band and the posterolateral arms’ ciliary band. The prominent features of the neurons are large size, the presence of a cilium, an electron-light cytoplasm filled with microvesicles with neurotransmitters, and a large nucleus with a predominant euchromatin. The ectoderm cells (except mucous cells) are characterized by the presence of a cilium surrounded by a collar of microvilli and a thin layer of apical extracellular matrix. The entodermal cells form the digestive tract epithelium and differentiate into four cell types: type I and II cells probably function in the nutrient uptake and assimilation; type III cells perhaps secrete digestive enzymes; and myoepithelial cells that constitute the cardiac and pyloric sphincters and the anus. Sclerenchymatous cells, which are the descendants of the primary mesenchyme, form a syncytium around the developing spicules. The biomineralization process is intrasyncytial, the ophioplutei spicules retain the cytoplasmic covering throughout the period of larval development. The secondary mesenchyme gives rise to smooth muscle cells and amebocytes. Muscle cells compose the circumesophageal musculature, the cell processes of each “muscle band” seem to fuse together. At the base of the preoral band are two symmetrically located groups of muscles, viz., the anterior dilators. Amebocytes function in excretion either near the epidermis or are able to penetrate through the epidermis and excrete wastes into the external environment. The mesoderm formed by the enterocoely gives rise to three pairs of coeloms; their cells remain unspecialized during the entire period of larval development. Results of this study are compared with the micro- and neuroanatomy of the larvae of other echinoderms.     

Ciliogenesis: building the cell's antenna

The cilium is a complex organelle, the assembly of which requires the coordination of motor-driven intraflagellar transport (IFT), membrane trafficking and selective import of cilium-specific proteins through a barrier at the ciliary transition zone. Recent findings provide insights into how cilia assemble and disassemble in synchrony with the cell cycle and how the balance of ciliary assembly and disassembly determines the steady-state ciliary length, with the inherent length-dependence of IFT rendering the ciliary assembly rate a decreasing function of length. As cilia are important in sensing and processing developmental signals and directing the flow of fluids such as mucus, defects in ciliogenesis and length control are likely to underlie a range of cilium-related human diseases.     

Development of the Nervous System of Sea Urchin Embryos: Formation of Ciliary Bands and the Appearance of Two Types of Ectoneural Cells in the Pluteus

An ultrastructural study of the larval integument of the sea urchin, Hemicentrotus pulcherrimus , was conducted with special emphasis on the development of the nervous system in relation to the formation of ciliary bands. In the integument of 4-armed pluteus larvae, cells associated with the ciliary band, which have 200 nm-thick projections at their apices, and cells in the squamous epithelium, which have a cilium and long, fine radiating processes in the apical region, were observed. Both cell types have axons at their basal ends that form nerve bundles beneath the ciliary bands, where the axons make contact with ectodermal effector cells with motile cilia. The cilia and other apical projections of these ectoneural cells run parallel to the surface of the cells, and are under the hyaline layer. The axoneme of the cilium has a typical "9 + 2" microtubular arrangement, but generally has no dynein arms. These ectoneural cells are more frequent on the oral surface than on the antioral surface.     

A Distinctive Nerve Cell Type Common to Diverse Deuterostome Larvae: Comparative Data from Echinoderms,Hemichordates and Amphioxus

Abstract The larval ciliary bands of echinoderm bipinnaria and pluteus larvae and the hemichordate tornaria contain similar multipolar or bipolar nerve cells with unusual apical processes that run across the surface of the band between the bases of its cilia. We report on some distinctive ultrastructural features of these cells. Among these are specialized junctions that occur between the cells' apical processes and adjacent ciliary band cells near the base of each cilium. Such structures are best developed in pluteus larvae. Many nerve cells in the larval spinal cord of amphioxus also have large apical processes that cross the central lumen of the cord. We interpret our observations on these cells in terms of Garstang's hypothesis, which derives the chordate neural tube from a larval ciliary band, and suggest that multipolar cells like those in echinoderm and tornaria bands may be the antecedents of some categories of neurons in the chordate spinal cord.     

