Singhvi, Aakanksha et al. (2015) International Worm Meeting "Glia engulf AFD neuron receptive endings."

Bae, Andrea, Shaham, Shai, Singhvi, Aakanksha

[International Worm Meeting, 2015]

Glia participate in pruning of neuronal receptive endings during development and in response to plastic changes in the nervous system. We present evidence that glial engulfment of neuronal receptive endings also takes place in C. elegans. We find that animals expressing GFP targeted to the sensory receptive ending of the AFD sensory neuron also display GFP fluorescence in punctate structures within the amphid sheath glia that wrap around these receptive endings. Importantly, ablation of the AFD neuron abrogates glial GFP accumulation, suggesting that glia are taking up neuronal material. We show that this engulfment process is independent of known apoptotic engulfment genes, suggesting that glia employ a novel machinery to regulate uptake of neuronal content. We have previously demonstrated (see abstract by Singhvi and Shaham, this meeting) that AFD-glia interactions are molecularly similar to interactions in the mammalian retina between photoreceptor neurons and the glia-like retinal pigmented epithelium (RPE). Strikingly, outer segments of photoreceptor cells are regularly engulfed by the RPE, suggesting deep parallels between the C. elegans and retinal sensory structures, and also suggesting that C. elegans could be used to understand retinal outer segment pruning. To begin to test this idea, we performed candidate and forward genetic screens that together uncover eleven independent mutants that disrupt glial engulfment of AFD neuron material. We present preliminary characterization and cloning of some of these mutants. Our work suggests that pruning of receptive endings may be an evolutionarily conserved function of sensory glia to regulate the structure and function of sensory receptive endings.

Katz, Menachem et al. (2017) International Worm Meeting "Repetitive behavior mediated by glutamate is controlled by astrocyte-like glia in C. elegans."

Keil, Wolfgang, Katz, Menachem, Singhal, Anupriya, Shaham, Shai, Liang, Yupu, Corson, Francis, Bae, Andrea, Lu, Yun

[International Worm Meeting, 2017]

Neural circuits control animal behavior, and their dysfunction promotes neurological diseases manifesting aberrant behaviors. Neural circuits are tightly associated with astroglial processes, yet glial roles in animal behavior are not well understood. C. elegans has a compact nervous system driving a limited and well-characterized behavioral repertoire. C. elegans glia are not essential for neuron viability, making this animal a useful setting for investigating glial influences on circuits and behavior. We have characterized roles of the brain-associated C. elegans CEPsh glia in animal locomotion. We demonstrate that, like astrocytes, CEPsh glia infiltrate the brain neuropil and abut glutamatergic synapses. Postembryonic ablation of CEPsh glia results in foraging defects, including an increased reversal rate. CEPsh glia transcriptome analysis reveals similarities to astrocytes, including expression of the glial glutamate transporter, glt-1. Behavior studies reveal that glt-1 mutants, unlike wild-type animals, have increased reversal frequency, as well as repetitive reversal bouts. AVA neurons control reversal initiation, and our in vivo measurements of synaptic glutamate adjacent to AVA reveal roles for glial GLT-1 in rapid glutamate clearance. Inappropriate clearance leads to high-frequencies oscillations in synaptic glutamate and AVA activation. We show that these oscillations are mediated by the presynaptic metabotropic glutamate receptor mgl-2/mGluR5, and that mutations in mgl-2 suppress glt-1-mediated repetitive reversals. Loss of murine astrocyte-expressed GLT1 has been shown to produce repetitive grooming, and inhibition of murine mGluR5 ameliorates repetitive behavior in an Obsessive Compulsive Disease (OCD) model. Our studies, therefore, suggest conserved roles for glutamate clearance by glia in controlling repetitive behavior, a feature of some human neuropsychiatric disorders.

De Vore, Deanna Michele et al. (2013) International Worm Meeting "Exploring the role of mechanosensory extracellular matrix components in the structure and function of primary cilia."

