Ciliary Organelles Research Articles

Role of endosomal pathway in the ciliary transport and the membrane organization of outer segment disc membrane in photoreceptors

A photoreceptor is a specialized neuron that is responsible for the conversion of light into an electrical signal. Photoreceptors are classified into rods and cones, and both photoreceptors possess light-sensing ciliary organelles called outer segments (OSs), anchored in the cells by a microtubule-based axoneme. The OS consists of a stack of disc membranes, which are abundant for the retinal phototransduction proteins such as rhodopsin. Recently, modern protein synchronization techniques using in vivo transfection in rodents revealed that rhodopsin transits through Rab11-positive recycling endosomes, preferentially entering the OS in the dark. Moreover, Peripherin-2 (PRPH2, also called retinal degeneration slow, RDS), a photoreceptor-specific tetraspanin protein essential for the morphogenesis of disc membranes, is delivered to the OS following complementary to that of rhodopsin. Various PRPH2 disease-causing mutations have been found in humans, and most of the mutations in the cytosolic C-terminus of PRPH2 are linked to cone-dominant macular dystrophies. It has been shown that the late endosome is the waystation that sorts newly synthesized PRPH2 into the cilium. The multiple C-terminal motifs of PRPH2 regulate its late endosome and ciliary targeting through ubiquitination and binding to an Endosomal Sorting Complexes Required for Transport (ESCRT) component, Hrs. These findings suggest that the late endosomes play an important role in the biosynthetic pathway of ciliary proteins and can be a new therapeutic target for the diseases caused by ciliary defects.

Sequence and Properties of Cagein, a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus.

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as "basal-body cages." The major protein comprising Euplotes cages has been previously identified and termed "cagein." Screening a E.aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear "nanochromosome" of ca. 1.5kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.

A Flow Cytometry-Based Approach for the Isolation and Characterization of Neural Stem Cell Primary Cilia.

In the adult mammalian brain, the apical surface of the subependymal zone (SEZ) is covered by many motile ependymal cilia and a few primary cilia originating from rare intermingled neural stem cells (NSCs). In NSCs the primary cilia are key for the transduction of essential extracellular signals such as Sonic hedgehog (SHH) and platelet-derived growth factor (PDGF). Despite their importance, the analysis of NSC primary cilia is greatly hampered by the fact that they are overwhelmingly outnumbered by the motile cilia. We here take advantage of flow cytometry to purify the two cilia types and allow their molecular characterization. Primary cilia were identified based on immunoreactivity to the marker adenylate cyclase type III (AC3) and differential levels of prominin-1 whereas motile cilia displayed immunoreactivity only to the latter. Consistent with the morphological differences between the two classes of cilia, enrichment of motile cilia positively correlated with size. Moreover, we observed age-dependent variations in the abundance of the two groups of ciliary organelles reflecting the changes associated with their development. The two cilia groups also differed with respect to the expression of signaling molecules, since PDGF receptor (PDGFR)α, smoothened (Smo) and CXC chemokine receptor (CXCR)4 were only detected in isolated primary but not motile cilia. Thus, our novel method of cilia isolation and characterization by flow cytometry has the potential to be extended to the study of cilia from different tissues and organs, providing a powerful tool for the investigation of primary cilia in physiological and pathological conditions.

Cortical Localization of α- and γ-Tubulin and the Assembly of Cortical Microtubule Cytoskeleton in Hypotrichous Ciliate Euplotes eurystomus

