Mechanisms of Weight Control by Primary Cilia

A primary cilium, a hair-like protrusion of the plasma membrane, is a pivotal organelle for sensing external environmental signals and transducing intracellular signaling. An interesting linkage between cilia and obesity has been revealed by studies of the human genetic ciliopathies Bardet-Biedl syndrome and Alström syndrome, in which obesity is a principal manifestation. Mouse models of cell type-specific cilia dysgenesis have subsequently demonstrated that ciliary defects restricted to specific hypothalamic neurons are sufficient to induce obesity and hyperphagia. A potential mechanism underlying hypothalamic neuron cilia-related obesity is impaired ciliary localization of G protein-coupled receptors involved in the regulation of appetite and energy metabolism. A well-studied example of this is melanocortin 4 receptor (MC4R), mutations in which are the most common cause of human monogenic obesity. In the paraventricular hypothalamus neurons, a blockade of ciliary trafficking of MC4R as well as its downstream ciliary signaling leads to hyperphagia and weight gain. Another potential mechanism is reduced leptin signaling in hypothalamic neurons with defective cilia. Leptin receptors traffic to the periciliary area upon leptin stimulation. Moreover, defects in cilia formation hamper leptin signaling and actions in both developing and differentiated hypothalamic neurons. The list of obesity-linked ciliary proteins is expending and this supports a tight association between cilia and obesity. This article provides a brief review on the mechanism of how ciliary defects in hypothalamic neurons facilitate obesity.     

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.     

Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li₂CO₃ were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.     

Disruption of intraflagellar transport in adult mice leads to obesity and slow-onset cystic kidney disease

The assembly of primary cilia is dependent on intraflagellar transport (IFT), which mediates the bidirectional movement of proteins between the base and tip of the cilium. In mice, congenic mutations disrupting genes required for IFT (e.g., Tg737 or the IFT kinesin Kif3a) are embryonic lethal, whereas kidney-specific disruption of IFT results in severe, rapidly progressing cystic pathology. Although the function of primary cilia in most tissues is unknown, in the kidney they are mechanosenstive organelles that detect fluid flow through the tubule lumen. The loss of this flow-induced signaling pathway is thought to be a major contributing factor to cyst formation. Recent data also suggest that there is a connection between ciliary dysfunction and obesity as evidenced by the discovery that proteins associated with human obesity syndromes such as Alstr?m and Bardet-Biedl localize to this organelle. To more directly assess the importance of cilia in postnatal life, we utilized conditional alleles of two ciliogenic genes (Tg737 and Kif3a) to systemically induce cilia loss in adults. Surprisingly, the cystic kidney pathology in these mutants is dependent on the time at which cilia loss was induced, suggesting that cyst formation is not simply caused by impaired mechanosensation. In addition to the cystic pathology, the conditional cilia mutant mice become obese, are hyperphagic, and have elevated levels of serum insulin, glucose, and leptin. We further defined where in the body cilia are required for normal energy homeostasis by disrupting cilia on neurons throughout the central nervous system and on pro-opiomelanocortin-expressing cells in the hypothalamus, both of which resulted in obesity. These data establish that neuronal cilia function in a pathway regulating satiety responses.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

The conserved proteins CHE-12 and DYF-11 are required for sensory cilium function in Caenorhabditis elegans

Sensory neuron cilia are evolutionarily conserved dendritic appendages that convert environmental stimuli into neuronal activity. Although several cilia components are known, the functions of many remain uncharacterized. Furthermore, the basis of morphological and functional differences between cilia remains largely unexplored. To understand the molecular basis of cilia morphogenesis and function, we studied the Caenorhabditis elegans mutants che-12 and dyf-11. These mutants fail to concentrate lipophilic dyes from their surroundings in sensory neurons and are chemotaxis defective. In che-12 mutants, sensory neuron cilia lack distal segments, while in dyf-11 animals, medial and distal segments are absent. CHE-12 and DYF-11 are conserved ciliary proteins that function cell-autonomously and are continuously required for maintenance of cilium morphology and function. CHE-12, composed primarily of HEAT repeats, may not be part of the intraflagellar transport (IFT) complex and is not required for the localization of some IFT components. DYF-11 undergoes IFT-like movement and may function at an early stage of IFT-B particle assembly. Intriguingly, while DYF-11 is expressed in all C. elegans ciliated neurons, CHE-12 expression is restricted to some amphid sensory neurons, suggesting a specific role in these neurons. Our results provide insight into general and neuron-specific aspects of cilium development and function.     

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.     

Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism

The assembly of the axoneme, the structural scaffold of cilia and flagella, requires translocation of a vast quantity of tubulin into the growing cilium, but the mechanisms that regulate the targeting, quantity, and timing of tubulin transport are largely unknown. In Chlamydomonas, GFP-tagged α-tubulin enters cilia as an intraflagellar transport (IFT) cargo and by diffusion. IFT-based transport of GFP-tubulin is elevated in growing cilia and IFT trains carry more tubulin. Cells possessing both nongrowing and growing cilia selectively target GFP-tubulin into the latter. The preferential delivery of tubulin boosts the concentration of soluble tubulin in the matrix of growing versus steady-state cilia. Cilia length mutants show abnormal kinetics of tubulin transport. We propose that cells regulate the extent of occupancy of IFT trains by tubulin cargoes. During ciliary growth, IFT concentrates soluble tubulin in cilia and thereby promotes elongation of the axonemal microtubules.     