Knobel, Karla, De Vore, Deanna Michele, Barr, Maureen

[International Worm Meeting, 2013]

PKD2 encodes a transient receptor potential polycystin (TRPP) channel receptor protein found in non-motile, primary cilia of mammalian cells 1. In humans, PKD2 mutations result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). In the nematode Caenorhabditis elegans, the polycystin-2 homolog, PKD-2, localizes to the primary cilia of male-specific sensory neurons (CEMs and RnBs in the head and tail respectively) where it is required for male mating behaviors. Given the ancient and evolutionarily conserved role for polycystin-2 in cilia, I am using C. elegans as a model to identify new genes required for ciliary receptor localization. Our laboratory performed a forward genetic screen for PKD-2::GFP ciliary localization (Cil) defective mutants (Bae et al 2008). The cil-2(my2) mutants exhibit excess unusual PKD-2 accumulation at the ciliary base, cilium proper, and around the distal dendrite. We performed three-factor and deficiency mapping, whole genome sequencing, and single gene rescue experiments and determined that the cil-2(my2) allele is a mutation in the gene, mec-5. MEC-5 is a unique collagen protein found in the extracellular matrix (ECM) surrounding touch receptor neurons (Du et al 1996). In touch receptor neurons (TRNs), MEC-1, MEC-5, and MEC-9 are found in the ECM, play a role in mechanosensation, and may localize degenerin/epithelial sodium channels (DEG/ENaCs) along the TRN processes (Du et al 1996, Emtage et al 2004). Our preliminary data indicates that mec-1 and mec-9 regulate PKD-2::GFP localization in male-specific sensory neurons. We are currently determining how these ECM proteins regulate localization and channel properties of the PKD-2 TRP polycystin channel. These studies will shed light on how sensory receptors like PKD-2 are targeted to cilia, and may advance the understanding and treatment of ADPKD. References: 1) Bae YK et al, Dev Dyn. 2008 Aug;237(8):2021-9. 2) Du H. et al, Neuron. 1996 Jan;16(1):183-94. 3) Emtage L et al, Neuron. 2004 Dec 2;44(5):795-807.

De Vore, Deanna Michele et al. (2011) International Worm Meeting "Mapping the PKD-2 localization defective mutant cil-2(my2)."

De Vore, Deanna Michele, Knobel, Karla, Wang, Juan

[International Worm Meeting, 2011]

PKD2 encodes a transient receptor potential polycystin (TRPP) channel receptor protein located in non-motile, primary cilia of mammalian cells. In humans, PKD2 mutations result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). In C. elegans, PKD-2 localizes to cilia of male-specific sensory neurons where it is required for male mating behaviors. Given the ancient and evolutionarily conserved role for the PKD-2 protein in cilia, we are using C. elegans as a tool to identify new genes required for ciliary receptor localization. To this end, we performed a forward genetics screen and identified several PKD-2::GFP ciliary localization (Cil) defective mutants (Bae et al 2008). Here, we focus on the cil-2(my2) mutant that exhibits accumulation of PKD2::GFP in the ciliary base and proximal dendrites of C. elegans male specific CEM neurons and exhibits temperature sensitive sterility in hermaphrodites. Three factor, SNP, and deficiency mapping in conjunction with whole genome sequencing were used to determine the gene mutated in cil-2(my2) animals. cil-2(my2) maps between +22.9 and +24.4 on linkage group X with mec-5, let-2, crb-1, and tps-1 as possible candidate genes. Complementation testing and single gene rescue experiments are in progress to determine the identity of cil-2. Once we clone cil-2, our future plans include determining whether cil-2 acts cell-autonomously in male-specific neurons and the mechanism by which CIL-2 affects PKD-2 ciliary receptor trafficking. cil-2(my2) hermaphrodite sterility suggests that CIL-2 plays a broader role in the animal, and we will also address this possibility. These studies will shed light on how sensory receptors like PKD-2 are targeted to cilia, and may advance the understanding and treatment of ADPKD. Reference: Bae YK. (2008) Identification of Genes Involved in the Ciliary Trafficking of C. elegans PKD-2 Developmental Dynamics 237:2021-2029.

