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Discovering the entire list of human ciliary genes would help in the diagnosis of cilia-related human disorders known as ciliopathy, but at present the genetic diagnosis of many ciliopathies (over 30%) is far from complete (Bachmann-Gagescu et al., 2015; Knopp et al., 2015; Paff et al., 2018). In a theory, many independent approaches may uncover the whole list of ciliary genes, but 30% of the genes on the ciliary gene list are still ciliary candidate genes (van Dam et al., 2019; Vasquez et al., 2021). All of these cutting-edge techniques, however, have relied on a different single strategy to discover ciliary candidate genes. Because different methodologies demonstrated distinct capabilities with varying quality, categorizing the ciliary candidate genes in the ciliary gene list without further evidence has been difficult. Here, we present a method for predicting ciliary capacity of each human gene that incorporates diverse methodologies (single-cell RNA sequencing, protein-protein interactions (PPIs), comparative genomics, transcription factor (TF)-network analysis, and text mining). By integrating multiple approaches, we reveal previously undiscovered ciliary genes. Our method, CilioGenics, outperforms other approaches that are dependent on a single method. Our top 500 gene list contains 256 new candidate ciliary genes, with 31 experimentally validated. Our work suggests that combining several techniques can give useful evidence for predicting the ciliary capability of all human genes.

Rapid and low-cost sequencing, as well as computer analysis, have facilitated the diagnosis of many genetic diseases, resulting in a substantial rise in the number of disease-associated genes. However, genetic diagnosis of many disorders remains problematic due to the lack of interpretation for many genetic variants, especially missenses, the infeasibility of high-throughput experiments on mammals, and the shortcomings of computational prediction technologies. Additionally, the available mutant databases are not well-utilized. Toward this end, we usedCaenorhabditis elegansmutant resources to delineate the functions of eight missense variants (V444I, V517D, E610K, L732F, E817K, H873P, R1105K, and G1205E) and two stop codons (W937stop and Q1434stop), including several matching variants (MatchVar) with human in ciliopathy associated IFT-140 (also called CHE-11)//IFT140 (intraflagellar transport protein 140). Moreover, MatchVars carryingC. elegansmutants, including IFT-140(G680S) and IFT-140(P702A) for the human (G704S) (dbSNP: rs150745099) and P726A (dbSNP: rs1057518064 and a conflicting variation) were created using CRISPR/Cas9. IFT140 is a key component of IFT complex A (IFT-A), which is involved in the retrograde transport of IFT along cilia and the entrance of G protein-coupled receptors (GPCRs) into cilia. Functional analysis of all ten variants revealed that P702A and W937stop, but not others phenocopied the ciliary phenotypes (short cilia, IFT accumulations, mislocalization of membrane proteins, and cilia entry of non-ciliary proteins) of the IFT-140 null mutant, indicating that both P702A and W937stop are phenotypic inC. elegans. Our functional data offered experimental support for interpreting human variants, by using ready-to-use mutants carrying MatchVars and generating MatchVars with CRISPR/Cas9.

Cilia are found in eukaryotic species ranging from single-celled organisms, such asChlamydomonas reinhardtii, to humans, but not in plants. The ability to respond to repellents and/or attractants, regulate cell proliferation and differentiation, and provide cellular mobility are just a few examples of how crucial cilia are to cells and organisms. Over 30 distinct rare disorders generally known as ciliopathy are caused by abnormalities or functional impairments in cilia and cilia-related compartments. Because of the complexity of ciliopathies and the rising number of ciliopathies and ciliopathy genes, a ciliopathy-oriented and up-to-date database is required. In addition, disorders not yet known as ciliopathy but have genes that produce cilia localizing proteins have yet to be classified. Here we present CiliaMiner, a manually curated ciliopathy database that includes ciliopathy lists collected from articles and databases. Analysis reveals that there are 55 distinct disorders likely related to ciliopathy, with over 4000 clinical manifestations. Based on comparative symptom analysis and subcellular localization data, diseases are classified as primary, secondary, or atypical ciliopathies. CiliaMiner provides easy access to all of these diseases and disease genes, as well as clinical features and gene-specific clinical features, as well as subcellular localization of each protein. Additionally, the orthologs of disease genes are also provided for mice, zebrafish, Xenopus, Drosophila, andC. elegans. CiliaMiner (https://kaplanlab.shinyapps.io/ciliaminer) aims to serve the cilia community with its comprehensive content, and highly enriched interactive heatmaps, and will be continually updated.

