NDP kinase moves into developing primary cilia

Inmunofluorescence staining of murine NIH3T3 fibroblasts grown at high density shows that conventional nucleoside diphosphate (NDP) kinases A and B localize to a sensory organelle, the primary cilium. Similar results are obtained with Xenopus A6 kidney epithelial cells, suggesting that NDP kinases are a universal component of the primary cilium. The translocation of NDP kinase into primary cilia depends on size, taking place only when cilia reach a critical length of 5-6 microm. In mature cilia, NDP kinases are distributed along the ciliary shaft in a punctate pattern that is distinct from the continuous staining observed with acetylated alpha-tubulin, a ciliary marker and axonemal component. Isolation of a fraction enriched in primary cilia from A6 cells led to the finding that ciliary NDP kinase is enzymatically active, and is associated with the membrane and the matrix, but not the axoneme. In contrast, acetylated alpha-tubulin is found in the axoneme and, to a lesser extent, in the membrane. Based on the tightly regulated translocation process and the subciliary distribution pattern of NDP kinase, we propose that it plays a role in the elongation and maintenance of primary cilia by its ability to regenerate the GTP utilized by ciliary microtubule turnover and transmembrane signaling.     

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

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

Primary cilium mechanotransduction of tensile strain in 3D culture: Finite element analyses of strain amplification caused by tensile strain applied to a primary cilium embedded in a collagen matrix

Human adipose-derived stem cells (hASC) exhibit multilineage differentiation potential with lineage specification that is dictated by both the chemical and mechanical stimuli to which they are exposed. We have previously shown that 10% cyclic tensile strain increases hASC osteogenesis and cell-mediated calcium accretion. We have also recently shown that primary cilia are present on hASC and that chemically-induced lineage specification of hASC concurrently results in length and conformation changes of the primary cilia. Further, we have observed cilia length changes in hASC cultured within a collagen I gel in response to 10% cyclic tensile strain. We therefore hypothesize that primary cilia may play a key mechanotransduction role for hASC exposed to tensile strain. The goal of this study was to use finite element analysis (FEA) to determine strains occurring within the ciliary membrane in response to 10% tensile strain applied parallel, or perpendicular, to cilia orientation. To elucidate the mechanical environment experienced by the cilium, several lengths were modeled and evaluated based on cilia lengths measured on hASC grown under varied culture conditions. Principal tensile strains in both hASC and ciliary membranes were calculated using FEA, and the magnitude and location of maximum principal tensile strain determined. We found that maximum principal tensile strain was concentrated at the base of the cilium. In the linear elastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane from 150% to 200%, while applying strain parallel to the cilium resulted in much higher strains, approximately 400%. In the hyperelastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane around 30%, while applying strain parallel to the cilium resulted in much higher strains ranging from 50% to 70%. Interestingly, FEA results indicated that primary cilium length was not directly related to ciliary membrane strain. Rather, it appears that cilium orientation may be more important than cilium length in determining sensitivity of hASC to tensile strain. This is the first study to model the effects of tensile strain on the primary cilium and provides newfound insight into the potential role of the primary cilium as a mechanosensor, particularly in tensile strain and potentially a multitude of other mechanical stimuli beyond fluid shear.     

Cobblestone HUVECs: a human model system for studying primary ciliogenesis

Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium.In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear.Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body.We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.     

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.     

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.     

The PCP effector Fuzzy controls cilial assembly and signaling by recruiting Rab8 and Dishevelled to the primary cilium

The planar cell polarity (PCP) pathway controls multiple cellular processes during vertebrate development. Recently the PCP pathway was implicated in ciliogenesis and in ciliary function. The primary cilium is an apically projecting solitary organelle that is generated via polarized intracellular trafficking. Because it acts as a signaling nexus, defects in ciliogenesis or cilial function cause multiple congenital anomalies in vertebrates. Loss of the PCP effector Fuzzy affects PCP signaling and formation of primary cilia; however, the mechanisms underlying these processes are largely unknown. Here we report that Fuzzy localizes to the basal body and ciliary axoneme and is essential for ciliogenesis by delivering Rab8 to the basal body and primary cilium. Fuzzy appears to control subcellular localization of the core PCP protein Dishevelled, recruiting it to Rab8-positive vesicles and to the basal body and cilium. We show that loss of Fuzzy results in inhibition of PCP signaling and hyperactivation of the canonical WNT pathway. We propose a mechanism by which Fuzzy participates in ciliogenesis and affects both canonical WNT and PCP signaling.     