Karla Knobel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identification of PKD-2 Cilioprotein Targeting Elements"

Karla Knobel, Maureen Barr

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Proper plasma membrane and subcellular localization of signaling receptors and ion channels is required for normal cell physiology and sensory behavior. Mistargeting or dysfunction of ciliary cystoproteins such as the polycystins is the leading cause of polycystic kidney disease (PKD) in humans. I am using the nematode Caenorhabditis elegans to reveal the molecular mechanisms regulating polycystin trafficking within sensory neurons. C. elegans polycystin-2 (PKD-2) is localized to the cilia of certain male sensory neurons (CEMs, RnBs, and HOB). PKD-2 localization and function are required for male mating. I am taking two complementary approaches to study the molecular mechanisms regulating PKD-2 cilioprotein localization in adult male worms. First, I have modified PKD-2 structure in order to identify dendritic and ciliary targeting elements. PKD-2 deletion analysis has revealed that distinct domains mediate ciliary targeting and protein function. The transmembrane (TM) region is sufficient for ciliary trafficking but both of the cytoplasmic N- and C- termini of PKD-2 are required for protein function. I tested the ability of human polycystin-2 (PC-2) to complement a pkd-2 null mutation. Full-length PC-2 retains partial function but is not visibly targeted to cilia and remains in the endoplasmic reticulum (ER). To test if ciliary targeting is essential for PKD-2 function I generated a chimeric protein containing the PKD-2 TM domain fused between the PC-2 cytoplasmic termini. This PC-2:PKD-2 chimera is transported to cilia but is not functional, indicating that PC-2 residual function requires its TM domain but not ciliary targeting. These data also suggest that the cytoplasmic tails of PKD-2 and PC-2 perform divergent functions. To identify new genes required for ciliogenesis or the ciliary localization of PKD-2, Young-Kyung Bae and I performed a genetic screen to identify mutations that interfere with the localization of GFP-tagged PKD-2. Several cil (ciliary localization) mutations that modify the expression or localization of GFP-tagged PKD-2 were identified and are currently under characterization. Please see the poster presented by Young-Kyung Bae at this meeting.

Pierleoni A et al. (2006) Bioinformatics "BaCelLo: a balanced subcellular localization predictor."

Casadio R, Martelli PL, Fariselli P, Pierleoni A

[Bioinformatics, 2006]

Motivation. The knowledge of the subcellular localization of a protein is fundamental for elucidating its function. It is difficult to determine the subcellular location for eukaryotic cells with experimental high-throughput procedures. Computational procedures are then needed for annotating the subcellular location of proteins in large scale genomic projects. Results. BaCelLo is a predictor for five classes of subcellular localization (secretory pathway, cytoplasm, nucleus, mitochondrion and chloroplast) and it is based on different SVMs organized in a decision tree. The system exploits the information derived from the residue sequence and from the evolutionary information contained in alignment profiles. It analyzes the whole sequence composition and the compositions of both the N- and C-termini. The training set is curated in order to avoid redundancy. For the first time a balancing procedure is introduced in order to mitigate the effect of biased training sets. Three kingdom-specific predictors are implemented: for animals, plants and fungi, respectively. When distributing the proteins from animals and fungi into four classes, accuracy of BaCelLo reach 74% and 76%, respectively; a score of 67% is obtained when proteins from plants are distributed into five classes. BaCelLo outperforms the other presently available methods for the same task and gives more balanced accuracy and coverage values for each class. We also predict the subcellular localization of five whole proteomes, Homo sapiens, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae and Arabidopsis thaliana, comparing the protein content in each different compartment. Availability. BaCelLo can be accessed at http://www.biocomp.unibo.it/bacello/ Contact. casadio@alma.unibo.it, andrea@biocomp.unibo.it, gigi@biocomp.unibo.it, piero@biocomp.unibo.it.

