Nguyen, Phuong Anh T et al. (2011) International Worm Meeting "The role of cilia in cGMP signaling in the AFD thermosensory neuron."

Johnson, Jacque-Lynne, Nguyen, Phuong Anh T, Leroux, Michel R

[International Worm Meeting, 2011]

Primary cilia act as the antennae of eukaryotic cells, being able to transduce light, mechanical and chemical stimuli from the environment. The importance of these organelles in human physiology and development is increasingly recognized, given their involvement in different signaling pathways and implication in human disorders (ciliopathies) that affect essentially all organs. We have just found through phylogenetic analysis that various cGMP signaling proteins are present in essentially all eukaryotes except for those that have secondarily lost cilia during evolution. As cyclic nucleotides are found in prokaryotes, this finding suggests that cGMP signaling could have been the first signal transduction pathway adopted by primary cilia during its emergence as a sensory device, preceding the advent of other ciliary signaling pathways in vertebrates. A prime example of the cilia-cGMP signaling connection can be gleaned in vertebrate photoreceptors; this specialized cilium concentrate cGMP signaling proteins essential for phototransduction, including guanylyl cyclases that produce cGMP, phosphodiesterases that break down cGMP, and cGMP-dependent cation channels. How the cGMP signaling cascade is established in the ciliary compartment is not understood, however. A well-studied behavior in C. elegans that involves cGMP signaling is thermotaxis. In this behavior, temperature is sensed mainly by AFD neurons, each of which possesses a cilium of unknown function. Our lab has showed that ciliary mutants are defective in thermotaxis. Because of the close link between cilia and cGMP signaling, we hypothesized that ciliary proteins are required to establish and maintain cGMP signaling in AFD. Indeed, we discovered that at AFD dendritic endings, cGMP signaling components are localized to different compartments, and proper localizations require specific ciliary proteins acting cell-autonomously. Microscopy studies of ciliary proteins in AFD suggest that its membranous 'fingers' are a bona fide ciliary compartment, because whereas most ciliary proteins are found in the canonical cilium, some are also present at the base of the fingers. This suggests ciliary proteins might form a barrier or 'transport hub' separating the finger membrane from the dendritic membrane. Our research will help establish the AFD neuron as a model system analogous to the ciliary photoreceptor, probing the important connection between ciliary trafficking and cGMP signaling.

Blacque Oliver (2007) International Worm Meeting "C. elegans functional genomics of sensory cilia identifies an evolutionarily conserved gene, DYF-11, as a novel intraflagellar transport protein."

Blacque Oliver

[International Worm Meeting, 2007]

Primary cilia play important roles in sensing the local chemical and physical environment, and are now linked to several critical signaling and developmental pathways. The building and maintenance of cilia and flagella requires intraflagellar transport (IFT), a conserved kinesin and dynein dependent bidirectional motility of multi-subunit protein complexes along ciliary axonemes. To investigate IFT in C. elegans, we employ a multi-disciplinary approach encompassing (1) transcriptomics- and bioinformatics-based experimentation to identify novel IFT genes, (2) fluorescence microscopy and in vivo IFT motility assays to examine IFT protein cellular localization and transport, and (3) analysis of mutant alleles to uncover the genetic basis of IFT gene function. In the last several years, we have identified several novel proteins associated with IFT, including a family of proteins associated with Bardet-Biedl syndrome, which in humans is characterised by numerous ailments, including obesity, blindness and polycystic kidneys. Here, we demonstrate that a C. elegans protein regulated by the ciliogenic DAF-19 transcription factor is a novel IFT-associated protein required for normal IFT and sensory cilia function. The protein, DYF-11, is encoded by a gene which we show is mutated in the dye-filling mutant strain dyf-11, whose phenotype is indicative of cilia dysfunction. Abrogation of DYF-11 results in truncated cilia and phenotypic behaviours associated with defects in sensory cilia function. In vivo analyses of GFP-tagged DYF-11 or other IFT proteins in wt or various mutant strains suggest that DYF-11 is associated with subcomplex B IFT components that were first isolated biochemically in Chlamydomonas. Consistent with the notion that DYF-11 is a conserved IFT protein, DYF-11 is present in the membrane and matrix fraction of Chlamydomonas cilia, a property exhibited by other IFT proteins. Our results therefore extend the repertoire of components known to be associated with IFT and cilia function. ********Update******** The authorship for this abstract was changed according to the request by Michel Leroux in Aug-2007. The updated author list: Peter N. Inglis, Chunmei Li, Evgeni Efimenko, Carmen C. Leitch, Nico Katsanis, Peter Swoboda, Michel Leroux