Primary cilium: an elaborate structure that blocks cell division?

A primary cilium is a microtubule-based membranous protrusion found in almost all cell types. A primary cilium has a “9 + 0” axoneme that distinguishes this ancient organelle from the canonical motile “9 + 2” cilium. A primary cilium is the sensory center of the cell that regulates cell proliferation and embryonic development. The primary ciliary pocket is a specialized endocytic membrane domain in the basal region. The basal body of a primary cilium exists as a form of the centriole during interphase of the cell cycle. Although conventional thinking suggests that the cell cycle regulates centrosomal changes, recent studies suggest the opposite, that is, centrosomal changes regulate the cell cycle. In this regard, centrosomal kinase Aurora kinase A (AurA), Polo-like kinase 1 (Plk1), and NIMA related Kinase (Nek or Nrk) propel cell cycle progression by promoting primary cilia disassembly which indicates a non-mitotic function. However, the persistence of primary cilia during spermatocyte division challenges the dominate idea of the incompatibility of primary cilia and cell division. In this review, we demonstrate the detailed structure of primary cilia and discuss the relationship between primary cilia disassembly and cell cycle progression on the background of various mitotic kinases.     

Localization of extracellular matrix receptors on the chondrocyte primary cilium.

A single primary cilium is found in chondrocytes and other connective tissue cells. We have previously shown that extracellular matrix (ECM) macromolecules such as collagen fibers closely associate with chondrocyte primary cilia, and their points of contact are characterized by electron-opaque plaques suggesting a direct link between the ECM and the cilium. This study examines the expression of receptors for ECM molecules on chondrocyte primary cilia. Embryonic chick sterna were fluorescently labeled with antibodies against alpha and beta integrins, NG2, CD44, and annexin V. Primary cilia were labeled using acetylated alpha-tubulin antibody. Expression of ECM receptors was examined on chondrocyte plasma membranes and their primary cilia using immunofluorescence and confocal microscopy. All receptors examined showed a punctate distribution on the plasma membrane. alpha2, alpha3, and beta1 integrins and NG2 were also present on primary cilia, whereas annexin V and CD44 were excluded. The number of receptor-positive cilia varied from 8/50 for NG2 to 43/50 for beta1 integrin. This is the first study to demonstrate the expression of integrins and NG2 on chondrocyte primary cilia. The data strongly suggest that chondrocyte primary cilia have the necessary machinery to act as mechanosensors, linking the ECM to cytoplasmic organelles responsible for matrix production and secretion.     

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.     

Analysis of the orientation of primary cilia in growth plate cartilage: a mathematical method based on multiphoton microscopical images

The chondrocytic primary cilium has been hypothesized to act as a mechano-sensor, analogously to primary cilium of cells in epithelial tissues. We hypothesize that mechanical inputs during growth, sensed through the primary cilium, result in directed secretion of the extracellular matrix, thereby establishing tissue anisotropy in growth plate cartilage. The cilium, through its orientation in three-dimensional space, is hypothesized to transmit to the chondrocyte the preferential direction for matrix secretion. This paper reports on the application of classical mathematical methods to develop an algorithm that addresses the particular challenges relative to the assessment of the orientation of the primary cilium in growth plate cartilage, based on image analysis of optical sections visualized by multiphoton microscopy. Specimens are prepared by rapid cold precipitation-based fixation to minimize possible artifactual post-mortem alterations of ciliary orientation. The ciliary axoneme is localized by immunocytochemistry with antibody acetylated-alpha-tubulin. The method is applicable to investigation of ciliary orientation in different zones of the growth plate, under either normal or altered biomechanical environments. The methodology is highly flexible and adaptable to other connective tissues where tissue anisotropy and directed secretion of extracellular matrix components are hypothesized to depend on the tissue's biomechanical environment during development and growth.     

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2‐dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog‐related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.     