Review series: The cell biology of vision

The small ciliary G protein Arl13b is required for cilium biogenesis and sonic hedgehog signaling and is mutated in patients with Joubert syndrome (JS). In this study, using Caenorhabditis elegans and mammalian cell culture systems, we investigated the poorly understood ciliary and molecular basis of Arl13b function. First, we show that Arl13b/ARL-13 localization is frequently restricted to a proximal ciliary compartment, where it associates with ciliary membranes via palmitoylation modification motifs. Next, we find that loss-of-function C. elegans arl-13 mutants possess defects in cilium morphology and ultrastructure, as well as defects in ciliary protein localization and transport; ciliary transmembrane proteins abnormally accumulate, PKD-2 ciliary abundance is elevated, and anterograde intraflagellar transport (IFT) is destabilized. Finally, we show that arl-13 interacts genetically with other ciliogenic and ciliary transport–associated genes in maintaining cilium structure/morphology and anterograde IFT stability. Together, these data implicate a role for JS-associated Arl13b at ciliary membranes, where it regulates ciliary transmembrane protein localizations and anterograde IFT assembly stability.     

Rhodopsin transport in the membrane of the connecting cilium of mammalian photoreceptor cells

The transport of the photopigment rhodopsin from the inner segment to the photosensitive outer segment of vertebrate photoreceptor cells has been one of the main remaining mysteries in photoreceptor cell biology. Because of the lack of any direct evidence for the pathway through the photoreceptor cilium, alternative extracellular pathways have been proposed. Our primary aim in the present study was to resolve rhodopsin trafficking from the inner to the outer segment. We demonstrate, predominantly by high-sensitive immunoelectron microscopy, that rhodopsin is also densely packed in the membrane of the photoreceptor connecting cilium. Present prominent labeling of rhodopsin in the ciliary membrane provides the first striking evidence that rhodopsin is translocated from the inner segment to the outer segment of wild type photoreceptors via the ciliary membrane. At the ciliary membrane rhodopsin co-localizes with the unconventional myosin VIIa, the product of human Usher syndrome 1B gene. Furthermore, axonemal actin was identified in the photoreceptor cilium, which is spatially co-localized with myosin VIIa and opsin. This actin cytoskeleton of the cilium may provide the structural bases for myosin VIIa-linked ciliary trafficking of membrane components, including rhodopsin.     

Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry

The primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, whereas resorption of the cilium precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein Nde1 (nuclear distribution gene E homologue 1) as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 have longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer's vesicle and left-right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle progression and have implications for understanding the transition from a quiescent to a proliferative state.     

Anterior neural centres in echinoderm bipinnaria and auricularia larvae: cell types and organization

Serial and interval electron micrograph series were used to examine the anterior part of the ciliary band system in the bipinnaria larva of Pisaster ochraceus and the auricularia larva of Stichopus californicus for evidence of ganglion‐like organization. The bipinnaria has paired concentrations of Multipolar with Apical Processes (MAP) cells in this region that correspond in position with previously identified clusters of serotonergic and peptidergic neurones. MAP cells located in the centre of the band have well‐developed apical processes, but no cilium. Those at the sides of the band have fewer processes, but some have recumbent cilia that extend under the glycocalyx, suggesting a sensory function. Comparable cell types are not found elsewhere in the band, a clear indication that the apical parts of the ciliary band system are organized in a distinctive fashion. Two neuronal cell types were identified in the apical region of the auricularia larva, a conventional bipolar neurone that corresponds with previously described serotonergic apical cells, and more numerous MAP cells for which there is no previous record and hence, no known transmitter. Previous immunocytochemical studies are summarized and re‐examined in the light of these results. Relevant evolutionary issues are also discussed, but the data fail to provide strong evidence either for or against Garstang’s hypothesis that the chordate brain and spinal cord derive from larval ciliary bands resembling those of modern echinoderms.     

