The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization

Both the basal body and the microtubule-based axoneme it nucleates have evolutionarily conserved subdomains crucial for cilium biogenesis, function and maintenance. Here, we focus on two conspicuous but underappreciated regions of these structures that make membrane connections. One is the basal body distal end, which includes transition fibres of largely undefined composition that link to the base of the ciliary membrane. Transition fibres seem to serve as docking sites for intraflagellar transport particles, which move proteins within the ciliary compartment and are required for cilium biogenesis and sustained function. The other is the proximal-most region of the axoneme, termed the transition zone, which is characterized by Y-shaped linkers that span from the axoneme to the ciliary necklace on the membrane surface. The transition zone comprises a growing number of ciliopathy proteins that function as modular components of a ciliary gate. This gate, which forms early during ciliogenesis, might function in part by regulating intraflagellar transport. Together with a recently described septin ring diffusion barrier at the ciliary base, the transition fibres and transition zone deserve attention for their varied roles in forming functional ciliary compartments.     

Ultrastructure of cilia and flagella - back to the future!

Eukaryotic cilia and flagella perform motility and sensory functions which are essential for cell survival in protozoans, and to organism development and homoeostasis in metazoans. Their ultrastructure has been studied from the early beginnings of electron microscopy, and these studies continue to contribute to much of our understanding about ciliary biology. In the light of the progress made in the visualization of cellular structures over the last decade, we revisit the ultrastructure of cilia and flagella. We briefly describe the typical features of a 9+2 axoneme before focusing extensively on the transition zone, the ciliary necklace, the singlet zone, the ciliary cap and the ciliary crown. We discuss how the singlet zone is linked to sensory and/or motile function, the contribution of the ciliary crown to ovocyte and mucosal propulsion, and the relationship between the ciliary cap and microtubule growth and shortening, and its relation to ciliary beat. We further examine the involvement of the transition zone/the ciliary necklace in axonemal stabilization, autotomy and as a diffusion barrier.     

TMEM231, mutated in orofaciodigital and Meckel syndromes,organizes the ciliary transition zone

The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.     

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.     

The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans

Cilia are cellular projections that perform sensory and motile functions. A key ciliary subdomain is the transition zone, which lies between basal body and axoneme. Previous work in Caenorhabditis elegans identified two ciliopathy-associated protein complexes or modules that direct assembly of transition zone Y-links. Here, we identify C. elegans CEP290 as a component of a third module required to form an inner scaffolding structure called the central cylinder. Co-inhibition of all three modules completely disrupted transition zone structure. Surprisingly, axoneme assembly was only mildly perturbed. However, dendrite extension by retrograde migration was strongly impaired, revealing an unexpected role for the transition zone in cell adhesion.     

Ciliary trafficking: CEP290 guards a gated community

A recent study reveals that the large coiled-coil protein CEP290 is an integral component of the transition zone between the cell body and the cilium and functions as a gatekeeper to regulate trafficking of ciliary proteins.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS‐5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y‐shaped axoneme‐to‐membrane connectors. Coiled‐coil and C2 domains within MKS‐5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body‐associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS‐5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP‐2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP₂ within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment.     

Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may participate in their pathogenesis. The ciliary transition zone contains two protein complexes affected in the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP). The BBSome is a third protein complex, affected in the ciliopathy Bardet-Biedl syndrome (BBS). We tested whether mutations in MKS, NPHP and BBS complex genes modify the phenotypic consequences of one another in both C. elegans and mice. To this end, we identified TCTN-1, the C. elegans ortholog of vertebrate MKS complex components called Tectonics, as an evolutionarily conserved transition zone protein. Neither disruption of TCTN-1 alone or together with MKS complex components abrogated ciliary structure in C. elegans. In contrast, disruption of TCTN-1 together with either of two NPHP complex components, NPHP-1 or NPHP-4, compromised ciliary structure. Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did. As in nematodes, disrupting two components of the mouse MKS complex did not cause additive phenotypes compared to single mutants. However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1. Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.     

Rpgrip1l controls ciliary gating by ensuring the proper amount of Cep290 at the vertebrate transition zone

A range of severe human diseases called ciliopathies is caused by the dysfunction of primary cilia. Primary cilia are cytoplasmic protrusions consisting of the basal body (BB), the axoneme, and the transition zone (TZ). The BB is a modified mother centriole from which the axoneme, the microtubule-based ciliary scaffold, is formed. At the proximal end of the axoneme, the TZ functions as the ciliary gate governing ciliary protein entry and exit. Since ciliopathies often develop due to mutations in genes encoding proteins that localize to the TZ, the understanding of the mechanisms underlying TZ function is of eminent importance. Here, we show that the ciliopathy protein Rpgrip1l governs ciliary gating by ensuring the proper amount of Cep290 at the vertebrate TZ. Further, we identified the flavonoid eupatilin as a potential agent to tackle ciliopathies caused by mutations in RPGRIP1L as it rescues ciliary gating in the absence of Rpgrip1l.     