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.     

Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells

Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.     

Kinesin-2 transports Orco into the olfactory cilium of Drosophila melanogaster at specific developmental stages

The cilium, the sensing centre for the cell, displays an extensive repertoire of receptors for various cell signalling processes. The dynamic nature of ciliary signalling indicates that the ciliary entry of receptors and associated proteins must be regulated and conditional. To understand this process, we studied the ciliary localisation of the odour-receptor coreceptor (Orco), a seven-pass transmembrane protein essential for insect olfaction. Little is known about when and how Orco gets into the cilia. Here, using Drosophila melanogaster, we show that the bulk of Orco selectively enters the cilia on adult olfactory sensory neurons in two discrete, one-hour intervals after eclosion. A conditional loss of heterotrimeric kinesin-2 during this period reduces the electrophysiological response to odours and affects olfactory behaviour. We further show that Orco binds to the C-terminal tail fragments of the heterotrimeric kinesin-2 motor, which is required to transfer Orco from the ciliary base to the outer segment and maintain within an approximately four-micron stretch at the distal portion of the ciliary outer-segment. The Orco transport was not affected by the loss of critical intraflagellar transport components, IFT172/Oseg2 and IFT88/NompB, respectively, during the adult stage. These results highlight a novel developmental regulation of seven-pass transmembrane receptor transport into the cilia and indicate that ciliary signalling is both developmentally and temporally regulated.

Jana, Dutta, Jain et al., show that the odour-receptor coreceptor only enters the cilia expressed on olfactory sensory neurons at specified developmental stages requiring heterotrimeric kinesin-2. The motor binds to the coreceptor and plays a crucial role in localising them to a compact, environment-exposed domain at the ciliary outer-segment.     

Neuronal primary cilia: a review

Primary cilia in neurons have often been regarded as rare, vestigial curiosities. However, neuronal cilia are now gaining recognition as ubiquitous organelles in the mammalian brain, raising speculation about what their functions may be. They might have some features tailored for the nervous system and others that serve needs shared by a spectrum of other cell types. Here we review clues from the literature and present new data supporting several possibilities for the significance of neuronal cilia. Our immunocytochemical results show regional heterogeneity in neuronal cilia. Brain regions nearer to the cerebral ventricles had longer cilia, suggesting that they might sense chemicals such as peptides, originating from cerebrospinal fluid. In mutant Tg737(orpk)mice, most brain regions appeared to be missing cilia. The importance of intraflagellar transport proteins establishes a functional link between neuronal cilia and other primary cilia.     

The proteome of the mouse photoreceptor sensory cilium complex

Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including approximately 1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.     

Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons

DCDC2 is one of the candidate susceptibility genes for dyslexia. It belongs to the superfamily of doublecortin domain containing proteins that bind to microtubules, and it has been shown to be involved in neuronal migration. We show that the Dcdc2 protein localizes to the primary cilium in primary rat hippocampal neurons and that it can be found within close proximity to the ciliary kinesin-2 subunit Kif3a. Overexpression of DCDC2 increases ciliary length and activates Shh signaling, whereas downregulation of Dcdc2 expression enhances Wnt signaling, consistent with a functional role in ciliary signaling. Moreover, DCDC2 overexpression in C. elegans causes an abnormal neuronal phenotype that can only be seen in ciliated neurons. Together our results suggest a potential role for DCDC2 in the structure and function of primary cilia.     

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis

Approximately 10% of the photoreceptor outer segment (OS) is turned over each day, requiring large amounts of lipid and protein to be moved from the inner segment to the OS. Defects in intraphotoreceptor transport can lead to retinal degeneration and blindness. The transport mechanisms are unknown, but because the OS is a modified cilium, intraflagellar transport (IFT) is a candidate mechanism. IFT involves movement of large protein complexes along ciliary microtubules and is required for assembly and maintenance of cilia. We show that IFT particle proteins are localized to photoreceptor connecting cilia. We further find that mice with a mutation in the IFT particle protein gene, Tg737/IFT88, have abnormal OS development and retinal degeneration. Thus, IFT is important for assembly and maintenance of the vertebrate OS.     

Subcellular spatial regulation of canonical Wnt signalling at the primary cilium

Mechanisms of signal transduction regulation remain a fundamental question in a variety of biological processes and diseases. Previous evidence indicates that the primary cilium can act as a signalling hub, but its exact role in many of the described pathways has remained elusive. Here, we investigate the mechanism of cilia-mediated regulation of the canonical Wnt pathway. We found that primary cilia dampen canonical Wnt signalling through a spatial mechanism involving compartmentalization of signalling components. The cilium, through regulated intraflagellar transport, diverts Jouberin (Jbn), a ciliopathy protein and context-specific Wnt pathway regulator, away from the nucleus and limits β-catenin nuclear entry. This repressive regulation does not silence the pathway, but instead maintains a discrete range of Wnt responsiveness; cells without cilia have potentiated Wnt responses, whereas cells with multiple cilia have inhibited responses. Furthermore, we show that this regulation occurs during embryonic development and is disrupted in cancer cell proliferation. Together these data explain a spatial mechanism of Wnt signalling regulation that may provide insight into ciliary regulation of other signalling pathways.     

Stages of ciliogenesis and regulation of ciliary length

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

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.