Mohan, Swetha et al. (2011) International Worm Meeting "C.elegans rootletin homolog, che-10, is required for intraflagellar transport and cilia maintenance."

Mohan, Swetha, Leroux, Michel

[International Worm Meeting, 2011]

Non-motile or primary cilia are specialized microtubule-based organelles that protrude from many cell types in metazoans. They are adapted to serve many sensory functions, including transducing mechanical, chemical and visual stimuli. The formation and maintenance of all cilia depends on a process termed intraflagellar transport (IFT), which mobilizes ciliary precursor proteins from the base of the cilia (basal body) to the growing end of the ciliary compartment. IFT involves two anterograde molecular motors, heterotrimeric Kinesin-II (KIF3) and homodimeric Kinesin-II (KIF17). The retrograde motor, cytoplasmic dynein 1b/2, is loaded on to the axoneme during anterograde transport and is activated upon arrival at the tip of the cilium, where it is required for retrograde transport of all IFT machinery. Primary cilia are mainly composed of the ciliary axoneme, the basal body from which the axoneme nucleates, the ciliary membrane and a cytoskeleton-like structure called ciliary (or striated) rootlets. Rootlets are associated with the basal body and extend proximally towards the cell nucleus. Striated rootlets are evolutionarily-conserved organelles whose components and function remain unclear. Rootletin is a core structural component of the striated rootlets; in the absence of the protein, the rootlets do not form. Although rootlets are known to be required for the stability of some cilia (e.g. photoreceptor cilia), their mechanism of function remains unknown. Here, we demonstrate that the C. elegans Rootletin homolog, che-10, is required for the maintenance of cilia. In the absence of CHE-10, only some cilia are present post-embryogenesis but these degenerate over time. che-10 mutants have varying lengths of cilia, wherein IFT is absent from shorter (<5 microns) cilia. Furthermore, velocity analyses of IFT proteins suggests that IFT is deregulated in che-10 mutants. The strict regulation of IFT velocities may be compromised due to defects in motor coordination. We hypothesize that CHE-10 (Rootletin) regulates IFT by assembling the IFT components at the base of the cilium, such that disruption of the cytoskeletal protein leads to improper assembly and eventual degeneration of cilia.

Park, Kwangjin et al. (2015) International Worm Meeting "CDKL-1, a protein related to the human CDKL5 kinase implicated in Rett syndrome and epilepsy, is a novel transition zone protein that controls cilium formation."

Leroux, Michel, Park, Kwangjin, Li, Chunmei

[International Worm Meeting, 2015]

The surfaces of most cells in humans have small antenna-like structures termed cilia. There are two types of cilia: motile cilia and non-motile cilia. Motile cilia facilitate cell locomotion or fluid flow and non-motile cilia mediate cellular signaling, critically important for development. We are studying a specialized subcompartment of cilia, called the ciliary transition zone (TZ). The role of the TZ is to create a 'barrier' that controls the protein composition of the cilium, a function that is critical for the ability of the cilium to capture and transduce signals. In this study, we identified cyclin-dependent kinase-like 1 (CDKL-1) as a novel TZ protein. To our surprise, CDKL-1 is not associated with the formation of the TZ barrier, but instead, regulates ciliary length. We observe elongated cilia in cdkl-1 null ADL neurons, and shorter ADL cilia when CDKL-1 is overexpressed, indicating the correct level of this kinase is crucial for cilium length control. Mutations of human CDKL5, one of five CDKL family members, often cause human neurological disorders such as Rett syndrome and epilepsy, and most mutations are located in the conserved kinase domain of CDKL family members. We therefore introduced three of these mutations into C. elegans CDKL-1 (the only C. elegans CDKL family member). All CDKL-1 variants harboring the mutations failed to localize to the TZ, implicating that CDKL5 mutations may result in ciliary dysfunction in humans. Altogether, our results suggest that CDKL family members are ciliary proteins that regulate ciliary length and their dysfunction may lead to neurological phenotypes in humans.

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

Ready, Bryn et al. (2013) International Worm Meeting "The role of the C. elegans Shugoshin homolog in sensory neurons."

Leroux, Michel, Baxi, Kunal, Carvalho, Carlos, Timbers, Tiffany, Ready, Bryn

[International Worm Meeting, 2013]

Members of the Shugoshin family of proteins have essential roles in cell division from yeast to humans. Shugoshin is necessary to prevent premature loss of sister chromatid association at the centromere during anaphase. Surprisingly, deletion mutants and RNAi knockdown of the sequence homolog of Shugoshin in C. elegans (sgo-1; C33H5.15) does not show significant loss of viability. Considering that C. elegans chromosomes lack discrete centromeres, these findings pose the question of what, if any, is the function of this protein in the worm. Here we present preliminary evidence for the expression and function of SGO-1 in sensory neurons with cilia, the microtubule-based sensory organelles. Our preliminary findings suggested that the subcellular localization of a GFP::SGO-1 fusion protein is at the base of, or within, cilia. We carried out functional assays to test for a potential role of SGO-1 in ciliary biogenesis, maintenance and/or function using a sgo-1 deletion mutant. We found that sgo-1(tm2443) worms display developmental and behavioral defects also observed in other true ciliary mutants: (1) worms are defective in dauer formation; (2) worms do not disperse on food but rather stay aggregated, (3) worms show a reduced and retarded avoidance response to CO2 and (4) worms fail to chemotax towards the attractant diacetyl. We are currently investigating the biological function of SGO-1 in cilia formation by looking at ciliary structure phenotypes of sgo-1(tm2443) mutants that may be explained by defects in Intraflagellar transport (IFT). IFT is a process involved in cilia biogenesis in which molecular motors transport ciliary cargo bidirectionally from the base of the cilia, along the microtubule-based axoneme, towards the growing end of the ciliary compartment. In light of evidence that suggests that Shugoshin may also have extra-nuclear roles in regulating the assembly or stability of microtubule-related structures such as the centrosome, our findings further reveal a previously unknown function for a Shugoshin homolog in the context of a fully differentiated cell type.

