Emerging mechanistic understanding of cilia function in cellular signalling

Abstract

Primary cilia are solitary, immotile sensory organelles present on most cells in the body that participate broadly in human health, physiology and disease. Cilia generate a unique environment for signal transduction with tight control of protein, lipid and second messenger concentrations within a relatively small compartment, enabling reception, transmission and integration of biological information. In this Review, we discuss how cilia function as signalling hubs in cell-cell communication using three signalling pathways as examples: ciliary G-protein-coupled receptors (GPCRs), the Hedgehog (Hh) pathway and polycystin ion channels. We review how defects in these ciliary signalling pathways lead to a heterogeneous group of conditions known as 'ciliopathies', including metabolic syndromes, birth defects and polycystic kidney disease. Emerging understanding of these pathways' transduction mechanisms reveals common themes between these cilia-based signalling pathways that may apply to other pathways as well. These mechanistic insights reveal how cilia orchestrate normal and pathophysiological signalling outputs broadly throughout human biology.

Fig. 1 |. The primary cilium is a specialized compartment for signal transduction.

Several signal transduction pathways organized within the primary cilium have been identified, including signalling by select G-proteincoupled receptors (GPCRs), Hedgehog (Hh) and polycystin. The primary cilium is nucleated by the basal body and composed of a microtubule-based axoneme and a ciliary membrane. The periciliary membrane, which often forms the ciliary pocket at the base of the primary cilium, connects the ciliary membrane to the plasma membrane. The transition zone creates a diffusion barrier at the base of the primary cilium, generating a unique composition critical for specialized signalling. a, Although contiguous with the plasma membrane, the ciliary membrane has a distinct composition. Through the action of the cilia-localized phosphoinositide 5-phosphatase type IV (INPP5E), the ciliary membrane is enriched in phosphatidylinositol 4-phosphate (PtdIns4P, also know as PI4P) and depleted of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂, also known as PI(4,5)P₂) relative to the adjoining plasma membrane. The ciliary membrane also contains sterols and sphingolipids (for example, cholesterol and sphingomyelin, respectively) critical for ciliary signalling. b, Similarly, the composition of the cilioplasm is distinct from that of the cytoplasm. Through the action of cilia-localized GPCRs and ion channels, ciliary cAMP and calcium (Ca²⁺) levels can be substantially different compared with the cytoplasm, enabling compartmentalized signalling. c, The intraflagellar transport (IFT) system mediates microtubule-driven transport of ciliary cargo along the axoneme. Powered by the microtubule motors kinesin 2 and cytoplasmic dynein 2, respectively, IFT complexes transport ciliary cargo towards the ciliary tip (anterograde transport) or back to the ciliary base (retrograde). d, In a multistep process, Tubby-like protein 3 (TULP3) helps transport membrane-bound proteins across the transition zone into the primary cilium. First, TULP3 binds both to the ciliary localization sequence of ciliary cargo and to PI(4,5)P₂ in the periciliary membrane (stage 1). Next, cargo-bound and PI(4,5)P₂-bound TULP3 binds to IFT, allowing TULP3 and its associated cargo to enter the primary cilium (stage 2). Finally, in the PI(4,5)P₂-depleted ciliary membrane, TULP3 releases the newly imported cargo into the ciliary membrane (stage 3). e, The BBSome is a complex consisting of several Bardet–Biedl syndrome (BBS) proteins and is required for the internalization of activated ciliary GPCRs into the cell body: activated GPCRs first become phosphorylated by a GPCR kinase (such as GPCR kinase 2 (GRK2)) (stage 1) and then bound by β-arrestin 2 (stage 2). This directs a ubiquitin ligase to add a Lys63 (K63)-linked ubiquitin chain onto the C-terminal tail of the activated GPCR (stage 3), allowing subsequent binding of the ubiquitinated tail by endosomal sorting complexes required for transport (ESCRT) protein, TOM1-like protein 2 (TOM1L2) (stage 4). Finally, the BBSome, associated with the membrane through GTP-bound ADP-ribosylation factor-like protein 6 (ARL6), binds TOM1L2 and functions as an adaptor for IFT to traffic the activated GPCR out of the primary cilium (stage 5). PKD1, polycystin-1; PKD2, polycystin-2; PTCH1, Patched 1.

