The mechanics of the primary cilium: an intricate structure with complex function

The primary cilium is a non-motile singular cellular structure that extends from the surface of nearly every cell in the body. The cilium has been shown to play numerous roles in maintaining tissue homeostasis, through regulating signaling pathways and sensing both biophysical and biochemical changes in the extracellular environment. The structural performance of the cilium is paramount to its function as defective cilia have been linked to numerous pathologies. In particular, the cilium has demonstrated a mechanosensory role in tissues such as the kidney, liver, endothelium and bone, where cilium deflection under mechanical loading triggers a cellular response. Understanding of how cilium structure and subsequent mechanical behavior contributes to the roles that cilium plays in regulating cellular behavior is a compelling question, yet is a relatively untouched research area. Recent advances in biophysical measurements have demonstrated the cilium to be a structurally intricate organelle containing an array of load bearing proteins. Furthermore advances in modeling of this organelle have revealed the importance of these proteins at regulating the cilium's mechanosensitivity. Remarkably, the cilium is capable of adapting its mechanical state, altering its length and possibly it's bending resistance, to regulate its mechanosensitivity demonstrating the importance of cilium mechanics in cellular responses. In this review, we introduce the cilium as a mechanosensor; discuss the advances in the mechanical modeling of cilia; explore the structural features of the cilium, which contribute to its mechanics and finish with possible mechanisms in which alteration in structure may affect ciliary mechanics, consequently affecting ciliary based mechanosensing.     

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.     

Ciliary behavior of a negatively phototactic Chlamydomonas reinhardtii

With an instrument that can record the motion of both cilia of the unicellular alga Chlamydomonas reinhardtii for many hours, the behavioral differences of its two cilia have been studied to determine their specific role in phototaxis. The organism was held on a fixed micropipette with the plane of ciliary beating rotated into the imaging plane of a quadrant photodetector. The responses to square-wave light patterns of a wide range of temporal frequencies were used to characterize the responses of each cilium. Eighty-one cells were examined showing an unexpectedly diverse range of responses. Plausible common signals for the linear and nonlinear signals from the cell body are suggested. Three independent ciliary measures--the beat frequency, stroke velocity, and phasing of the two cilia--have been identified. The cell body communicates to the cilia the direction of phototaxis the cell desires to go, the absolute light intensity, and the appropriate graded transient response for tracking the light source. The complexity revealed by each measure of the ciliary response indicates many independent variables are involved in the net phototactic response. In spite of their morphological similarity, the two cilia of Chlamydomonas respond uniquely. Probably the signals from the cell body fan out to independent pathways in the cilia. Each cilium modifies the input in its own way. The change in the pattern of the effective and recovery strokes of each cilium associated with negative phototaxis has been demonstrated and its involvement in phototactic turning is described.     

A Computational Model of Dynein Activation Patterns that Can Explain Nodal Cilia Rotation

Normal left-right patterning in vertebrates depends on the rotational movement of nodal cilia. In order to produce this ciliary motion, the activity of axonemal dyneins must be tightly regulated in a temporal and spatial manner; the specific activation pattern of the dynein motors in the nodal cilia has not been reported. Contemporary imaging techniques cannot directly assess dynein activity in a living cilium. In this study, we establish a three-dimensional model to mimic the ciliary ultrastructure and assume that the activation of dynein proteins is related to the interdoublet distance. By employing finite-element analysis and grid deformation techniques, we simulate the mechanical function of dyneins by pairs of point loads, investigate the time-variant interdoublet distance, and simulate the dynein-triggered ciliary motion. The computational results indicate that, to produce the rotational movement of nodal cilia, the dynein activity is transferred clockwise (looking from the tip) between the nine doublet microtubules, and along each microtubule, the dynein activation should occur faster at the basal region and slower when it is close to the ciliary tip. Moreover, the time cost by all the dyneins along one microtubule to be activated can be used to deduce the dynein activation pattern; it implies that, as an alternative method, measuring this time can indirectly reveal the dynein activity. The proposed protein-structure model can simulate the ciliary motion triggered by various dynein activation patterns explicitly and may contribute to furthering the studies on axonemal dynein activity.     

Computation of the internal forces in cilia: application to ciliary motion, the effects of viscosity, and cilia interactions.

This paper presents a simple and reasonable method for generating a phenomenological model of the internal mechanism of cilia. The model uses a relatively small number of parameters whose values can be obtained by fitting to ciliary beat shapes. Here, we use beat patterns observed in Paramecium. The forces that generate these beats are computed and fit to a simple functional form called the "engine." This engine is incorporated into a recently developed hydrodynamic model that accounts for interactions between neighboring cilia and between the cilia and the surface from which they emerge. The model results are compared to data on ciliary beat patterns of Paramecium obtained under conditions where the beats are two-dimensional. Many essential features of the motion, including several properties that are not built in explicitly, are shown to be captured. In particular, the model displays a realistic change in beat pattern and frequency in response to increased viscosity and to the presence of neighboring cilia in configurations such as rows of cilia and two-dimensional arrays of cilia. We found that when two adjacent model cilia start beating at different phases they become synchronized within several beat periods, as observed in experiments where two flagella are brought into close proximity. Furthermore, examination of various multiciliary configurations shows that an approximately antiplectic wave pattern evolves autonomously. This modeling evidence supports earlier conjectures that metachronism may occur, at least partially, as a self-organized phenomenon due to hydrodynamic interactions between neighboring cilia.     