Dynamics of the primary cilium in shear flow

In this work, the equilibrium shape and dynamics of a primary cilium under flow are investigated by using both theoretical modeling and experiment. The cilium is modeled as an elastic beam that may undergo large deflection due to the hydrodynamic load. Equilibrium results show that the anchoring effects of the basal body on the cilium axoneme behave as a nonlinear rotational spring. Details of the rotational spring are elucidated by coupling the elastic beam with an elastic shell. We further study the dynamics of cilium under shear flow with the cilium base angle determined from the nonlinear rotational spring, and obtain good agreement in cilium bending and relaxing dynamics when comparing between modeling and experimental results. These results potentially shed light on the physics underlying the mechanosensitive ion channel transport through the ciliary membrane.     

The Resorption of Cilia in Cyathodinium piriforme*

SYNOPSIS. The fine structure of the cilium, kinetosome, kinetodesmal fiber, and basal microtubules has been described in Cyathodinium piriforme. The ciliary axoneme is encased in an electron-dense jacket termed the axonemal jacket. This jacket surrounds the axoneme and is found midway between the axoneme and the ciliary membrane when viewed in cross section. Before division or reorganization the cilia are withdrawn into the cell. Intact cilia surrounded by their jackets are found in the cytoplasm during the early phases of retraction. Degradation of the axonemal microtubules precedes the dissolution of the axonemal jacket. Profiles of the jackets are observed after the microtubules have been resorbed. The cilia appear to detach from the kinetosomes. Barren kinetosomes are seen below the cell surface frequently with kinetodesmal fibers still attached. Whether all or some of these barren kinetosomes contribute to the formation of the new ciliary anlage cannot be ascertained.     

The ultrastructure of primary cilia in the endocrine and excretory duct cells of the pancreas of mice and rats

A primary cilium was frequently observed in the endocrine alpha, beta and delta cells, as well as in the excretory duct cells of the pancreas of normal mice and rats. The characteristic components of the cilium including the basal body, axoneme (shaft), and terminal part were clearly recognizable. The basal body or distal centriole surrounded by Golgi vesicles was perpendicularly oriented to the proximal centriole, and a dense striated band was seen filling the gap between them. The microtubules of the basal body consisted of nine peripheral triplets exhibiting a 9 + 0 pattern, an appearance similar to that of the proximal centriole. Rootlets, basal feet and alar sheets associated with the basal body were occasionally seen. The axoneme usually consisted of a 9 + 0 pattern of microtubule doublets, but other irregular patterns of 7 + 2, 7 + 3, and 8 + 1 were also seen. The microtubules in the terminal part of the cilium became fewer in number and had no peculiar arrangement. The cilium of the endocrine cells always projected into the intercellular canaliculus and was covered by the ciliary sheath, and occasionally, double cilia were visualized in the vicinity of beta cells. In the excretory duct cells, the cilium showed similar features, but it was slightly longer and always projected into the dense secretory content of duct lumen. On the other hand, no primary cilium was ever observed in the acinar cells of mouse and rat pancreas. In conclusion, the present study describes the morphology of primary cilia and its associated components in the endocrine and excretory duct cells of the pancreas of mice and rats. The findings suggest that the primary cilium should be considered as a constant intracellular organelle though its function and significance remain speculative.     

The ependymal surface of the fourth ventricle of the rat: a combined scanning and transmission electron microscopic study.

The morphological features of the ependymal surface and supraependymal elements of the fourth ventricle of the rat were examined by scanning electron microscopy (SEM) and by the transmission electron microscopy (TEM). The results confirm the following aspects: 1) The presence of supraependymal elements and microvilli in the ependymal territories, including the sites where the cilia completely cover the ependymal surface; 2) The existence of cilia with oval or spherical thickenings together with supraependymal bulbs similar in size to those of the larger ciliary swellings; 3) Identification of the long supraependymal fibres with intermittent fusiform dilations observed under the SEM with the nerve fibres seen under the TEM; 4) The existence of intraventricular axodendritic synapses.     

The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly

Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.     

Intraflagellar transport motors in cilia: moving along the cell's antenna

Intraflagellar transport (IFT), the motor-dependent movement of IFT particles along the axoneme, is critical for the assembly, maintenance, and function of motile and sensory cilia, and, consequently, this process underlies ciliary motility, cilium-based signaling, and ciliopathies. Here, I present my perspective on IFT as a model system for studying motor-driven cargo transport. I review evidence that kinesin-2 motors physically transport IFT particles as cargo and hypothesize that several accessory kinesins confer cilia-specific functions by augmenting the action of the two core IFT motors, kinesin-2 and dynein 1b, which assemble the cilium foundation.