Jensen, Victor L. et al. (2013) International Worm Meeting "Analysis of developmental RNA-Seq libraries reveals signature profile for cilia-related genes."

Jensen, Victor L., Leroux, Michel R., Li, Chunmei, Timbers, Tiffany A., Morin, Ryan D.

[International Worm Meeting, 2013]

Ciliogenesis in C. elegans hermaphrodites occurs during late embryogenesis, with most cilia being formed within hours of each other. This makes C. elegans well suited to expression analyses to identify novel cilia genes. Using the available RNA-Seq libraries we identify a signature expression pattern specific to cilia-related genes, which includes a large spike in expression at late embryo with a reduction at mid-L1 (first larval stage) and low levels of expression at all other stages. P-values for Pearson correlation for each gene in the genome are compared to 41 known ciliary genes and each gene is then assigned the minimum calculated p-value. Using a 1E-5 cutoff and only including genes with human orthologues, we identify 635 genes with the specific pattern. Comparisons to CilDb (an online database of previously published ciliary genomics/proteomics experiments) reveals an 15-fold enrichment of genes with greater than seven or more cilia references. Further clustering of this list based on individual expression patterns increases the enrichment by greater than 40 times. Extending this expression analysis method, we also find a shared-but different from the above-expression pattern among serpentine olfactory G-protein-coupled receptors, many of which are known to be targeted to cilia. Using this approach, we identify evolutionarily-conserved proteins that function within three distinct ciliary regions or modules. One, a recently-characterized protein linked to retinitis pigmentosa, is found in the proximal-most segment of the axoneme, which is implicated as a ciliary gate needed for effective signalling. Another protein is present at the basal body, which is required for the formation of cilia. Finally, a third protein represents a previously undescribed component of the intraflagellar transport (IFT) machinery, which plays a critical role in ciliogenesis and delivering cargo (e.g. signalling proteins) to cilia.

Jensen, Victor L. et al. (2017) International Worm Meeting "Role of metazoan dynein in transition zone formation, ciliary maintenance, and processive intraflagellar transport."

Mohan, Swetha, Timbers, Tiffany A., Leroux, Michel R., Cai, Jerry, Jensen, Victor L.

[International Worm Meeting, 2017]

Cilia are ubiquitous microtubule-based organelles found at surface of metazoan cells. Non-motile (sensory/primary) cilia act to receive signals from the environment, and motile cilia either move cells or the surrounding fluid. Cilia are built and maintained using a cargo-trafficking intraflagellar transport (IFT) machinery powered by kinesin (anterograde) and dynein (retrograde) molecular motors. How IFT-dynein contributes mechanistically to ciliary processes in metazoans remains poorly studied, in part because null mutants exhibit a severe terminal phenotype with IFT protein accumulations in the bulbous tips of highly truncated cilia. We carried out a screen for retrograde IFT transport defects in C. elegans, and identified the first temperature-sensitive IFT mutant in a metazoan. Using this strain, we show that the IFT-dynein (CHE-3) heavy chain is essential for cilium maintenance and correct formation of the transition zone (TZ) ciliary gate. Cilia resorb upon shift to restrictive temperature (inactive IFT-dynein), and are restored upon return to permissive temperature (active IFT-dynein). Importantly, this resorption and regrowth is observed for primary cilia in terminally differentiated, sensory neurons. We next investigated how IFT-dynein enables processive IFT, in contrast to the bidirectional tug-of-war mechanism of transport used by kinesin and dynein elsewhere in the cell. We find that the IFT-dynein heavy, intermediate and two light chains are trafficked by the IFT-subcomplex B, unlike the light intermediate chain, which is transported independent of the canonical IFT complex. This differential transport provides an effective mechanism by which the retrograde transport machinery is maintained in an inactive state during anterograde transport. Together, our findings reveal that IFT-dynein is required for ciliary maintenance and TZ formation, and regulates the processivity of anterograde IFT by spatial separation and thus inactivation of its components.