Jauregui, Andrew et al. (2009) International Worm Meeting "C. elegans Nephrocystin-2 functionally interacts with the other nephrocystins to regulate cilia shape and position."

Warburton-Pitt, Simon, Leroux, Michel, Barr, Maureen, Jauregui, Andrew, Li, Chunmei

[International Worm Meeting, 2009]

Nephronophthisis is a rare autosomal recessive nephropathy causing progressive renal failure in children and adolescents and is caused by mutation in one of nine different genes (NPHP1-NPHP9), which account for approximately 30% of all cases. Many NPHP genes are evolutionarily conserved, and the NPHP gene products localize to cilia in diverse organisms, however their role in cilia is largely unclear. We previously showed that C. elegans NPHP-1 and NPHP-4 regulate ciliary length and shape, and proposed that the nephrocystins are components of a transition zone (TZ) complex that regulates ciliary protein import and export. Our goal is develop C. elegans as a model organism to study the role the nephrocystins play in cilia and to define their molecular and genetic interactions. We have begun to characterize the C. elegans Inversin homolog NPHP-2, to define its role in cilia, and to examine its interactions with the other nephrocystins. Nephronophthisis type 2 is caused by mutation in a gene called Inversin. In humans, Inversin/NPHP2 is associated with the infantile form of nephronophthisis causing situs inversus, enlarged kidneys, cyst formation and renal failure by the age of five. The Inversin protein localizes to cilia and is involved in the switch between canonical and non-canonical Wnt signaling, thus implicating the cilium in the regulation of Wnt signaling. In C. elegans, nphp-2 is expressed in the ciliated sensory nervous system, and encodes at least two splice forms with overlapping, yet distinct, localization patterns. nphp-2 mutants have TZ placement and orientation defects that are not due to perturbation of the intraflagellar transport machinery required to build all cilia and flagella. While TZ localization of NPHP-1 or NPHP-4 was not altered in nphp-2 mutants, a dramatic increase in NPHP-2::GFP fluorescence was seen in nphp-4 mutants. nphp-2 mRNA levels are not increased, suggesting increased stability or decreased degradation of NPHP-2 in nphp-4 mutants. We are currently exploring the basis of this observation and hypothesize that NPHP-4 may be involved in the switch between the canonical and non-canonical Wnt pathways. Finally, using a yeast-two hybrid approach to study interactions between the nephrocystins, we have identified a nucleoporin that physically interacts with NPHP-4, NPHP-2, and OSM-6 and localizes to the ciliary base. We are currently exploring the function of this candidate and testing the ciliary TZ complex hypothesis.

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.

Li, Chunmei et al. (2009) International Worm Meeting "Ciliopathy-associated proteins maintain the structural and functional integrity of the ciliary gate."

Bialas, Nathan, Inglis, Peter, Kida, Katarzyna, Leroux, Michel, Li, Chunmei, Blacque, Oliver

[International Worm Meeting, 2009]

Cilia are membrane-ensheathed organelles with motile and/or sensory functions that are important for a wide array of physiological and developmental processes. Many of the proteins destined for localisation to cilia are trafficked on post-Golgi vesicles that must fuse at the base. The proteins may then be captured by a non-vesicular intraflagellar transport (IFT) machinery that docks at transitional fibers and is mobilised within the organelle by virtue of kinesin motor(s). The transitional fibers, along with a neighbouring region (transition zone) possessing Y-shaped crosslinks, join the microtubule axoneme to the membrane and as such form a ''ciliary gate'' that restricts vesicle entry and perhaps also acts as a molecular gatekeeper akin to the nuclear pore complex. However, the components that form or maintain such a ciliary gate are unknown. Candidates include approximately 12 different proteins, all of which are implicated in ciliopathies collectively characterised by retinal degeneration, kidney disease and other ailments; these proteins localise at or near the transition zone and perform essentially unknown roles related to cilia function. Here, we demonstrate using C. elegans that a functional interaction between a novel C2 domain-containing protein (MKS-6), recently implicated in the ciliopathy Meckel syndrome, and the Nephrocystin protein NPH-4, is required for the structural integrity of this ciliary gate. The loss of these two proteins causes a disruption in the ultrastructure of the transition zone region, characterised by an expanded membrane, absence of Y-links and the accumulation of membranous-type material. Our findings expand the interaction network of proteins present within the ciliary transition zone region, and point to the importance of ciliopathy-associated proteins in maintaining a functional ciliary gate.

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.

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.