This study aimed to investigate the assembly characteristics of ciliature and cortical microtubules and the localization of tubulins in different depths and regions of the cortex. The hypotrichous ciliates have closely arranged cilia and a highly complex microtubular system. Direct fluorescence and immunofluorescence labeling were used to observe ciliary organelles and cortical microtubular cytoskeleton in Euplotes eurystomus. An immunofluorescence analysis demonstrated that α-tubulin localized to the ventral and dorsal ciliary organelles and their associated microtubules, while γ-tubulin localized to the basal bodies of ciliary organelles, macronuclear membrane, and excretory pore of a contractile vacuole in the interphase. A direct fluorescence analysis showed that the ciliature and cortical microtubules in the deep cortex were more clearly marked by fluorescent taxoid (FLUTAX). Interestingly, α- and γ-tubulins also colocalized to the ringlike ciliary base–associated microtubules of dorsal kineties. The short microtubular bundles between the bases of transverse cirri could be marked by FLUTAX and γ-tubulin rather than α-tubulin, suggesting that tubulins in cortical microtubules in E. eurystomus varied, and the differentiation of cortical microtubules in the hypotrichous ciliate tended to be versatile. Also, during morphogenesis, γ-tubulin also localized to the base of ciliary primordium, where new basal bodies were formed, suggesting that the endocellular position of γ-tubulin in ciliates was related to basal bodies and regulated by the cell cycle. This study might help understand the assembly characteristics and tubulin composition of microtubules in different depths and regions of the cortex in hypotrichous ciliate E. eurystomus.

Sensory mechanisms in dentine: A literature review of light microscopy (LM), transmission microscopy (TEM), scanning microscopy (SEM) & electro physiological (EP) tooth sensitivity: Is the ciliary organelle on the odontoblast the elusive primary nociceptor?

Sensory mechanisms in dentine: A literature review of light microscopy (LM), transmission microscopy (TEM), scanning microscopy (SEM) & electro physiological (EP) tooth sensitivity: Is the ciliary organelle on the odontoblast the elusive primary nociceptor?

The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling.

Left-right asymmetries in brains are usually minor or cryptic. We report brain asymmetries in the tiny, dorsal tubular nervous system of the ascidian tadpole larva, Ciona intestinalis. Chordate in body plan and development, the larva provides an outstanding example of brain asymmetry. Although early neural development is well studied, detailed cellular organization of the swimming larva's CNS remains unreported. Using serial-section EM we document the synaptic connectome of the larva's 177 CNS neurons. These formed 6618 synapses including 1772 neuromuscular junctions, augmented by 1206 gap junctions. Neurons are unipolar with at most a single dendrite, and few synapses. Some synapses are unpolarised, others form reciprocal or serial motifs; 922 were polyadic. Axo-axonal synapses predominate. Most neurons have ciliary organelles, and many features lack structural specialization. Despite equal cell numbers on both sides, neuron identities and pathways differ left/right. Brain vesicle asymmetries include a right ocellus and left coronet cells.

Evolution of Cilia

Anton van Leeuwenhoek's startling microscopic observations in the 1600s first stimulated fascination with the way that cells use cilia to generate currents and to swim in a fluid environment. Research in recent decades has yielded deep knowledge about the mechanical and biochemical nature of these organelles but only opened a greater fascination about how such beautifully intricate and multifunctional structures arose during evolution. Answers to this evolutionary puzzle are not only sought to satisfy basic curiosity, but also, as stated so eloquently by Dobzhansky (Am Zool 4: 443 [1964]), because "nothing in biology makes sense except in the light of evolution." Here I attempt to summarize current knowledge of what ciliary organelles of the last eukaryotic common ancestor (LECA) were like, explore the ways in which cilia have evolved since that time, and speculate on the selective processes that might have generated these organelles during early eukaryotic evolution.

Molecular basis for photoreceptor outer segment architecture.

To serve vision, vertebrate rod and cone photoreceptors must detect photons, convert the light stimuli into cellular signals, and then convey the encoded information to downstream neurons. Rods and cones are sensory neurons that each rely on specialized ciliary organelles to detect light. These organelles, called outer segments, possess elaborate architectures that include many hundreds of light-sensitive membranous disks arrayed one atop another in precise register. These stacked disks capture light and initiate the chain of molecular and cellular events that underlie normal vision. Outer segment organization is challenged by an inherently dynamic nature; these organelles are subject to a renewal process that replaces a significant fraction of their disks (up to ∼10%) on a daily basis. In addition, a broad range of environmental and genetic insults can disrupt outer segment morphology to impair photoreceptor function and viability. In this chapter, we survey the major progress that has been made for understanding the molecular basis of outer segment architecture. We also discuss key aspects of organelle lipid and protein composition, and highlight distributions, interactions, and potential structural functions of key OS-resident molecules, including: kinesin-2, actin, RP1, prominin-1, protocadherin 21, peripherin-2/rds, rom-1, glutamic acid-rich proteins, and rhodopsin. Finally, we identify key knowledge gaps and challenges that remain for understanding how normal outer segment architecture is established and maintained.