Daniel Poole et al. (2006) Development & Evolution Meeting "DIP-1, a Potential DYN-1-Interacting Protein, Is Critical for Cleavage Furrow Completion"

Daniel Poole, Yunsik Kang, Ahna Skop, Margaret Forrestal

[Development & Evolution Meeting, 2006]

Cytokinesis is the most fundamental process in the development of all living organisms. The mechanisms that control cytokinesis are unknown, yet we know that membrane-cytoskeletal events are crucial for the cellular dynamics that occur. The process is not only of considerable intellectual interest but also of relevance to a number of physiological and pathological conditions. Members of the dynamin protein family have been shown to play essential roles in cell division in both plant and animal systems. Yet outstanding questions remain as to how dynamin is regulated during mitosis and what role dynamin plays during furrow ingression and completion. We would like to identify proteins that regulate dynamin/DYN-1 function during mitosis. We have initially characterized DIP-1 (Dynamin Interacting Protein-1), a protein identified to interact with DYN-1 from a genome-wide yeast two-hybrid screen (Li et al. 2004). DIP-1 is an uncharacterized transmembrane, ankryin, and BRCT domain-containing protein similar to BARD1 in its C-terminus. BRCT domain-containing proteins are commonly involved in cell cycle control, and BRCT domains are found in proteins such as BRCA1 and ECT2, both of which are also involved in cytokinesis (Bae et al. 2005; Saito et al. 2003). Preliminary data suggests that DIP-1 functions in cleavage furrow completion, similar to what we observe with dyn-1 fRNAi treated embryos. Defects in cell cycle progression, microtubule organization, and chromatin bridging are also observed. DIP-1 protein may exist in several isoforms as dip-1 is alternatively spliced. We are also currently testing whether DYN-1 and DIP-1 biochemically interact. Determining the mitotic role of DIP-1 may provide us with a unique approach by which we can investigate the role and regulation of dynamin/DYN-1 during mitosis.

Rachael A Nimmo et al. (2003) International Worm Meeting "Worm Tales! Molecular Genetic Analysis of Male Tail Development in C. elegans."

Rachael A Nimmo, Alison Woollard, Adam Antebi

[International Worm Meeting, 2003]

The male tail of C. elegans is a specialised structure consisting of a cuticular fan in which are embedded 18 symmetrically placed mechanosensory sensilla called rays, which each have distinct characteristics due to their morphology, position and neurotransmitter usage. The mab-2 mutation causes loss of both the V and T cell derived rays1. We have found this mutation to be allelic with a rnt-1 knockout and to be rescued by the cosmid B0414 which contains rnt-1. We will further analyse the ray phenotype by lineage analysis using suitable markers. In addition, mutations appear to be temperature sensitive for sterility and an intestinal mitotic defect. It is interesting that a rnt-1::gfp reporter construct has been shown previously to be expressed in the intestine as well as seam cells2 so lending support to the hypothesis that both the ray loss and intestinal phenotypes may be caused by mutation of rnt-1.Initial attempts at cloning mab-2 involved screening genes mapping to the same region by feeding RNAi (Chromosome I feeding library was a gift from J. Ahringer). As a result of the screen two genes which phenocopied mab-2 mutants were identified. In light of our subsequent identification of mab-2, it is interesting to find that 3 genes giving a virtually identical ray loss phenotype are located within a 0.21 cM region of LGI.The two novel Mab genes are situated in a putative operon and so are presumably co-regulated. Do worm operons represent functional cassettes of genes as in bacteria? The common RNAi phenotypes of these two co-regulated genes imply interesting co-functionality. Further support for their co-functionality is provided by the fact that homologues of these two genes plus another in the operon are involved in rRNA processing in yeast. In addition, orthologues of one of these Mab genes and another gene in the operon are thought to modulate the Notch signalling pathway in mammals. We are in the process of investigating the possibility that these functions are conserved in C. elegans.References: 1) Hodgkin, J. Genetics (1983) 103: 43-64; 2) Nam S, Jin YH, Li QL, Lee KY, Jeong GB, Ito Y, Lee J, Bae SC. Mol Cell Biol. (2002) 22(2):547-54.

Young-Kyung Bae et al. (2007) International Worm Meeting "The male connection: A Cil-Spe mutant."

