Kaplan, Oktay et al. (2009) International Worm Meeting "Distinct roles for C. elegans clathrin adaptin complexes AP1 and AP2 in mediating protein trafficking to cilia."

Blacque, Oliver, Kaplan, Oktay, Kida, Katarzyna

[International Worm Meeting, 2009]

Clathrin in association with four adaptor complexes (AP1-4) facilitates membrane trafficking and protein sorting between various cellular compartments. Despite increasing evidence implicating these systems in ciliary processes, the role of metazoan clathrin and AP complexes in cilium formation and function remains poorly understood. Here we addressed this question in C. elegans sensory neurons. First, we found that clathrin heavy chain (CHC-1), AP1 (UNC-101, APS-1) and AP2 (DPY-23, APA-2) subunits, but not AP3 subunits, are required for normal cilia integrity, morphology and ultrastructure. Subcellular localisation analysis using rescuing GFP reporters revealed that AP1 and AP2 subunits display distinct non-ciliary localisations, with UNC-101 and APS-1 predominantly within the perinuclear region, whereas DPY-23 is found more diffusely throughout the cell, including a region near the ciliary base. Loss of CHC-1, AP1 and AP2 subunit function results in overlapping yet distinct defects in ciliary protein trafficking. In chc-1 and unc-101 mutants, ODR-10 (transmembrane protein) is trapped at the plasma membrane and fails to undergo dendritic translocation, whereas in dpy-23 mutants, ODR-10 translocates normally along dendrites but frequently accumulates within the distal dendrite region. Consistent with differential effects of AP1 and AP2 subunits on membrane trafficking to cilia, the assembly of enlarged AWB ciliary membrane fans in grk-2 mutants requires unc-101, but not dpy-23. Similarly, loss of CHC-1 and AP1/2 function differentially affects intraflagellar transport, with IFT grossly normal in unc-101 mutants, partially slowed in dpy-23 mutants and absent altogether in chc-1(RNAi) animals. Finally, loss of chc-1 and dpy-23 function, but not unc-101, results in the distal dendrite accumulation of IFT proteins. Together, these data demonstrate roles for metazoan CHC, AP1 and AP2, but not AP3, in cilium formation. In addition, these findings suggest that AP1 and AP2 operate at distinct ciliary protein sorting compartments, mediating differential effects on membrane trafficking to cilia and intraflagellar transport.

Cevik, Sebiha et al. (2009) International Worm Meeting "A C. elegans homologue of Joubert syndrome-associated Arl13B associates with the ciliary membrane and is required for proper targeting and motilities of ciliary transmembrane and IFT proteins."

Kaplan, Oktay, Blacque, Oliver, Cottell, David, Toivenon, Tiina, Kida, Katarzyna, Cevik, Sebiha

[International Worm Meeting, 2009]

Joubert syndrome (JS) and JS-related disorders (JSRD) are clinically heterogeneous, characterised by cerebellar malformation and hypervariable phenotypes such as cystic kidneys, retinitis pigmentosa and polydactyly. Emerging evidence indicate that JS/JSRD are caused by defects in primary cilia, which serve diverse sensory and signaling roles during chemo/mechano-sensation, phototransduction, and development. Although multiple JS/JSRD proteins are known, the molecular bases of their functions remain poorly understood. Here, we investigate the molecular functions of C. elegans ARL-13, a homologue of the small Ras G-protein Arl13b, which causes classical JS and is required for vertebrate cilium formation and sonic hedgehog signaling. First, we find that ARL-13 is mostly restricted to proximal (middle) segments of ciliary axonemes, does not undergo intraflagellar transport (IFT), and requires N-terminal palmitoylation lipid modification for its ciliary targeting/retention. Loss of ARL-13 function disrupts cilium chemosensory function, morphology, and ultrastructure, with defects including truncated axonemes, microtubule misplacement/malformation, enlarged middle segments and ciliary accumulation of matrix/membranous material. Consistent with these phenotypes, arl-13mutants are defective in localisation and/or translocation of ciliary proteins, with ciliary transmembrane proteins partially mislocalised and IFT proteins exhibiting anterograde IFT motility defects, indicative of kinesin2 motor uncoupling, differential separation of IFT complex A/B components, and reduced OSM-3 translocation rates. Finally, arl-13 interacts synthetically with bbs-8, dyf-5 and nph-4, with double mutants exhibiting enhanced cilia integrity and IFT defects. Taken together, we propose that ARL-13 associates with the ciliary membrane where it maintains cilium structure/function and the localisation and motilities of ciliary transmembrane and IFT proteins, perhaps by regulating aspects of ciliary membrane biogenesis and/or turnover. We also suggest that defects in this function underlie the molecular basis of Arl13b-associated Joubert syndrome.

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.

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.

Jensen, Victor et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25"

Bishop-Hurley, Sharon, Molday, Robert, Leroux, Michel, Bialas, Nathan, Nguyen, Phuong, Jensen, Victor, Blacque, Oliver, Kida, Katarzyna, Riddle, Don L, Molday, Laurie

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In harsh conditions Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-? and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation, and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10 /IFT122 mutations (which disrupt ciliogenesis), but not by daf-6 /PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order and expression profile of daf-25 are similar to daf-11 , which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88 and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.