Fig. 2 |. G-protein-coupled receptor signalling in primary cilia.

a, G-protein-coupled receptors (GPCRs) contain seven transmembrane domains with an extracellular N terminus and an intracellular C terminus. They bind heterotrimeric G proteins, composed of an α-subunit, β-subunit and γ-subunit, with their intracellular regions. Canonically, ligand binding to the extracellular domain of a GPCR induces a conformational change, which in turn promotes the exchange of bound GDP for GTP in the coupled G proteins. Once GTP-bound, the Gα-subunit dissociates from the Gβ- and Gγ-subunits, enabling intracellular signal transduction. G proteins exist in a great variety of heterotrimeric combinations, resulting in the activation of distinct downstream signalling depending on which G protein transducer the GPCR is coupled to. Gα is the best-characterized G protein subunit and can be subdivided into four functional families: Gα_s stimulates the activity of adenylyl cyclases (ADCYs) leading to a localized concentration increase of the second messenger cAMP, Gα_i/o inhibits the activity of ADCYs leading to decreased levels of cAMP, Gα_q/11 activates phospholipase C (PLC) to hydrolyse phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) into inositol 1,4,5-trisphosphate (IP₃) and diacylglyerol (DAG), and Gα_12/13 regulates Rho guanine exchange factors (GEFs) such as RhoA. In addition, GPCRs can signal through β-arrestins. Localization of GPCRs to the primary cilium can affect G protein transducer coupling in several ciliated cell types. For example, energy homeostasis in the body is coordinately regulated through GPCRs in multiple tissues with ciliated cells. b, Primary cilia on hypothalamic neurons control feeding behaviour. Feeding is inhibited when the ciliary GPCR melanocortin receptor 4 (MC4R) is activated by α-melanocyte-stimulating hormone (α-MSH), which is secreted by pro-opiomelanocortin (POMC) neurons. Activation of ciliary MC4R results in an increase of cilia-localized cAMP. By contrast, physiological hunger signals result in activation of agouti-related protein (AgRP)-expressing and neuropeptide Y (NPY)-expressing neurons, which inhibit MC4R neurons. c, Primary cilia on adipocyte stem and progenitor cells (ASPCs) regulate adipocyte differentiation. Activation of the ciliary GPCR free fatty acid receptor 4 (FFAR4) by its ligand, ω-3 fatty acid, induces the adipogenic transcriptional programme by increasing ciliary cAMP. d, Primary cilia on pancreatic α-islet and β-islet cells modulate the secretion of glucagon and insulin, respectively, to regulate blood glucose levels. Activation of ciliary FFAR4 and ciliary prostaglandin E₂ receptor EP₄ by their ligands, ω-3 fatty acid and prostaglandin E₂ (PGE₂), respectively, promote glucose-stimulated glucagon and insulin secretion by increasing ciliary cAMP.

Fig. 3 |. Hedgehog signal transduction in primary cilia.

a, When the Hedgehog (Hh) pathway is off, Patched 1 (PTCH1) blocks activation of Smoothened (SMO) by transporting or redistributing sterols within the membrane, thereby restricting SMO access to its activating sterol ligand(s). As a result, ciliary protein kinase A (PKA), composed of regulatory (R) and catalytic (C) subunits, is active and phosphorylates and inactivates GLI2 and GLI3 transcription factors, stimulating their proteolysis into transcriptional repressors (GLI^R) that block expression of Hh-regulated genes. This process is regulated indirectly through the action of the G-protein-coupled receptor (GPCR) GPR161, which promotes adenylyl cyclase (ADCY) activity (via its ability to couple to Gα_s proteins) and also binds PKA holoenzymes through its C-terminal A kinase-anchoring protein (AKAP) domain, thus contributing to ciliary PKA-C activity. In addition, in the inactive state, SMO becomes ubiquitylated (Ub) and transported out of the cilium. b, When nearby cells secrete Hh ligands, Hh proteins bind to and inhibit PTCH1, enabling SMO to encounter and be activated by its sterol ligands. The resulting active conformation of SMO, characterized by an outward movement of the transmembrane helices TM5 and TM6 (inset), is rapidly recognized and phosphorylated by GPCR kinase 2 (GRK2), triggering SMO to recruit and inactivate PKA-C (‘direct PKA-C inhibition’) through its PKA inhibitor (PKI) motif. Consequently, GLI phosphorylation is inhibited, leading to GLI protein conversion to activator forms (GLI^A). Activated SMO also promotes transport of GPR161 out of the cilium and may couple to Gα_i, which inhibits ADCY activity. Thus, SMO not only inactivates PKA directly but also contributes to lowered levels of cAMP, thus preventing PKA-C activity. c, GLI, thus activated, can induce transcription of Hh target genes. Ultimately, both PTCH1 and GPR161 undergo ubiquitylation and ciliary exit.