Development of ciliary bands in larvae of the living isocrinid sea lily Metacrinus rotundus

Embryos and larvae of an isocrinid sea lily, Metacrinus rotundus, are described by scanning electron microscopy. Around hatching (35 h after fertilization), the outer surface of the gastrula becomes ubiquitously covered with short cilia. At 40 h, the hatched swimming embryo develops a cilia‐free zone of ectoderm on the ventral side. By 3 days, the very early dipleurula larva develops a cilia‐free zone ventrally, densely ciliated regions laterally, and a sparsely ciliated region dorsally. At this stage, the posterior and anterior ciliary bands first appear: the former runs along a low ridge separating the densely from the sparsely ciliated epidermal regions, while the latter is visible, at first discontinuously, along the boundary between the densely ciliated lateral regions and the cilia‐free ventral zone. In the late dipleurula larva (5 days after fertilization), the anterior and posterior loops of ciliary bands are well defined. The transition from the dipleurula to the semidoliolaria larva occurs at 6 days as the posterior loop becomes rearranged to form incompletely circumferential ciliary bands. The larva becomes competent to settle at this stage. The arrangement of the ciliary bands on the semidoliolaria is maintained during the second week of development, while the larva retains its competence to settle. The larval ciliary patterns described here are compared with those of stalkless crinoids and eleutherozoan echinoderms. The closest morphological similarities are between M. rotundus and the basal eleutherozoan class Asteroidea.     

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.     

Scanning Electron Microscopy of the Adoral Ciliary Zone of Cycloposthium Bundle (Ciliophora,Entodiniomorphida)1

The adoral ciliary zone of Cycloposthium spp., inhabiting the large intestine of the horse, was studied by scanning electron microscopy. It could be divided into four parts: outer, inner, left, and right zones. The outer zone, extending on the anterior periphery of the apical cone of the body, had 20 tuft-like syncilia arranged radially around the longitudinal axis. Each ciliary tuft consisted of about 170 cilia, and in cross section it had a rectangular shape. The cilia of the inner zone, situated at the top of the apical cone, were aggregated irregularly to form shorter bundles than the tufts of the outer zone. The innermost cilia of this zone were shorter than the outermost. There was a distinct non-ciliated border between the outer and inner zones. A horseshoe-like operculum having no cilia was present at the center of the adoral ciliary zone, and the opening of the vestibulum was situated as a cleft crossing from the center to the right periphery of this zone. No cilia extended onto the vestibular wall. The left ciliary zone was situated beneath the outer zone and consisted of five short rows of barren kinetosomes of which only the central row possessed very short cilia. The right ciliary zone, consisting of a few rows of cilia situated at the bottom of the inner adoral lip, was also easily distinguished from the other ciliary zones. This zone was interpreted as an extension of the outer adoral zone passing along the right side of the apical cone. These ciliary zones were considered to be highly differentiated and useful for both movement of and ingestion of food.     

The centrosomal kinase Plk1 localizes to the transition zone of primary cilia and induces phosphorylation of nephrocystin-1

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.     

Scanning electron microscopy of the adoral ciliary zone of Cycloposthium bundle (Ciliophora, Entodiniomorphida)

The adoral ciliary zone of Cycloposthium spp., inhabiting the large intestine of the horse, was studied by scanning electron microscopy. It could be divided into four parts: outer, inner, left, and right zones. The outer zone, extending on the anterior periphery of the apical cone of the body, had 20 tuft-like syncilia arranged radially around the longitudinal axis. Each ciliary tuft consisted of about 170 cilia, and in cross section it had a rectangular shape. The cilia of the inner zone, situated at the top of the apical cone, were aggregated irregularly to form shorter bundles than the tufts of the outer zone. The innermost ciliar of this zone were shorter than the outermost. There was a distinct non-ciliated border between the outer and inner zones. A horseshoe-like operculum having no cilia was present at the center of the adoral ciliary zone, and the opening of the vestibulum was situated as a cleft crossing from the center to the right periphery of this zone. No cilia extended onto the vestibular wall. The left ciliary zone was situated beneath the outer zone and consisted of five short rows of barren kinetosomes of which only the central row possessed very short cilia. The right ciliary zone, consisting of a few rows of cilia situated at the bottom of the inner adoral lip, was also easily distinguished from the other ciliary zones. This zone was interpreted as an extension of the outer adoral zone passing along the right side of the apical cone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antennal receptors in the blowfly Calliphora erythrocephala: II. Fine structure of the large pedicellar campaniform sensillum