Warburton-Pitt, Simon RF et al. (2011) International Worm Meeting "Ciliogenesis in Caenorhabditis elegans requires genetic interactions between nphp-2 and transition zone associated genes."

Leroux, Michel R, Warburton-Pitt, Simon RF, Barr, Maureen M, Juaregui, Andrew R, Li, Chunmei

[International Worm Meeting, 2011]

The cystic kidney diseases Nephronophthisis (NPHP), Meckel Syndrome (MKS), and Joubert Syndrome (JBTS) share an underlying etiology of dysfunctional cilia. INVS, mutated in NPHP type II, encodes inversin, a cilia localizing protein. Here we show that the C. elegans inversin homolog, nphp-2, localizes to the transition zone (TZ) and middle segments of sensory cilia, and is partially redundant with the NPHP4/nephrocystin-4 homolog nphp-4 for the proper biogenesis of cilia, namely positioning and axoneme formation. nphp-2 is not required for correct localization of other TZ proteins. nphp-2 genetically interacts with other ciliopathy gene homologs in a cell-dependent manner to control cilia formation. We conclude that nphp-2 plays an important role in C. elegans cilia, and that this gene defines a third cell-type specific pathway that acts in conjunction with previously described NPHP and MKS pathways in cilia formation and development.

Jensen, Victor L. et al. (2009) International Worm Meeting "The novel cilia protein DAF-25 is required for DAF-11 cilia localization."

Riddle, Donald L., Bishop-Hurley, Sharon, Bialas, Nathan J., Jensen, Victor L., Leroux, Michel R.

[International Worm Meeting, 2009]

Four alleles of daf-25 were isolated from screens for new temperature-sensitive Daf-c (dauer-constitutive) mutants. At restrictive temperature, daf-25 mutants form dauer larvae even when food is abundant. The Daf-c phenotype can be rescued by maternally supplied daf-25(+). Compared to N2, daf-25 adults failed to avoid sucrose and high concentrations of NaCl, indicating that they are defective in sensing osmotic gradients. There is also a reduction of brood size when worms are shifted to the restrictive temperature (25 deg C) at L4, despite normal levels of progeny at the permissive temperature (15 deg C). The Daf-c phenotype of daf-25 is suppressed by daf-10/IFT122 but not daf-6/PTCHD3. This puts DAF-25 function downstream of DAF-6 but upstream of DAF-10. This implies that DAF-25 function requires cilia because DAF-10 is required for proper cilia development, whereas DAF-6 is required for proper formation of the receptor channel and exposure of the ciliated neurons to the environment. DAF-25 does not appear to be required for ciliogenesis, because there is a normal pattern of DiI staining in all stages, including the dauer. A rescuing DAF-25::GFP fusion protein is expressed in ciliated neurons, as well as some interneurons, and shows sub-cellular localization to the cilia. Because the phenotype, epistatic order and expression profile of daf-25 are similar to daf-11 (encoding a membrane-bound guanyl cyclase), we thought there may be an effect on DAF-11 in a daf-25 mutant background. Indeed, DAF-25 is required for proper DAF-11::GFP localization to the cilia. This may be a specific interaction because daf-25 mutations do not affect the cilia localization of GFP translational fusions for TAX-4/CNGA1 or OSM-9/TRPV4. We show that DAF-25 is a novel cilia protein. We are currently exploring the possible role of the mammalian ortholog of DAF-25 in human disease.