ConVarT (https://convart.org/) is a search engine for searching for conjugate variants between humans and other species. The search engine is based on matching conjugate variants called MatchVars between species. Matching equivalent variants requires correct alignment of orthologous proteins with the use of multiple sequence alignments (MSA). Indeed, the ConVarT pipeline has performed over a million MSAs and integrated variants and variant-specific annotations (pathogenicity, phenotypic variants; etc.) into the corresponding positions on MSAs. When a clinically relevant variant is discovered whose functional relevance is unknown, ConVarT offers clinician scientists the possibility to search for a MatchVar in other species and to look for functional data on that variant. Fortunately, ConVarT enables users to paste a protein sequence in FASTA format to search for human orthologous proteins. A pairwise sequence alignment (PSA) is then performed between the provided protein sequence and the human orthologous protein, allowing users to visualize human variants on the PSA. Here, we describe the step-by-step usage of ConVarT. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Searching matching variants (MatchVar) with gene/protein identifiers. Basic Protocol 2: Searching with a FASTA sequence. Alternate Protocol: Search with gene name in multiple species. Basic Protocol 3: Search genes associated with a disease.

The availability of genetic variants, together with phenotypic annotations from model organisms, facilitates comparing these variants with equivalent variants in humans. However, existing databases and search tools do not make it easy to scan for equivalent variants, namely “matching variants” (MatchVars) between humans and other organisms. Therefore, we developed an integrated search engine called ConVarT (http://www.convart.org/) for matching variants between humans, mice, andC. elegans. ConVarT incorporates annotations (including phenotypic and pathogenic) into variants, and these previously unexploited phenotypic MatchVars from mice andC. eleganscan give clues about the functional consequence of human genetic variants. Our analysis shows that many phenotypic variants in different genes from mice andC. elegans, so far, have no counterparts in humans, and thus, can be useful resources when evaluating a relationship between a new human mutation and a disease.

The correct intraflagellar transport (IFT) assembly at the ciliary base and the IFT turnaround at the ciliary tip are key for the IFT to perform its function, but we still have poor understanding about how these processes are regulated. Here, we identify WDR31 as a new ciliary protein, and analysis from zebrafish and Caeno-rhabditis elegans reveals the role of WDR31 in regulating the cilia morphology. We find that loss of WDR-31 together with RP-2 and ELMD-1 (the sole ortholog ELMOD1-3) results in ciliary accumu-lations of IFT Complex B components and KIF17 kinesin, with fewer IFT/BBSome particles traveling along cilia in both anterograde and retrograde directions, suggesting that the IFT/BBSome entry into the cilia and exit from the cilia are impacted. Furthermore, anterograde IFT in the middle segment travels at increased speed in wdr-31;rpi-2;elmd-1. Remarkably, a non-ciliary protein leaks into the cilia of wdr-31;rpi-2;elmd-1, possibly because of IFT de-fects. This work reveals WDR31-RP-2-ELMD-1 as IFT and BBSome trafficking regulators. National Institutes of Health (NIH) - USA P40 OD010440 Abdullah Gul University Scienti fic Research Project Coordination Unit TOA-2018-110 German Research Foundation (DFG) INST 2388/62-1

The term “ciliopathy” refers to a group of over 35 rare disorders characterized by defective cilia and many overlapping clinical features, such as hydrocephalus, cerebellar vermis hypoplasia, polydactyly, and retinopathy. Even though many genes have been implicated in ciliopathies, the genetic pathogenesis in certain cases remains still undisclosed. Here, we identified a homozygous truncating variant in WDR31 in a patient with a typical ciliopathy phenotype encompassing congenital hydrocephalus, polydactyly, and renal agenesis. WDR31 is an evolutionarily conserved protein that localizes to the cilium and cilia-related compartment. Analysis from zebrafish supports the role of WDR31 in regulating the cilia morphology. The CRISPR/Cas9 knock-in (p.Arg261del) C. elegans model of the patient variant (p.Arg268*) reproduced several cilia-related defects observed in wdr-31 null mutants. Mechanistic analysis from C. elegans revealed that WDR-31 functions redundantly with ELDM-1 (ELMOD protein) and RPI-2 (RP2) to regulate the IFT trafficking through controlling the cilia entry of the BBSome. This work revealed WDR31 as a new ciliopathy protein that regulates IFT and BBSome trafficking.

