Transport and barrier mechanisms that regulate ciliary compartmentalization and ciliopathies.

Primary cilia act as cell surface antennae, coordinating cellular responses to sensory inputs and signalling molecules that regulate developmental and homeostatic pathways. Cilia are therefore critical to physiological processes, and defects in ciliary components are associated with a large group of inherited pleiotropic disorders - known collectively as ciliopathies - that have a broad spectrum of phenotypes and affect many or most tissues, including the kidney. A central feature of the cilium is its compartmentalized structure, which imparts its unique molecular composition and signalling environment despite its membrane and cytosol being contiguous with those of the cell. Such compartmentalization is achieved via active transport pathways that bring protein cargoes to and from the cilium, as well as gating pathways at the ciliary base that establish diffusion barriers to protein exchange into and out of the organelle. Many ciliopathy-linked proteins, including those involved in kidney development and homeostasis, are components of the compartmentalizing machinery. New insights into the major compartmentalizing pathways at the cilium, namely, ciliary gating, intraflagellar transport, lipidated protein flagellar transport and ciliary extracellular vesicle release pathways, have improved our understanding of the mechanisms that underpin ciliary disease and associated renal disorders.

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision.

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.

Dawn and dusk peaks of outer segment phagocytosis, and visual cycle function require Rab28.

RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28-OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11-cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae (p < .05). These data give further understanding on the molecular mechanisms of RAB28-associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling.

Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans.

Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of an animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function (neuronal dye filling, roaming and chemotaxis assays) measured how the variants impacted mks-3 gene function. Effects of the variants on mks-3 function were further investigated by looking at MKS-3::GFP localization and cilia ultrastructure. The quantitative assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile and Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg and Ser961Tyr). Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signalling function. We conclude that efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model for rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia.

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

CDKL kinase regulates the length of the ciliary proximal segment.

Cilia are organelles found throughout most unicellular eukaryotes and different metazoan cell types. To accomplish their essential roles in cell motility, fluid flow, and signaling, cilia are divided into subcompartments with variable structures, compositions, and functions. How these specific subcompartments are built remains almost completely unexplored. Here, we show that C. elegans CDKL-1, related to the human CDKL kinase family (CDKL1/CDKL2/CDKL3/CDKL4/CDKL5), specifically controls the length of the proximal segment, a ciliary subdomain conserved in evolution from Tetrahymena motile cilia to C. elegans chemosensory, mammalian olfactory, and photoreceptor non-motile cilia. CDKL-1 associates with intraflagellar transport (IFT), influences the distribution of the IFT anterograde motors heterotrimeric kinesin-II and homodimeric OSM-3-kinesin/KIF17 in the proximal segment, and shifts the boundary between the proximal and distal segments (PS/DS boundary). CDKL-1 appears to function independently from several factors that influence cilium length, namely the kinases DYF-5 (mammalian CILK1/MAK) and NEKL-1 (NEK9), as well as the depolymerizing kinesins KLP-13 (KIF19) and KLP-7 (KIF2). However, a different kinase, DYF-18 (CCRK), is needed for the correct localization and function of CDKL-1 and similarly influences the length of the proximal segment. Loss of CDKL-1, which affects proximal segment length without impairing overall ciliary microtubule structural integrity, also impairs cilium-dependent processes, namely cGMP-signaling-dependent body length control and CO2 avoidance. Collectively, our findings suggest that cilium length is regulated by various pathways and that the IFT-associated kinase CDKL-1 is essential for the construction of a specific ciliary compartment and contributes to development and sensory physiology.

Interpreting the pathogenicity of Joubert syndrome missense variants in Caenorhabditis elegans.

Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have used a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S and G155S) in mksr-2/B9D2 associated with Joubert syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment and regulates the molecular composition and sensory/signalling functions of the cilium. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm that both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and MKS-2/TMEM216. Together, these data establish C. elegans as a model for interpreting JBTS mutations and provide further insight into MKS module organisation. This article has an associated First Person interview with the first author of the paper.

Genetic Deletion of Zebrafish Rab28 Causes Defective Outer Segment Shedding, but Not Retinal Degeneration.

The photoreceptor outer segment is the canonical example of a modified and highly specialized cilium, with an expanded membrane surface area in the form of disks or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localization of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display significantly reduced shed outer segment material/phagosomes in the RPE at 1 month post fertilization (mpf), but otherwise normal visual function up to 21 dpf and retinal structure up to 12 mpf. Cone photoreceptor-specific transgenic reporter lines show Rab28 localizes almost exclusively to outer segments, independently of GTP/GDP nucleotide binding. Co-immunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, phosphodiesterase 6C and guanylate cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy.

Ciliary Rab28 and the BBSome negatively regulate extracellular vesicle shedding.

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.

ERICH3 in Primary Cilia Regulates Cilium Formation and the Localisations of Ciliary Transport and Sonic Hedgehog Signaling Proteins.

Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. In this study, we identify the mostly uncharacterised ERICH3 protein as a component of the mammalian primary cilium. Loss of ERICH3 causes abnormally short cilia and results in the accumulation of IFT-A/B proteins at the ciliary tip, together with reduced ciliary levels of retrograde transport regulators, ARL13B, INPP5E and BBS5. We also show that ERICH3 ciliary localisations require ARL13B and BBSome components. Finally, ERICH3 loss causes positive (Smoothened) and negative (GPR161) regulators of sonic hedgehog signaling (Shh) to accumulate at abnormally high levels in the cilia of pathway-stimulated cells. Together, these findings identify ERICH3 as a novel component of the primary cilium that regulates cilium length and the ciliary levels of Shh signaling molecules. We propose that ERICH3 functions within retrograde IFT-associated pathways to remove signaling proteins from cilia.

Rab35 controls cilium length, function and membrane composition.

Rab and Arl guanine nucleotide-binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G protein with endocytic recycling, actin remodelling and cytokinesis roles. Rab35 loss via siRNAs, morpholinos or knockout reduces cilium length in mammalian cells and the zebrafish left-right organiser (Kupffer's vesicle) and causes motile cilia-associated left-right asymmetry defects. Consistent with these observations, GFP-Rab35 localises to cilia, as do GEF (DENND1B) and GAP (TBC1D10A) Rab35 regulators, which also regulate ciliary length and Rab35 ciliary localisation. Mammalian Rab35 also controls the ciliary membrane levels of Shh signalling regulators, promoting ciliary targeting of Smoothened, limiting ciliary accumulation of Arl13b and the inositol polyphosphate 5-phosphatase (INPP5E). Rab35 additionally regulates ciliary PI(4,5)P2 levels and interacts with Arl13b. Together, our findings demonstrate roles for Rab35 in regulating cilium length, function and membrane composition and implicate Rab35 in pathways controlling the ciliary levels of Shh signal regulators.

CiliaCarta: An integrated and validated compendium of ciliary genes.

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.

Membrane retrieval, recycling and release pathways that organise and sculpt the ciliary membrane.

The microtubule-based cilium that extends from the surface of most eukaryotic cell types serves motility, sensory reception and cell-cell signaling functions, and is disrupted in wide-ranging ciliopathy disorders. The cilium is heavily reliant on dynamic and tuneable intracellular trafficking systems such as intraflagellar transport and Golgi-derived secretory pathways, which control the organelle's structure, function and molecular composition. More recently, endosomal retrieval and recycling, as well as extracellular vesicle (EV) release, pathways have been associated with ciliary membrane control. Here, we discuss the emerging role of these pathways in the control of ciliary membrane homeostasis. The new findings provide a deeper and more integrated understanding of how the ciliary membrane is organised.

EFHC1, implicated in juvenile myoclonic epilepsy, functions at the cilium and synapse to modulate dopamine signaling.

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy.

Role for intraflagellar transport in building a functional transition zone.

Genetic disorders caused by cilia dysfunction, termed ciliopathies, frequently involve the intraflagellar transport (IFT) system. Mutations in IFT subunits-including IFT-dynein motor DYNC2H1-impair ciliary structures and Hedgehog signalling, typically leading to "skeletal" ciliopathies such as Jeune asphyxiating thoracic dystrophy. Intriguingly, IFT gene mutations also cause eye, kidney and brain ciliopathies often linked to defects in the transition zone (TZ), a ciliary gate implicated in Hedgehog signalling. Here, we identify a C. elegans temperature-sensitive (ts) IFT-dynein mutant (che-3; human DYNC2H1) and use it to show a role for retrograde IFT in anterograde transport and ciliary maintenance. Unexpectedly, correct TZ assembly and gating function for periciliary proteins also require IFT-dynein. Using the reversibility of the novel ts-IFT-dynein, we show that restoring IFT in adults (post-developmentally) reverses defects in ciliary structure, TZ protein localisation and ciliary gating. Notably, this ability to reverse TZ defects declines as animals age. Together, our findings reveal a previously unknown role for IFT in TZ assembly in metazoans, providing new insights into the pathomechanism and potential phenotypic overlap between IFT- and TZ-associated ciliopathies.

Intraflagellar Transport Complex A Genes Differentially Regulate Cilium Formation and Transition Zone Gating.

Cilia are found on most eukaryotic cell types, serving motility, environment sensing, and signaling (cell-cell) functions, and defects cause genetic diseases (ciliopathies), affecting the development of many tissues [1]. Cilia are built by intraflagellar transport (IFT), a bidirectional microtubule-based motility driven by kinesin-2 anterograde (toward ciliary tip) and IFT-dynein retrograde (toward ciliary base) motors together with IFT-A and IFT-B cargo adaptor complexes that control retrograde and anterograde IFT, respectively [2]. Ciliary composition is also facilitated by the transition zone (TZ) at the ciliary base and the associated Meckel-Gruber syndrome (MKS) and nephronophthisis (NPHP) modules that establish protein diffusion barriers and regulate cilium structure [3]. Although the molecular architecture of the IFT machine is emerging [2], how individual components contribute to cilium subtype formation and IFT remains relatively unexplored, especially in vivo. In addition, little is known about functional interactions between IFT and TZ modules. Here, in Caenorhabditis elegans (roundworms), we identify cell-type-specific mechanisms by which IFT-A sculpts the structures of discrete ciliary subtypes and regulates IFT. We also uncover differential roles for IFT-A subunits in controlling the TZ restriction of MKS module components and ciliary exclusion (gating) of periciliary membrane proteins, with IFT-140 controlling their ciliary entry and IFT-43/121/139 controlling their ciliary removal. Furthermore, we determine that IFT-A and MKS module components synergistically interact to determine cilium structure. Overall, this work provides insight into the functional architecture of a metazoan IFT-A complex in different cell types and uncovers new relationships between ciliopathy-associated IFT-A and TZ modules.

Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway.

Glutamylation is a post-translational modification found on tubulin that can alter the interaction between microtubules (MTs) and associated proteins. The molecular mechanisms regulating tubulin glutamylation in response to the environment are not well understood. Here, we show that in the sensory cilia of Caenorhabditis elegans, tubulin glutamylation is upregulated in response to various signals such as temperature, osmolality, and dietary conditions. Similarly, tubulin glutamylation is modified in mammalian photoreceptor cells following light adaptation. A tubulin glutamate ligase gene ttll-4, which is essential for tubulin glutamylation of axonemal MTs in sensory cilia, is activated by p38 MAPK. Amino acid substitution of TTLL-4 has revealed that a Thr residue (a putative MAPK-phosphorylation site) is required for enhancement of tubulin glutamylation. Intraflagellar transport (IFT), a bidirectional trafficking system specifically observed along axonemal MTs, is required for the formation, maintenance, and function of sensory cilia. Measurement of the velocity of IFT particles revealed that starvation accelerates IFT, which was also dependent on the Thr residue of TTLL-4. Similarly, starvation-induced attenuation of avoidance behaviour from high osmolality conditions was also dependent on ttll-4. Our data suggest that a novel evolutionarily conserved regulatory system exists for tubulin glutamylation in sensory cilia in response to the environment.

Endosome maturation factors Rabenosyn-5/VPS45 and caveolin-1 regulate ciliary membrane and polycystin-2 homeostasis.

Primary cilium structure and function relies on control of ciliary membrane homeostasis, regulated by membrane trafficking processes that deliver and retrieve ciliary components at the periciliary membrane. However, the molecular mechanisms controlling ciliary membrane establishment and maintenance, especially in relation to endocytosis, remain poorly understood. Here, using Caenorhabditis elegans, we describe closely linked functions for early endosome (EE) maturation factors RABS-5 (Rabenosyn-5) and VPS-45 (VPS45) in regulating cilium length and morphology, ciliary and periciliary membrane volume, and ciliary signalling-related sensory behaviour. We demonstrate that RABS-5 and VPS-45 control periciliary vesicle number and levels of select EE/endocytic markers (WDFY-2, CAV-1) and the ciliopathy membrane receptor PKD-2 (polycystin-2). Moreover, we show that CAV-1 (caveolin-1) also controls PKD-2 ciliary levels and associated sensory behaviour. These data link RABS-5 and VPS-45 ciliary functions to the processing of periciliary-derived endocytic vesicles and regulation of ciliary membrane homeostasis. Our findings also provide insight into the regulation of PKD-2 ciliary levels via integrated endosomal sorting and CAV-1-mediated endocytosis.

Rab GTPases in cilium formation and function.

Cilia are microtubule-based organelles extending from a basal body at the surface of eukaryotic cells. Cilia regulate cell and fluid motility, sensation and developmental signaling, and ciliary defects cause human diseases (ciliopathies) affecting the formation and function of many tissues and organs. Over the past decade, various Rab and Rab-like membrane trafficking proteins have been shown to regulate cilia-related processes such as basal body maturation, ciliary axoneme extension, intraflagellar transport and ciliary signaling. In this review, we provide a comprehensive overview of Rab protein ciliary associations, drawing on findings from multiple model systems, including mammalian cell culture, mice, zebrafish, C. elegans, trypanosomes, and green algae. We also discuss several emerging mechanistic themes related to ciliary Rab cascades and functional redundancy.

Primary Cilium Formation and Ciliary Protein Trafficking Is Regulated by the Atypical MAP Kinase MAPK15 in Caenorhabditis elegans and Human Cells.

Motile and immotile (or primary) cilia are microtubule-based structures that mediate multiple cellular functions, including the transduction of environmental cues, developmental signaling, cellular motility, and modulation of fluid flow. Although their core architectures are similar, motile and primary cilia exhibit marked structural differences that underlie distinct functional properties. However, the extent to which ciliogenesis mechanisms are shared between these different cilia types is not fully described. Here, we report that the atypical MAP kinase MAPK15 (ERK7/8), implicated in the formation of vertebrate motile cilia, also regulates the formation of primary cilia in Caenorhabditis elegans sensory neurons and human cells. We find that MAPK15 localizes to a basal body subdomain with the ciliopathy protein BBS7 and to cell-cell junctions. MAPK15 also regulates the localization of ciliary proteins involved in cilium structure, transport, and signaling. Our results describe a primary cilia-related role for this poorly studied member of the MAPK family in vivo, and indicate a broad requirement for MAPK15 in the formation of multiple ciliary classes across species.