A large mechanosensory campaniform sensillum (LCS) is found close to the flagellum/pedicellus joint in the antennae of the blowfly Calliphora erythrocephala. The LCS possesses a single sensory cell, enveloping cells and a cuticular stimulus-conducting structure. The distal part of the sensory process is developed as a tubular body and is connected to the two parts of the stimulusconducting apparatus. The sensory cell is characterized by the complete absence of ciliary structures in the transition zone between dendrite and sensory process.     

Ultrastructure of gill cilia and ciliary rootlets of Chaetoderma nitidulum Lovén 1844 (Mollusca, Chaetodermomorpha)

Cilia and associated structures on the gill lamellae on the ctenidum of Chaetoderma nitidulum were studied. The gill cilia are very long and have a whip-like narrow portion distally, where only three microtubule doublets continue to the distal tip. In the transition zone between the cilium and the centriolar triplet section of the basal body there is a dense plate, an aggregation of granules and a ciliary necklace with four strands. Further down there is a short cross-striated basal foot and two conical cross-striated ciliary rootlets. The first rootlet is flattened and directed forward. It connects distally with the basal feet of other adjacent cilia. The second rootlet is rounded in cross-section and vertically directed. The epithelial structures of Chaetoderma show similarities with other Mollusca. We found no structural characters that could support the current hypothesis of a close relationship of Xenoturbella to the Mollusca.     

TCTN3 Mutations Cause Mohr-Majewski Syndrome

Orofaciodigital syndromes (OFDSs) consist of a group of heterogeneous disorders characterized by abnormalities in the oral cavity, face, and digits and associated phenotypic abnormalities that lead to the delineation of 13 OFDS subtypes. Here, by a combined approach of homozygozity mapping and exome ciliary sequencing, we identified truncating TCTN3 mutations as the cause of an extreme form of OFD associated with bone dysplasia, tibial defect, cystic kidneys, and brain anomalies (OFD IV, Mohr-Majewski syndrome). Analysis of 184 individuals with various ciliopathies (OFD, Meckel, Joubert, and short rib polydactyly syndromes) led us to identify four additional truncating TCTN3 mutations in unrelated fetal cases with overlapping Meckel and OFD IV syndromes and one homozygous missense mutation in a family with Joubert syndrome. By exploring roles of TCTN3 in human ciliary related functions, we found that TCTN3 is necessary for transduction of the sonic hedgehog (SHH) signaling pathway, as revealed by abnormal processing of GLI3 in patient cells. These results are consistent with the suggested role of its murine ortholog, which forms a complex at the ciliary transition zone with TCTN1 and TCTN2, both of which are also implicated in the transduction of SHH signaling. Overall, our data show the involvement of the transition zone protein TCTN3 in the regulation of the key SHH signaling pathway and that its disruption causes a severe form of ciliopathy, combining features of Meckel and OFD IV syndromes.     

Rabl2 GTP hydrolysis licenses BBSome‐mediated export to fine‐tune ciliary signaling

Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome‐mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2‐GTP enters cilia by binding to IFT‐B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT‐B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP‐locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine‐tune cilia‐dependent signaling for normal embryonic development and organismic homeostasis.     

Scanning electron microscopy of ciliary zones of the ciliate protozoa in the large intestine of the horse.

The surface structure of the ciliary zone in 13 species of ciliates found in the large intestine of the horse was observed by scanning electron microscopy. In Holophryoides ovalis many fine depressions considered to be a result of phagocytosis or pinocytosis in the naked cytostome were noticed. In Blepharocorys spp. a distinct section was present between the portion with cilia and that without cilia. It was not present, however, in some species of the family Buetschliidae, such as Bundleia postciliata and Didesmis spp. The species of Entodiniomorphida had a lip around the ciliary zone with cilia forming synciliary tufts. In Spirodinium equi and Tetratoxum unifasciculatum the ciliary zone revolved counter-clockwise in an en face view. Some differences in the surface structure of the ciliary zone between the entodiniomorphid and spirotrich ciliates are discussed.