The correct intraflagellar transport (IFT) assembly at the ciliary base and the IFT turnaround at the ciliary tip are key for the IFT to perform its function, but we still have poor understanding about how these processes are regulated. Here, we identify WDR31 as a new ciliary protein, and analysis from zebrafish andCaenorhabditis elegansreveals the role ofWDR31in regulating the cilia morphology. We find that loss of WDR-31 together with RP-2 and ELMD-1 (the sole ortholog ELMOD1-3) results in ciliary accumulations of IFT Complex B components and KIF17 kinesin, with fewer IFT/BBSome particles traveling along cilia in both anterograde and retrograde directions, suggesting that the IFT/BBSome entry into cilia and exit from cilia are impacted. Furthermore, anterograde IFT in the middle segment travel at increased speed inwdr-31;rpi-2;elmd-1. Remarkably, a non-ciliary protein leaks into cilia ofwdr-31;rpi-2;elmd-1possible due to IFT defects. This work reveals WDR31-RP-2-ELMD-1 as IFT and BBSome trafficking regulators.

C. elegans EGL-36 displays extensive similarity to human KCNC proteins, the conserved potassium channel subfamily KV3 (the Shaw subfamily). This potassium channel subfamily contains four genes KCNC1, KCNC2, KCNC3, and KCNC4 encoding KV3.1 (Shaker), KV3.2 (Shab), KV3.3 (Shaw), and KV3.4 (Shal), respectively. Previous studies have demonstrated that C. elegans EGL-36 is expressed in the egg-laying muscles, ventral cord neurons, and the subset of ciliated sensory neurons including ADEL, ADER, PHB through its subcellular localization in the ciliated sensory neurons remains unknown (Elkes et al. 1997; Johnstone et al. 1997). To observe the subcellular localization of EGL-36 in the ciliated sensory neurons in C. elegans, we tagged green fluorescent protein (GFP) at the C-termini of EGL-36 with a cilia-specific promoter. We observed overexpressed EGL-36::GFP in the subsets of sensory neurons in the head and tail including PHB sensory neurons in the tail but not in the PHA sensory neuron, which is in line with previous findings (Elkes et al. 1997; Johnstone et al. 1997) (Figure 1A). Confocal laser scanning microscopy shows that EGL-36::GFP is predominantly located at the base of the cilium. The EGL-36::GFP signal can be found in the neuronal cell body, with weak dendrite staining, where they may function in the dendritic excitability (Figure 1A).

To provide accurate biological hypotheses and elucidate global properties of cellular networks, systematic identification of protein-protein interactions must meet high quality standards.We present an expanded C. elegans protein-protein interaction network, or 'interactome' map, derived from testing a matrix of approximately 10,000 x approximately 10,000 proteins using a highly specific, high-throughput yeast two-hybrid system. Through a new empirical quality control framework, we show that the resulting data set (Worm Interactome 2007, or WI-2007) was similar in quality to low-throughput data curated from the literature. We filtered previous interaction data sets and integrated them with WI-2007 to generate a high-confidence consolidated map (Worm Interactome version 8, or WI8). This work allowed us to estimate the size of the worm interactome at approximately 116,000 interactions. Comparison with other types of functional genomic data shows the complementarity of distinct experimental approaches in predicting different functional relationships between genes or proteins, Journal Article, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., info:eu-repo/semantics/published

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments. One such compartment is the cilium, whose dysfunction underlies disorders classified as ciliopathies. Although AP-1mu subunit (UNC-101) is linked to cilium formation and targeting of transmembrane proteins (ODR-10) to nematode sensory cilia at distal dendrite tips, these functions remain poorly understood. Here, using Caenorhabditis elegans sensory neurons and mammalian cell culture models, we find conservation of AP-1 function in facilitating cilium morphology, positioning and orientation, and microtubule stability and acetylation. These defects appear to be independent of IFT, because AP-1-depleted cells possess normal IFT protein localisation and motility. By contrast, disruption of chc-1 (clathrin) or rab-8 phenocopies unc-101 worms, preventing ODR-10 vesicle formation and causing misrouting of ODR-10 to all plasma membrane destinations. Finally, ODR-10 colocalises with RAB-8 in cell soma and they cotranslocate along dendrites, whereas ODR-10 and UNC-101 signals do not overlap. Together, these data implicate conserved roles for metazoan AP-1 in facilitating cilium structure and function, and suggest cooperation with RAB-8 to coordinate distinct early steps in neuronal ciliary membrane sorting and trafficking.