Contractile events in the cilia of Paramecium, Opalina, Mytilus, and Phragmatopoma.

The motion of the abnormal cilia of Opalina and Mytilus can be described by the recently developed model for ciliary motion, provided the activation of the contractility during the effective stroke is reduced by three- to fivefold compared with that in the recovery stroke. The stiffness of the Mytilus cilium during the effective stroke is found several hundred times larger than that predicted by the model, however. The stiffness of the cilia of Paramecium, Opalina, Phragmatopoma, and of Mytilus in the recovery phase, is predicted approximately correctly by the model. The activation of contractility in Mytilus and Phragmatopoma cilia increases with the viscosity of the medium, as the velocity of the ciliary motion slows down. This leads to the equivalent of a force-velocity relation. The velocity of propagation of the bend in the cilia during the recovery stroke is shown to be dependent only on the elastic properties of the ciliary shaft, and to be independent of the contractile activiey.     

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.     

An electro-optic monitor of the behavior of Chlamydomonas reinhardtii cilia

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.     

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.     

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.     

Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins

Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras superfamily of GTPases, is required for ciliary structure but has poorly defined ciliary functions. In this paper, we further characterize the role of Arl13b in cilia by examining mutant cilia in vitro and determining the localization and dynamics of Arl13b within the cilium. Previously, we showed that mice lacking Arl13b have abnormal Sonic hedgehog (Shh) signaling; in this study, we show the dynamics of Shh signaling component localization to the cilium are disrupted in the absence of Arl13b. Significantly, we found Smoothened (Smo) is enriched in Arl13b-null cilia regardless of Shh pathway stimulation, indicating Arl13b regulates the ciliary entry of Smo. Furthermore, our analysis defines a role for Arl13b in regulating the distribution of Smo within the cilium. These results suggest that abnormal Shh signaling in Arl13b mutant embryos may result from defects in protein localization and distribution within the cilium.     

Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ

Hyaline cartilage chondrocytes express one primary cilium per cell, but its function remains unknown. We examined the ultrastructure of chick embryo sternal chondrocyte cilia and their interaction with extracellular matrix molecules by transmission electron microscopy (TEM) and, for the first time, double-tilt electron tomography. Ciliary bending was also examined by confocal immunohistochemistry. Tomography and TEM showed the ciliary axoneme to interdigitate amongst collagen fibres and condensed proteoglycans. TEM also revealed the presence of electron-opaque particles in the proximal axoneme which may represent intraciliary-transport (ICT) particles. We observed a wide range of ciliary bending patterns. Some conformed to a heavy elastica model associated with shear stress. Others were acutely deformed, suggesting ciliary deflection by collagen fibres and proteoglycans with which the cilia make contact. We conclude that mechanical forces transmitted through these matrix macromolecules bend the primary cilium, identifying it as a potential mechanosensor involved in skeletal patterning and growth.     

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.     

Arf4 Is Required for Mammalian Development but Dispensable for Ciliary Assembly

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.     

All along the watchtower: is the cilium a tumor suppressor organelle?

Cilia or flagella have been around since almost the beginning of life, and have now developed specialized cell-type specific functions from locomotion to acting as environmental sensors participating in cell signalling. Genetic defects affecting cilia result in a myriad of pathological instances, including infertility, obesity, blindness, deafness, skeletal malformations, and lung problems. However, the consistency in which the common kidney cyst is coupled with cilia dysfunction has raised interest in the possibility that ciliary dysfunction might contribute to other neoplasms as well. A suite of recent papers convincingly linking cilia to hedgehog signalling, platelet-derived growth factor signalling, Wnt signalling and the von Hippel-Lindau tumor suppressor protein has rapidly expanded the knowledge base connecting cilia to cancer. We propose that these data support the notion of the cilium as a cellular Watchtower, whose absence can be an initiating event in neoplastic growth. Furthermore, we predict that we are just now seeing the tip of the iceberg, and that the list of cancers associated with altered ciliary signalling will grow exponentially in the next few years.     

Ciliary Extracellular Vesicles: Txt Msg Organelles

Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV–cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia–EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies.     

Heteromerization of Ciliary G Protein-Coupled Receptors in the Mouse Brain

Nearly every cell type in the mammalian body projects from its cell surface a primary cilium that provides important sensory and signaling functions. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and adenylyl cyclase type 3, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling. Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin-concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling.     

Ciliogenesis in sea urchin embryos – a subroutine in the program of development

One major milestone in the development of the sea urchin embryo is the assembly of a single cilium on each blastomere just before hatching. These cilia are constructed both from pre-existing protein building blocks, such as tubulin and dynein, and from a number of 9+2 architectural elements that are synthesized de novo at ciliogenesis. The finite or quantal synthesis of certain key architectural proteins is coincident with ciliary elongation and proportional to ciliary length. Upon deciliation, the synthesis of architectural proteins occurs anew, a new cilium grows, and the stores of various building blocks are replenished. This routine of coordinated ciliary gene expression may be replayed experimentally many times without delaying normal development. The ability to regenerate cilia has allowed elucidation of these various protein synthetic relationships and has led to the discovery of the pathways by which membrane-associated tubulin and axoneme-associated architectural proteins are conveyed into the highly compartmentalized growing cilium. The sea urchin embryo thus provides a very convenient model system for studies of ciliary assembly and maintenance, coordinate gene expression and membrane dynamics.