Sadler PL et al. (1995) International C. elegans Meeting "CENTROSOME RELATED MUTANTS DISRUPT MULTIPLE ASPECTS OF SPERMATOGENESIS IN C. ELEGANS"

Sadler PL, Shakes DC

[International C. elegans Meeting, 1995]

Originally classified as temperature sensitive maternal and paternal effect lethal mutants, emb-27 and emb-30 share highly similar phenotypes. Due to the general rarity of paternal effect phenotypes, we began analyzing gametogenic defects which might underlie the very striking 100 - 400 cell stage arrest of the mutants. Our results show that emb-27 and emb-30 disrupt multiple aspects of spermatogenesis in C. elegans. Spermatids from homozygous mutants show a range of morphological defects in cell size, nuclear distribution, and nuclear position. Under the most restrictive conditions, DNA missegregation is severely affected such that the majority of sperm are anucleate. Analysis of mutant meiotic figures indicates that most of these defects arise during the meiotic segregation of the haploid spermatids from the residual body. In addition, hemizygous males exhibit a wide range of germline defects including a breakdown in overall gonad polarity and DNA degradation in the more distal region of the gonad. Interestingly, synthesis and distribution of the major sperm proteins in both homozygote and hemizygote gonads is normal despite the chromosomal segregation defects. In terms of molecular identity, Tabbish et al (WBG Vol 13,#4,57 and at this meeting) recently described the rescue of emb-30 by a novel gamma tubulin gene. Given the similarities of emb-27 and emb-30 ,we are currently engaged in an analysis of possible centrosome related candidate genes for emb-27. One possibility is a t-complex gene residing on cosmid T05C12. A collaboration with Michel Leroux has been established to test the theory that emb-27 may encode tcp-1.

Timbers, Tiffany A. et al. (2013) International Worm Meeting "Screening a Million Mutations to Identify Novel Ciliary Proteins."

Leroux, Michel R., Garland, Stephanie, Lee, Katherine, Moerman, Donald G., Timbers, Tiffany A., Jensen, Victor L., Edgley, Mark

[International Worm Meeting, 2013]

Cilia are cellular organelles that enable sensory physiology (e.g. vision) and modulate various developmental signaling pathways (e.g. Wnt). Disrupting the function of these organelles results in a wide range of ailments, and is associated with many human genetic disorders, including polycystic kidney disease and Bardet-Biedl syndrome. To identify novel proteins required for the formation and function of primary cilia we have undertaken an innovative high-throughput screen of the "Million Mutation Project" (MMP; http://genome.sfu.ca/mmp) C. elegans library for phenotypes related to the dysfunction of cilia. This resource consists of 2007 chemically mutagenized worms, all of which have been sequenced to a depth of 15x genome equivalents, and collectively harbor 826,810 single nucleotide variants in 20,115 genes (Thompson et al. submitted). As the nature of the disrupted gene is known the library facilitates gene cloning, the greatest bottleneck in traditional mapping-cloning approaches after chemical mutagenesis. From screening the library we have identified strains with previously unidentified mutations that result in gross defects in cilia structure. These were uncovered using a dye-filling assay. Abrogated ciliary functions were revealed by probing cilia-dependent behaviors such as CO2 avoidance and dauer formation. To date, we have screened 20% of the library (400 strains) using the dye-filling assay and have identified 10 strains in which no neurons fill with DiI, and 30 strains in which the staining of the dye-filled neurons is weaker. 10 strains appear to have novel mutations. SNP mapping and rescue experiments will be used to identify the causative mutation. Detailed molecular analyses of these novel ciliary proteins will provide new insights into the factors required for cilia formation and function, fundamental cellular processes with important biomedical implications.