The small GTPase Arl13b regulates ciliary transmembrane protein localizations and anterograde IFT assembly stability., The small ciliary G protein Arl13b is required for cilium biogenesis and sonic hedgehog signaling and is mutated in patients with Joubert syndrome (JS). In this study, using Caenorhabditis elegans and mammalian cell culture systems, we investigated the poorly understood ciliary and molecular basis of Arl13b function. First, we show that Arl13b/ARL-13 localization is frequently restricted to a proximal ciliary compartment, where it associates with ciliary membranes via palmitoylation modification motifs. Next, we find that loss-of-function C. elegans arl-13 mutants possess defects in cilium morphology and ultrastructure, as well as defects in ciliary protein localization and transport; ciliary transmembrane proteins abnormally accumulate, PKD-2 ciliary abundance is elevated, and anterograde intraflagellar transport (IFT) is destabilized. Finally, we show that arl-13 interacts genetically with other ciliogenic and ciliary transport–associated genes in maintaining cilium structure/morphology and anterograde IFT stability. Together, these data implicate a role for JS-associated Arl13b at ciliary membranes, where it regulates ciliary transmembrane protein localizations and anterograde IFT assembly stability.

Several attempts have been made to systematically map protein-protein interaction, or 'interactome', networks. However, it remains difficult to assess the quality and coverage of existing data sets. Here we describe a framework that uses an empirically-based approach to rigorously dissect quality parameters of currently available human interactome maps. Our results indicate that high-throughput yeast two-hybrid (HT-Y2H) interactions for human proteins are more precise than literature-curated interactions supported by a single publication, suggesting that HT-Y2H is suitable to map a significant portion of the human interactome. We estimate that the human interactome contains approximately 130,000 binary interactions, most of which remain to be mapped. Similar to estimates of DNA sequence data quality and genome size early in the Human Genome Project, estimates of protein interaction data quality and interactome size are crucial to establish the magnitude of the task of comprehensive human interactome mapping and to elucidate a path toward this goal., Journal Article, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., info:eu-repo/semantics/published

Research from a wide range of model systems such as Chlamydomonas, C. elegans and mice have shown that intraflagellar transport (IFT) is a bidirectional motility of large protein complexes along cilia and flagella that is essential for building and maintaining these organelles. Since its discovery in 1993, much progress has been made in uncovering the molecular and functional basis of IFT. Presently, many components of the core IFT machinery are known, including the anterograde kinesin 2 motor(s), the IFT-dynein retrograde motor and the collection of at least 17 proteins that makes up the IFT particle. Most significantly, discoveries linking IFT to polycystic kidney disease and other developmental phenotypes have broadened the context of IFT research by demonstrating that primary cilia and IFT are required for processes such as kidney tubule and retinal tissue development, limb bud morphogenesis and organ patterning. Central to the functional basis of IFT is its ability to traffic various ciliary protein cargos, which include structural ciliary subunits, as well as non-structural proteins such as transmembrane channels/receptors and sensory signalling molecules. Indeed, exciting data over the past 3-4 years, linking IFT and primary cilia to developmental and growth factor signalling, as well as the cell cycle, indicates that the current repertoire of IFT cargos is likely to expand. Here we present a comprehensive review of IFT, with particular emphasis on its molecular composition and mechanism of action.

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms., Author Summary Protruding from most cells surfaces is a hair-like extension called the primary cilium. This organelle functions as a cellular antenna, receiving physical and chemical signals such as light, odorants, and molecules that coordinate cell growth, differentiation and migration. Underscoring their importance, cilium defects underlie an expanding spectrum of diseases termed ciliopathies, characterised by wide-ranging symptoms such as cystic kidneys, blindness and bone abnormalities. A key question is how ciliary proteins are targeted to and retained within cilia. The best understood system is intraflagellar transport (IFT), thought to ferry proteins between the ciliary base and tip. Also, ciliopathy protein modules organise protein diffusion barriers at the ciliary base transition zone (TZ). Despite major advances, it remains poorly understood how proteins are targeted to cilia, and ciliary membrane subdomains in particular. Here, we investigated how Joubert syndrome-associated ARL13B/ARL-13 is compartmentalized at subciliary membranes. Using C. elegans nematodes and mammalian cell experimental systems, we uncovered differential requirements for sequence motifs, IFT and ciliopathy modules in regulating ARL-13 ciliary restriction, mobility and compartment length. Also, we provide essential insight into how IFT and ciliopathy-associated protein complexes and modules influence ciliary membrane protein transport, diffusion across the TZ, the integrity of the ciliary membrane, and subciliary protein composition.