Electron microscope study on the development of ciliary components of the neural epithelium of the chick embryo

Summary Ciliary development was studied in the cells of the neural canal of chick embryos incubated from 60 hours to 7 days.It was found that centrioles move after the last mitosis to the cell periphery where one of them enters into contact (terminal contact) with the cell membrane; the other centriole remains close by, its axis aligned along the axis of the former.The cell membrane was seen afterwards bulging at the contact point, and the content of the ciliary bud thus formed is only constituted at the beginning of a varied number of vesicles of about 140 Å diameter.The ciliary bud becomes elongated shortly after filaments start becoming organized in the bud matrix.Roughly coinciding with the initiation of filament organization the centrioles move inward and the cilium becomes deeply invaginated in the cell. At the end of ciliary growth the centriole moves again toward the surface and the cilium emerges in the neural canal lumen.     

Structural studies of ciliary components

Cilia are organelles found on most eukaryotic cells, where they serve important functions in motility, sensory reception, and signaling. Recent advances in electron tomography have facilitated a number of ultrastructural studies of ciliary components that have significantly improved our knowledge of cilium architecture. These studies have produced nanometer-resolution structures of axonemal dynein complexes, microtubule doublets and triplets, basal bodies, radial spokes, and nexin complexes. In addition to these electron tomography studies, several recently published crystal structures provide insights into the architecture and mechanism of dynein as well as the centriolar protein SAS-6, important for establishing the 9-fold symmetry of centrioles. Ciliary assembly requires intraflagellar transport (IFT), a process that moves macromolecules between the tip of the cilium and the cell body. IFT relies on a large 20-subunit protein complex that is thought to mediate the contacts between ciliary motor and cargo proteins. Structural investigations of IFT complexes are starting to emerge, including the first three-dimensional models of IFT material in situ, revealing how IFT particles organize into larger train-like arrays, and the high-resolution structure of the IFT25/27 subcomplex. In this review, we cover recent advances in the structural and mechanistic understanding of ciliary components and IFT complexes.     

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.     

Biogenesis of the ciliary roots: participation of the dictyosomes of Golgi's complex.

In this study, the hypothesis of a possible biogenesis of the ciliary roots is suggested, after observing the cilia neurons under the electron microscope, which were found as an exception in the periaqueductal nucleus of the mesencephalon in the domestic cat, conserving the potential to differentiate the cilia, basal bodies and ciliary roots. The dictyosomes of Golgi's complex and Golgi's vesicles participated in this biogenesis. Vesicles of approximately 71.6 nm in diameter had become separated from the periphery of the flattened discoid cisterns of the dictyosome and were aligned normally, in tangential contact with each other, forming rows of vesicles or 'ringed chains', whose points of contact formed the beginning of the 'periodic striation' of a thin ciliary root. Later, the lateral walls of the vesicles and the molecules of the intracisternal proteins gave rise to the interperiodic microfilaments, when the carrier proteins were transformed into structural proteins of the ciliary roots. The parallel apposition of several ringed chains or thin ciliary roots, with their rings joined at the same level (or transversal striations), gave rise to thicker striated roots. This hypothesis of an ultrastructural biogenesis of the striated ciliary roots involves the following six stages: stage I = separation of Golgi's vesicles from the periphery of the flattened disk of dictyosomes near the basal body, with a diameter of over 71.6 nm; stage II = reinforcement of the membrane of the vesicles at the two opposite polar ends of its larger diameter; stage III = alignment of vesicles to form ringed chains, due to the tangential contact between their reinforced points; initiation of the 71.6-nm striation period, roots ringed linearly; stage IV = formation of joining microfilaments between periods (69.2 nm) with the lateral walls of the vesicles and the molecules of the proteins in their content; stage V = lengthening of the thin ciliary roots due to the coupling of new Golgi's vesicles at their ends so that their length increases as a result of the addition of terminal vesicles; stage VI = increase in thickness of the thin ciliary roots, due to the parallel apposition of several ringed chains or thin ringed ciliary roots, at the point where their transversal striation points coincide.