Metabolites from the Euryhaline Ciliate Pseudokeronopsis erythrina

AbstractThree new secondary metabolites (named erythrolactones A2, B2 and C2), that are characterized by a central 4‐hydroxy‐unsaturated δ lactone ring bearing an alkyl saturated chain at C(2) and a butyl‐benzenoid group at C(5), together with their respective sulfate esters (erythrolactones A1, B1 and C1), have been isolated from cell cultures of Pseudokeronopsis erythrina, clone TL‐1. The structures are assigned on the basis of extensive spectroscopic measurements (1D and 2D NMR, UV, IR and HR‐MALDI‐TOF). A plausible biogenetic route for their formation is also suggested. Cold‐shock treatment was performed in order to induce the discharge of the metabolites contained in pigment granules lying on the ciliary organelles of this microorganism. HPLC‐ESI‐MS analysis of this granule discharge reveals that erythrolactones A2–C2 are actually therein contained, strongly suggesting a possible role for these metabolites in the chemical defence strategy of P. erythrina.

The roles of evolutionarily conserved functional modules in cilia-related trafficking

Cilia are present across most eukaryotic phyla and have diverse sensory and motility roles in animal physiology, cell signalling and development. Their biogenesis and maintenance depend on vesicular and intraciliary (intraflagellar) trafficking pathways that share conserved structural and functional modules. The functional units of the interconnected pathways, which include proteins involved in membrane coating as well as small GTPases and their accessory factors, were first experimentally associated with canonical vesicular trafficking. These components are, however, ancient, having been co-opted by the ancestral eukaryote to establish the ciliary organelle, and their study can inform us about ciliary biology in higher organisms.

The interplay between RPGR, PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo

Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR-interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo-loaded PDEδ and exposes the Arl2/Arl3-binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo-loaded PDEδ and Arl3 to release lipidated cargo into cilia.

Structure and Protein Composition of a Basal‐Body Scaffold (“Cage”) in the Hypotrich Ciliate Euplotes

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal-body "cage". Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45-kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45-kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double-layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or "basal" cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or "medial" cage layer. The isolated cage thus resembles a miniature test-tube rack, able to accommodate varying arrangements of basal-body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal-body cages in Euplotes, we have termed this novel 45-kDa protein "cagein".

Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model

Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.

Re‐Evaluation on the Diversity of the Polyphyletic Genus Metaurostylopsis (Ciliophora, Hypotricha): Ontogenetic, Morphologic, and Molecular Data Suggest the Establishment of a New Genus Apourostylopsis n. g.

The urostylid genus Metaurostylopsis Song et al., 2001 was considered to be a well-outlined taxon. Nevertheless, recent evidence, including morphological, ontogenetic, and molecular information, have consistently revealed conflicts among congeners, regarding their systematic relationships, ciliature patterns, and origins of ciliary organelles. In the present work, the morphogenetic and morphogenetic features were re-checked and compared, and the phylogeny of nominal species was analysed based on information inferred from the small subunit ribosomal RNA (SS rRNA) gene sequence. In addition, the binary divisional process in a new isolate of Metaurostylopsis struederkypkeae Shao et al., 2008 is described. All results obtained reveal that the genus is a polyphyletic assemblage whose nominal congeners fall into three clades within the core Urostylida, based on SS rRNA gene sequences. These three clades not match the groups inferred from morphological/morphogenetical evidences. Some conflicting data from molecular and ontogenetic studies also indicate that single-gene information might not be consistently reliable in detecting the phylogenetic relationships among closely related groups and comprehensive multi-gene analyses are necessary to give a more exact evaluation for this divergent assemblage. According to our new understandings, five forms are confirmed to be true Metaurostylopsis. The morphotype Metaurostylopsis sinica Shao et al., 2008 should be excluded from the genus and represents a distinct type, and, thus, a new genus Apourostylopsis n. g. with it as the type specie, i.e. Apourostylopsis sinica (Shao et al., 2008) n. comb.