Li, Chunmei et al. (2011) International Worm Meeting "Sensing the environment: novel molecular pathway for the formation of sensory cilia."

Kida, Katarzyna, Blacque, Oliver E., Williams, Corey L., Leroux, Michel R., Yoder, Bradley K., Li, Chunmei, Jensen, Victor L.

[International Worm Meeting, 2011]

C. elegans perceives its environment mainly by way of sensory neurons which have cilia at the distal ends of dendrites, much like mammals can smell with the use of olfactory cilia or can see with photoreceptor cilia. In studying several human disorders involving cilia dysfunction (ciliopathies), we have uncovered a novel molecular pathway necessary for ciliogenesis. The disorders in question-nephronophthisis (NPHP), Meckel syndrome (MKS), Joubert syndrome (JBTS), Senior-Loken syndrome (SLSN), and Leber congenital amaurosis (LCA)-present with overlapping ailments, such as retinopathy, kidney disease, liver fibrosis and brain malformations. They also show considerable allelism between at least twelve causative genes, suggesting a common molecular aetiology that remains unexplained. We demonstrate using C. elegans that the recently-identified MKS-6 and MKS-2 proteins, together with MKS-1, MKSR-1, MKSR-2, MKS-5, NPHP-1 and NPHP-4, collectively function at the base of cilia, in a region termed transition zone (TZ), to orchestrate cilium formation. Specifically, the proteins act as two distinct modules, which we term MKS and NPHP, to facilitate basal body-transition zone anchoring to the membrane; disruption of the TZ proteins results in defects in prominent ciliary TZ and axoneme formation defects, and thus, chemosensory anomalies. This first pathway is independent of a second pathway specifically required for the formation and function of the ciliary organelles, involving intraflagellar transport (IFT) and Bardet-Biedl syndrome (BBS) proteins. Our genetic and cell biology analyses reveal a hierarchical organisation of the TZ proteins, with MKS-5 as the central anchor, followed by B9 domain-containing proteins (MKS-1, MKSR-1, MKSR-2). Together, our findings expand the interaction network of ciliopathy-associated proteins and suggest a two-stage ciliogenic pathway that first involves transition zone proteins, followed by an intraflagellar transport (IFT)-dependent formation of the remaining axoneme.

Waddell, Brandon et al. (2021) International Worm Meeting "The C. elegans Shugoshin (SGO-1) is a cilia resident protein that interacts with TAC-1/TACC"

Li, Chunmei, Giesbrecht, Lesa, Timbers, Tiffany A., Park, Kwangjin, Waddell, Brandon, Quick, Evan, Egydio de Carvalho, Carlos, Leroux, Michel R.

[International Worm Meeting, 2021]

In organisms with monocentric chromosomes, the Shugoshin family of cell cycle regulators have conserved functions in spindle attachment and sister chromatid cohesion during mitosis and meiosis. In worms, the sole Shugoshin homolog (sgo-1) has early roles in meiosis but is otherwise dispensable for gamete and embryo viability. We have identified a novel function of SGO-1 in sensory neurons. sgo-1 is expressed in a series of adult amphid and phasmid neurons where the protein localizes to the base of cilia. sgo-1 mutants display behavioural and dauer entry defects consistent with dysfunctional cilia. These phenotypes are in turn rescued by a sgo-1(+) construct specifically expressed in cilia-producing neurons. In a screen for SGO-1-interacting proteins, we identified TAC-1, a regulator of spindle microtubule dynamics. TAC-1 co-localizes with SGO-1 in the base of cilia and a tac-1 mutation suppresses sgo-1 sensory and dauer entry defects. We are exploring the hypothesis that SGO-1 influences microtubule maintenance in the ciliary axoneme through the recruitment of TAC-1 to the basal body. These results add to a growing body of evidence suggesting that kinetochore and centrosomal proteins are transiently targeted to cilia and represent the first description of a non-nuclear role of Shugoshin in a post-differentiated cell type.