Maureen Barr, Young-Kyung Bae

[International Worm Meeting, 2007]

The C. elegans male has one reason to live: to transmit his genetic material by mating. Accordingly, the male-specific nervous system is devoted to copulatory behaviors. The polycystin-1 (PC1) receptor LOV-1 and transient receptor potential polycystin-2 (TRPP2) channel PKD-2 mediate mating behaviors (Barr and Sternberg,1999). In humans, defects in PC1 or TRPP2 result in autosomal dominant polycystic kidney disease (ADPKD). Cilia are the site of action for both the human and worm polycystins, but how membrane proteins are targeted to cilia is largely unknown. To identify new genes involved in localizing ciliary receptors, we performed a genetic screen looking for PKD-2::GFP ciliary localization (Cil) defects. We focus here on the my15 mutant. In wild type, PKD-2::GFP localizes to cilia and neuronal cell bodies. In the my15 mutant, both PKD-2::GFP and LOV-1::GFP are evenly distributed throughout the entire neuron including the cilium, dendrite, cell body, and axon. The my15 Cil phenotype is reminiscent of PKD-2 mislocalization in an unc-101 AP-1 mu1 subunit mutant (Bae et al., 2006), which also disrupts targeting of the ciliary membrane proteins TRPV OSM-9 and GPCR ODR-10 (Dwyer et al., 2001). However, OSM-9 and ODR-10 distribution is not altered in the my15 mutant, suggesting that my15 may affect a male-specific trafficking component required for polarized trafficking to the distal dendrite/cilia. Interestingly, my15 mutants also exhibit a sperm defect (Spe) in both male and hermaphrodite. my15 males produce normal spermatids but fail to activate, resulting in immotile and nonfunctional spermatozoa. Sperm activation involves membrane fusion. Our data suggest that my15 may be required for ciliary targeting/insertion of PKD-2 containing vesicles in sensory neurons and for membrane fusion during sperm activation. my15 is a nonsense mutation in a phosphatidylinositide (PI) 5-phosphatase gene. PI metabolism is important for many cellular processes including membrane trafficking. We are testing the hypothesis that a common PI-mediated pathway regulates membrane trafficking in both male-specific sensory neurons and sperm. Our study will provide understating of how a C. elegans male successfully navigates the complexities of mating, from copulatory behavior to sperm activation.

Karla M Knobel et al. (2003) International Worm Meeting "Cilia are where it's at: LOV-1 and PKD-2 localization"

Young-Kyung Bae, Dave Saber, Maureen M Barr, Karla M Knobel

[International Worm Meeting, 2003]

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic ailment that affects all humans. Mutations in polycystin-1 or -2 (PC1 and PC2) cause ADPKD. PC1 and PC2 form a receptor/channel complex. The C. elegans proteins LOV-1 and PKD-2 are structurally related to PC1 and PC2. Like LOV-1 and PKD-2, PC1 and PC2 localize to primary cilia of kidney tubules (Pazour 2002, Yoder 2002). That worm and mammalian polycystins act in the same genetic pathway and localize to cilia suggests conservation of both function and sorting mechanisms. Expression of GFP-tagged PKD-2 is a dynamic process. We have characterized the expression and localization of a GFP-tagged PKD-2 that rescues the mutant pkd-2 mating defects. L3 hermaphrodites and males express the fusion protein in the cytoplasm of several head and tail neurons. We are determining the identity of these neurons and whether PKD-2 functions outside the male sensory nervous system. During L4, the non-sex specific expression diminishes and the GFP-tagged PKD-2 is increasingly expressed in the cytoplasm of the male CEM neurons, certain ray neurons, and the HOB hook neuron. By adulthood, the GFP-tagged protein is enriched in the cell body and cilia of these neurons. We are determining what cis elements in LOV-1 and PKD-2 are required for ciliary sorting. In contrast to the C-terminal GFP-tagged PKD-2 fusion protein just described, an N-terminal GFP-tagged PKD-2 is mislocalized, suggesting that the cytoplasmic amino terminus of PKD-2 is required for ciliary localization. Deletion of the carboxy terminus of LOV-1 results in a GFP fusion that is restricted to the cell body (Barr and Sternberg, 1999). We would like to determine what features of the LOV-1 cytoplasmic tail contribute to its localization. To identify gene products required for the proper localization of PKD-2 we will perform a GFP mislocalization screen. We anticipate identifying mutations that delay or modify the expression of GFP-tagged PKD-2 and in genes like unc-101 that are required for the ciliary localization of PKD-2 (see abstract by Y-K. Bae).