Cisplatin induces modifications in the development of cell surface patterns of ciliates.

Cisplatin [cis-dichlorodiammineplatinum (II)] brings about significant quantitative modifications in the development of cell surface patterns in two unrelated ciliates: Stylonychia and Tetrahymena. Cells cultured in the presence of cisplatin exhibit the formation of supernumerary surface structures in the form of extra cilia/cirri (fused cilia) and other organized ciliary organelles. The metal-induced formation of extra primordia and their differentiation into supernumerary ciliary structures is governed by the normal rules of development. Additional structures are accommodated within the framework of a defined pattern, suggesting the existence of an overall global control of pattern formation. The modified pattern is rectified to its normal state during post-treatment fission cycles, suggesting the role of the cell membrane in correcting developmental errors.

A novel cilia-based feature within the food grooves of the ctenophore Mnemiopsis mccradyi Mayer

We describe a novel compound ciliary structure (g-cilium) from the food groove of the lobate ctenophore Mnemiopsis mccradyi. G-cilia are small, flat compound ciliary organelles that are oriented with their tips pointing toward the mouth. Typically three to four rows of g-cilia line the inner surface of the tentacular groove, which together with the transport groove, make up the food groove. G-cilium cells are ∼11.4 μm long and ∼4.2 μm wide at the g-cilium base. The g-cilium itself is ∼3.4 μm long and tapers to a flat, sharp tip. G-cilia are not motile but are surrounded by many hundreds of smaller, actively motile cilia that beat with orally-directed effective strokes. G-cilia contain ∼50 conventional `9+2' cilia embedded in a fibrous core that arises from the cell body. In addition, g-cilia contain mitochondria, thousands of small membrane-bounded vesicles and rod bacteria. G-cilia basal bodies are anchored by large, strongly-banded rootlets that extend approximately the entire length of the cell. G-cilia may have organizational, sensory and/or secretory function within the feeding apparatus. Their placement strongly suggests that they play critical roles in feeding. They may enhance the efficiency of prey capture and so contribute to M. mccradyi's well-known voracious appetite. By enhancing prey capture they probably play a critical role in the capacity of this organism to follow prey dynamics, so contributing to dense blooms in mid-late summer in coastal regions.

Investigating Phagocytosis in Tetrahymena: An Experimental System Suitable for Introductory & Advanced Instruction

Tetrahymena, a freshwater ciliated protozoan, is in many ways an ideal candidate for the study of cells by students. Readily obtained and cultured, Tetrahymena can be used to study ciliary motion, organelle structure and function, cell morphology, cell proliferation, and cell behaviors (Leick & Hellung-Larsen 1992; Wheatley et al. 1994). Because of the ease with which Tetrahymena can be grown and manipulated, it is especially suitable for use in inquiry-based labs and student projects. Phagocytosis in Tetrahymena is an especially dramatic cell behavior that students can observe directly with a compound microscope. When hungry Tetrahymena encounter food, they use their cilia to sweep it into each cell's oral groove. This process can be visualized by feeding stained yeast cells India ink (Keenan 1984) or Chlorella to Tetrahymena. Phagocytosis can be quantitated by counting the number of vacuoles that form in a defined time period. In this paper, I describe how our students use the India ink method, described below, to investigate phagocytosis and vacuole formation in Tetrahymena first to learn the basis of microscope use, and then to pursue various experimental projects.

