ConVarT: a search engine for matching human genetic variants with variants from non-human species.

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, and Caenorhabditis elegans. ConVarT incorporates annotations (including phenotypic and pathogenic) into variants, and these previously unexploited phenotypic MatchVars from mice and C. elegans can give clues about the functional consequence of human genetic variants. Our analysis shows that many phenotypic variants in different genes from mice and C. 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.

Active transport and diffusion barriers restrict Joubert Syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain.

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.

CiliaMiner: an integrated database for ciliopathy genes and ciliopathies.

Cilia are found in eukaryotic species ranging from single-celled organisms, such as Chlamydomonas 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. 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, Caenorhabditis elegans and Chlamydomonas reinhardtii. 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. Database URL: https://kaplanlab.shinyapps.io/ciliaminer/.

Matching variants for functional characterization of genetic variants.

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 used Caenorhabditis elegans mutant 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 carrying C. elegans mutants, 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 into cilia. Functional analysis of all 10 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 nonciliary proteins) of the IFT-140 null mutant, indicating that both P702A and W937stop are phenotypic in C. 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.

WDR31 displays functional redundancy with GTPase-activating proteins (GAPs) ELMOD and RP2 in regulating IFT complex and recruiting the BBSome to cilium.

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 Caenorhabditis 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 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 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 defects. This work reveals WDR31-RP-2-ELMD-1 as IFT and BBSome trafficking regulators.

The AP-1 clathrin adaptor facilitates cilium formation and functions with RAB-8 in C. elegans ciliary membrane transport.

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.

An empirical framework for binary interactome mapping.

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.

Empirically controlled mapping of the Caenorhabditis elegans protein-protein interactome network.

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

ConVarT: Search Engine for Missense Variants Between Humans and Other Organisms.

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.

Protocol for determining the average speed and frequency of kinesin and dynein-driven intraflagellar transport (IFT) in C. elegans.

Here, we present a protocol to image a fluorescent-labeled intraflagellar transport (IFT) component in Caenorhabditis elegans with fluorescence microscopy, including steps of sample preparations, in vivo live-cell imaging, and post-microscopy analysis with kymographs. This protocol breaks down all processes into three categories: (1) pre-imaging preparations, (2) preparations for the time of image acquisition, and (3) post-imaging analyses. The protocol can be applied to determine the speed and frequency of moving particles. For complete details on the use and execution of this protocol, please refer to Cevik et al. (2021).

Towards a proteome-scale map of the human protein-protein interaction network.

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.

The Joubert syndrome protein CEP41 is excluded from the distal segment of cilia in C. elegans.

Rare diseases are a fundamental issue in today's world, affecting more than 300 million individuals worldwide. According to data from Orphanet and OMIM, about 50-60 new conditions are added to the list of over 6,000 clinically distinct diseases each year, rendering disease diagnosis and treatment even more challenging. Ciliopathies comprise a heterogeneous category of rare diseases made up of over 35 distinct diseases, including Joubert syndrome (JBTS; OMIM 213300), that are caused by functional and structural defects in cilia. JBTS is an autosomal recessive condition characterized by a range of symptoms, including cerebellar vermis hypoplasia and poor muscle tone. There are now a total of 38 genes that cause JBTS, almost all of which encode protein products that are found in cilia and cilia-associated compartments, such as the basal body and transition zone. CEP41 is a JBTS-associated protein that is found in cilia and the basal body of mammals, but its localization in other ciliary organisms remains elusive. C. elegans is an excellent model organism for studying the molecular mechanisms of rare diseases like JBTS. We, therefore, decided to use C. elegans to identify the localization of CEP41. Our microscopy analysis revealed that CEPH-41(CEntrosomal Protein Homolog 41) not only localizes to cilia but is excluded from the distal segment of the amphid and phasmid cilia in C. elegans. Furthermore, we discovered a putative X-box motif located in the promoter of ceph-41 and the expression of ceph-41 is regulated by DAF-19, a sole Regulatory Factor X (RFX) transcription factor.

Subcellular localization of the voltage-gated K+ channel EGL-36 , a member of the KV3 subfamily, in the ciliated sensory neurons in C. elegans.

Delineated as the first cellular organelle in 1675 by Antonie van Leeuwenhoek, cilia did not receive much attention until the 2000s, when it became apparent that cilia played a key role in the development of embryos, a variety of signaling pathways. Therefore, collective efforts by many scientists have led to the identification of many novel ciliopathy and cilia genes, while we are still far from disclosing the complete components of cilia.Here we used the ciliated sensory neurons in C. elegans as a model system that revealed the voltage-gated K+ channel EGL-36 (a member of the Shaw subfamily) as a new component associated with cilia. The confocal microscopy examination of fluorescence tagged EGL-36 together with ciliary (IFT-140) or transition zone (MKS-6) markers reveal that EGL-36 is only expressed in subsets of the ciliated sensory neurons, where it partially overlaps with the basal body signals and predominantly localizes to the periciliary membrane compartment. This expression pattern along with studies of egl-36 gain-of-function variants indicates that egl-36 is not essential for ciliogenesis in C. elegans. Our data identify the voltage-gated K+ channel EGL-36 as a new cilia-associated protein, and future studies should reveal the functional significance of EGL-36 in cilia biogenesis.

Intraflagellar transport: from molecular characterisation to mechanism.

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.

Endocytosis genes facilitate protein and membrane transport in C. elegans sensory cilia.

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.

Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans.

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.