Multiple proteomics and genomics approaches have been used to identify the molecular parts list of cilia and flagella. However, the specific cilia-related functions of many of these components remain unknown. Previously, K04F10.2 was identified as a candidate cilia-related gene in C. elegans, exhibiting specific expression in ciliated sensory neurons. We now show that GFP-tagged K04F10.2 is highly enriched at the transition zone (TZ) compartment at the base of the ciliary axoneme, and possibly to the more proximal transition fiber/basal body region. Fluorescence microscopy and transmission electron microscopy show that K04F10.2 null mutants possess grossly normal cilium structure and ultrastructure. In contrast, wild-type worms overexpressing a K04F10.2::gfp transgene possess cilium integrity defects such as a dye-filling abnormality (Dyf) and phasmid cilia that are frequently short and abnormally separated. In addition, phasmid neuronal dendrites are abnormally short in these worms. Since GFP-tagged K04F10.2 is enriched at the TZ, we examined possible genetic relationships with known TZ-associated ciliary disease genes such as those causing Meckel-Gruber syndrome (MKS) and Nephronophthisis (NPHP). Unlike the synthetic Dyf (SynDyf) phenotypes known for alleles of various MKS and NPHP genes, no such phenotype was observed in K04F10.2;mks-5 and K04F10.2;nphp-4 double mutants. However, K04F10.2 was found to synthetically interact (SynDyf) with Joubert syndrome-associated Arl13b/arl-13, which interestingly does not localise at the TZ. Together, these data implicate K04F10.2 as a novel TZ-enriched protein with functions that are distinct from canonical MKS and NPHP module components.

Cilium dysfunction causes disorders such as Meckel syndrome (MKS), Bardet-Biedl syndrome (BBS), Nephronophthisis (NPHP) and Joubert syndrome (JS). Most ciliopathy proteins localize to cilia where they regulate developmental signaling and transport. Previously we showed that JS-associated Arl13b localises at proximal ciliary membranes, where it regulates transport in C. elegans. Now we employ C. elegans to investigate the molecular basis of Arl13b ciliary compartmentalisation. First we find that the localisation of C. elegans Arl13b (ARL-13) to proximal ciliary regions does not include transition zones. This localisation, reminiscent of the mammalian Inversin (Inv) ciliary compartment, requires an RVxP motif. Using quantitative imaging, ciliopathy and ciliary transport genes were found to differentially restrict ARL-13 to cilia, with IFT, BBS and MKS gene mutants displaying varying ARL-13 accumulations explicitly at periciliary membranes. Specifically, we observed strong accumulations in IFT-B complex mutants, moderate accumulations in IFT-A, BBS and MKS mutants, and no accumulations in NPHP, septin or kinesin-2 mutants. Interestingly, within IFT/BBS/MKS mutant cilia, ARL-13 distributions were unaffected, as was ARL-13 ciliary mobility. Finally, using a TAP-tag immunoprecipitation approach from mammalian HEK293T cells and state-of-the-art mass spectrometry, we identified components of mammalian Arl13b-associated complexes, including the IFT-B subcomplex. These data show that ARL-13 localisation to an Inv-like ciliary compartment requires an RVxP motif and differential contributions by ciliary transport and ciliopathy modules. Our data also suggest that IFT/BBS/MKS modules are not required for ARL-13 distribution and mobility within cilia, but rather its entry into cilia, perhaps by overcoming a transport barrier at the ciliary base.

Summary Background Multiple intracellular transport pathways drive the formation, maintenance, and function of cilia, a compartmentalized organelle associated with motility, chemo-/mechano-/photosensation, and developmental signaling. These pathways include cilium-based intraflagellar transport (IFT) and poorly understood membrane trafficking events. Defects in ciliary transport contribute to the etiology of human ciliary disease such as Bardet-Biedl syndrome (BBS). In this study, we employ the genetically tractable nematode Caenorhabditis elegans to investigate whether endocytosis genes function in cilium formation and/or the transport of ciliary membrane or ciliary proteins. Results Here we show that localization of the clathrin light chain, AP-2 clathrin adaptor, dynamin, and RAB-5 endocytic proteins overlaps with a morphologically discrete periciliary membrane compartment associated with sensory cilia. In addition, ciliary transmembrane proteins such as G protein-coupled receptors concentrate at periciliary membranes. Disruption of endocytic gene function causes expansion of ciliary and/or periciliary membranes as well as defects in the ciliary targeting and/or transport dynamics of ciliary transmembrane and IFT proteins. Finally, genetic analyses reveal that the ciliary membrane expansions in dynamin and AP-2 mutants require bbs-8 and rab-8 function and that sensory signaling and endocytic genes may function in a common pathway to regulate ciliary membrane volume. Conclusions These data implicate C. elegans endocytosis proteins localized at the ciliary base in regulating ciliary and periciliary membrane volume and suggest that membrane retrieval from these compartments is counterbalanced by BBS-8 and RAB-8-mediated membrane delivery.