Ciliary beating in three dimensions: Steps of a quantitative description

We document a novel approach for quantitative assessment of ciliary activity, exemplified in rapid three-dimensional cyclic motion of the frontal cirri of Stylonychia. Cells held under voltage-clamp control are stimulated by step pulses to elicit reproducible hyperpolarization- or depolarization-induced ciliary motor responses. High-speed video recording at 200 fields per second is used for imaging ciliary organelles of the same cell in two perspectives: the axial view and, following cell rotation by 90 degrees, the lateral view. From video sequences of typically 1 s, the contours of the cirral images are determined and digitized. Computer programs are established to (1) reduce an observed image to a "ciliary axis", (2) sort series of axes by template to generate an averaged ciliary cycle in 2D-projection, and (3) to associate the generalized axial and lateral 2D-images for generation of a sequence of three-dimensional images, which quantitatively represent the cycle in space and time. The method allows us to produce predetermined perspectives of images selected from the ciliary cycle, and to generate stereo views for graphical representation of ciliary motion. The approach includes a potential for extraction of the complete microtubular sliding program of a cilium under reproducible electric stimulation of the ciliary membrane.

Somatic kineties or paroral membrane: which came first in ciliate evolution?

The ciliate species which lack a distinctive oral ciliature are considered to represent an ancestral state in ciliate evolution. Consequently, the somatic kineties composed of kinetids (kinetosomes plus cilia and associated fibrillar systems) are thought to be the ancestral ciliature. Results on stomatogenesis in ‘gymnostomial ciliates’ have shown that these ciliates probably have evolved from ancestors already equipped with an oral ciliature. Thus instead of the somatic, the oral ciliature may be regarded as ancestral. Based on these ideas a hypothesis on the evolution of the ciliate kinetome (assembly of all kinetids covering the body of a given ciliate) is presented. The first step in the evolution of the kinetome was the formation of a paroral membrane, a compound ciliary organelle lying along the right side of the oral area which historically but falsely is termed membrane. It was composed of kinetosomal dyads (dikinetids), derived from the kinetid of a dinoflagellate-like ancestor. From the beginning the paroral membrane was responsible for locomotion, ingestion and for the formation of a cytopharyngeal tube which the first ciliate probably had inherited from its flagellate ancestor. In the second step a first somatic kinety was formed from the right row of kinetosomes of the paroral membrane as a result of a longitudinal splitting of the paroral membrane and a subsequent migration of the forming kinety to the right into the somatic cortex. To increase the number of somatic kineties this process was repeated until the kinety produced first reached the left border of the oral area. By this step the locomotive and the nutritional functions were differentiated between somatic and oral structures. In a third step the adoral organelles were formed from somatic kinetids left of the oral area. The primitive type of stomatogenesis was a buccokinetal one derived from the mode the flagellate ancestor used to distribute its replicated kinetosomes to the offspring cells (buccokinetal means that at least parts of the oral anlage for the posterior offspring cell has its origin in the parental oral apparatus). This hypothesis, based on comparative studies on ciliate morphogenesis, is corroborated by molecular data from other laboratories.

A Novel Type of Ciliary Activity in Stylonychia Mytilus: Analysis of Potential-Coupled Motor Responses in the Transverse Cirri

ABSTRACT The motor responses of the transverse cirri of Stylonychia mytilus were investigated by applying high-speed microcinematography and step voltageclamp. As a response to hyperpolarization, the transverse cirri began to swing posteriorly from an inactive upright posture at rest. The deeply inclined posture was maintained as long as the hyperpolarizing pulse was on. Upon depolarization, the cirri began to swing towards the anterior end of the cell and continued regular cyclic beating, orienting the effective stroke anteriorly. Motor responses of the transverse cirri occurred in quasi-planar motion, allowing analysis of the bend configuration along the full length of the cirri. Beating activity induced during sustained depolarization was virtually stable, with different oscillation profiles at base and tip. Cyclic movement of the distal region was enhanced at large amplitudes of depolarization. Termination of a hyperpolarizing voltage step induced a transient depolarization-type anterior beating, and termination of a depolarizing step induced a transient posteriad inclination of the transverse cirri. In both hyperpolarization- and depolarization-induced motor responses, a shear angle analysis of the initiation of the response indicated that sliding displacement of doublet microtubules was initiated at the base and propagated towards the tip. The discovery in a ciliary organelle of a very distinct response to hyperpolarizing and depolarizing stimulation is highly useful for the analysis of ciliary electromotor coupling. The functions of intraciliary Ca2+ in the regulation of the motor responses are discussed.