Fig. 4 |. Models of polycystin function in vertebrate development and homeostasis.

a, Polycystin signalling maintains homeostasis of the kidney epithelium, and disruption of polycystin function results in aggressive cystogenesis. Genetic disruption of cilia formation in the kidney epithelium also causes cystogenesis, although with a milder phenotype than that caused by loss of polycystin. Interestingly, deletion of both cilia and polycystin function in the mouse kidney epithelium induces cysts that are similar to those caused by loss of cilia alone rather than having an additive effect. A parsimonious explanation for these results is that cilia are important for the anti-cystogenic effect of polycystins but are also important for cilia-dependent cyst activation, a cystogenic effect that is repressed by polycystin function. b, Polycystin-2 (PKD2) is a six-pass transmembrane protein similar to other transient receptor potential (TRP) channel components and can form homotetramers. PKD2 comprises a voltage sensor domain (encompassing the N-terminal four transmembrane domains) and a pore domain (encompassing the C-terminal four transmembrane domains). An extracellular domain (the tetragonal opening for polycystins (TOP) domain) participates in subunit interactions. The shown model is based on the Protein Data Bank (PDB) accessions 5T4D (PKD2 homotetramer) and 3HRO (PKD2 C-terminal coiled-coil). c, PKD2 can form homotetramers or a 3:1 heteromer with polycystin-1 (PKD1) (PDB code 6A70). In comparison with PKD2, PKD1 possesses an extensive N-terminal addition which includes a long extracellular domain. This extracellular domain is cleaved within the G-protein-coupled receptor (GPCR) autoproteolysis-inducing (GAIN) domain (modelled on the GAIN domain of latrophilin-1 (PDB code 4DLQ)). Distal to the GAIN domain are an receptor for egg jelly (REJ) domain, 14 polycystic kidney disease (PKD) domains that are unique to PKD1 and polycystin-1-like 1 (PKD1L1), a Lectin domain (modelled on the Bothrops jararacussu C-type lectin domain (PDB code 5F2Q)), another lone PKD domain, a cell wall integrity and stress response components (WSC) domain (modelled on Saccharomyces cerevisiae WSC (PDB: 7PZ2]) and a leucine-rich repeat (LRR). This heteromer is likely to be the complex whose function is essential to restraining cystogenesis in the kidney. Models of polycystin complexes are based on the above PDB accessions combined with Alphafold2 (ref. 250) prediction of the remaining domains and assembled using PyMol 2.5.5. d, Lateral view of a late headfold-stage mouse embryo. The node is a transient and ciliated tissue, which is critical for the specification of the left–right axis. e, Posterior view of a late headfold-stage embryo, indicating that the node directs the early left–right asymmetric expression of Nodal on the left (L) periphery of the node and Dand5 on the right (R) periphery of the node. Nodal encodes a morphogen and Dand5 encodes a secreted Nodal antagonist; both are required for left–right axis patterning. Their expression is regulated by cilia, PKD2 and PKD1L1. f, Scanning electron micrograph of the node of an embryonic day 7.5 mouse embryo. Cilia are false-coloured in green. Scale bar, 2 μm. g, A schematic of a node depicting that the cilia are posteriorly tilted and rotate clockwise. Together, these features generate active strokes towards the left and fluid flow towards the left side of the node. Flow direction determines left in mice. h, Higher-magnification view of the node cilia. Scale bar, 500 nm. i, Model of a heteromeric polycystin channel on cilia at the node periphery. Left–right axis patterning depends on cilia, PKD2 and PKD1L1. A, anterior; P, posterior.

Fig. 5 |. Crosstalk between ciliary signalling pathways.

a, G-protein-coupled receptor (GPCR) and polycystin signalling can fine-tune the Hedgehog (Hh) signalling pathway. As detailed in Fig. 3, protein kinase A (PKA) is a key node in the regulation of GLI transcription factors and signalling pathways that augment ciliary PKA activity result in increased phosphorylation and inhibition of GLI, thus reducing Hh signal transduction. Ciliary GPCRs coupled to Gα_s activate ciliary adenylyl cyclase (ADCY), resulting in increased ciliary cAMP levels and expanding the size of the active PKA pool available to inhibit GLI. Similarly, calcium (Ca²⁺) influx through activated polycystin channels can activate calcium-sensitive ADCY. Conversely, ciliary GPCRs coupled to Gα_i inhibit ciliary ADCY, thus lowering the levels of ciliary cAMP and blocking PKA from inhibiting GLI activation. b, Patched 1 (PTCH1) is a sterol transporter and reduces sterol concentrations in the inner leaflet of the ciliary membrane when Hh signalling is off. Upon binding of an Hh ligand, PTCH1 exits the primary cilium, resulting in the redistribution of sterols within the ciliary membrane. This may include cholesterol and oxysterols such as 7β,27-DHC. These redistributed sterols are critical for the activation of Smoothened (SMO). It is possible that these redistributed ciliary sterols allosterically regulate other ciliary pathways. PKD1/2, polycystin-1/2.