Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

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PD Wes et al. (1999) International C. elegans Meeting "Information Coding in the C. elegans Olfactory System"

PD Wes, CI Bargmann

[International C. elegans Meeting, 1999]

Different signal transduction pathways coexist within the same cell, interacting in complex ways to produce novel responses, yet preserving fidelity. The olfactory system of C. elegans can be used to study the molecular basis of information processing within a single cell. C. elegans senses a broad array of attractive odors with just two pairs of sensory neurons, AWA and AWC. A number of odorants activate each neuron. Isoamyl alcohol (iaa), 2,3-pentanedione (pd), butanone (bt) and benzaldehyde (bz) all activate AWC, and, furthermore, each odorant utilizes the same cGMP signaling cascade. Nevertheless, animals are able to distinguish amongst some of these odorants. For instance, when placed in a uniform field of bt, animals will no longer chemotax toward a point source of bt (defined as saturation), yet will chemotax toward bz. A screen was conducted to identify mutants that responded normally to bz in the absence of bt, but failed to chemotax toward a point source of bz in a field of bt (defined as cross-saturation). 20,000 haploid genomes were screened, yielding 25 candidates. A range of phenotypes was observed, with the strongest mutant, ky542 , displaying total cross-saturation of bz by bt. ky542 exhibits other defects in odor recognition or processing. It has reduced sensitivity to pd, though its sensitivity to all others odorants remains undiminished. ky542 also has enhanced discrimination between iaa and bz. Olfactory discrimination is also enhanced by starvation, a form of plasticity modulated by serotonin 1 . In addition, ky542 and other cross-saturation mutants display a defect in olfactory adaptation, a long-lasting form of signal desensitization (see abstract by Noelle l'Etoile and C.B.). Several models that may shed light on these olfactory phenotypes are being tested. Understanding the principles behind olfactory discrimination may provide new insights into information coding within single cells, and may also elucidate some forms of behavioral plasticity that are modulated by C. elegans olfaction. 1 Colbert, H.A., Bargmann, C.I. 1997.

Oktay Ismail Kaplan et al. (2008) European Worm Meeting "Investigation of protein targeting to C. elegans sensory cilia"

Oktay Ismail Kaplan, Oliver Blacque

[European Worm Meeting, 2008]

Primary cilia are microtubule-based organelles found on the surface of. eukaryotic cells, where they play critical roles in chemosensation,. signalling and vertebrate development. Mutations in genes associated with. cilia biogenesis and function underlie several human disease phenotypes. including cystic kidneys, retinal degeneration and Bardet-Biedl syndrome. (BBS). Cilia are built and maintained by intraflagellar transport (IFT), a. bi-directional motor-driven motility of protein complexes along ciliary. axonemes that is thought to deliver various protein cargos (e.g.,. structural ciliary components, signalling molecules, membrane. receptors/channels) to the organelle. Although IFT likely serves as a. downstream distribution mechanism of targeting proteins to discrete parts. of ciliary structures, the upstream events of how proteins are targeted. from their site of synthesis (in the cell body) to the ciliary base region. is very poorly understood.. To investigate this question, we employ C. elegans, where neuronal. sensory cilia extend from the distal tip of long dendrite structures. In. this study, we are performing microscopy-based genetic screens (using EMS. mutagenesis) to identify mutations that disrupt trafficking of GFP-tagged. ODR-10 to AWB cilia. ODR-10 is a 7-transmembrane protein that functions as. a receptor to the volatile odorant diacetyl, and as shown previously, ODR-. 10::GFP localises very strongly in an unc-101-dependedent manner to wild-. type AWB and is observed to translocate along the dendritic compartment. [1]. So far in this study, approximately 1000 mutagenised genomes have been. screened, and 14 mutants recovered with defects in ODR-10::GFP ciliary. localisation. A number of these mutants are Dyf(+), indicating that the. inability of ODR-10 to localise to cilia is not due to a significant loss. of ciliary structure and may instead be due to defects in the targeting. mechanism.. 1. Dwyer ND, Adler CE, Crump JG, L''Etoile ND, Bargmann CI. Polarized. dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize. odorant receptors to olfactory cilia. Neuron 31, 277-87 (2001).

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.

M Bickle et al. (1999) International C. elegans Meeting "Investigating the sex-specific expression and function of the putative chemosensor srd-1."

M Bickle, D Hughes, J Hodgkin

[International C. elegans Meeting, 1999]

We are analysing the function and expression of the putative chemosensor encoded by srd-1 . srd-1 was identified as a predicted seven transmembrane receptor by Troemel et al in 1995 1 . A fusion of 3750 bp of promoter region of srd-1 to GFP was expressed in the ASI amphid neurons in hermaphrodites whereas in males it was expressed in the ASI, ADF and what appeared to be the R8/9 ray neurons of the tail. We have identified five putative TRA-1A binding sites in the promoter of srd-1 at approximately -810, -910, -2020, -2600 and -3480. TRA-1A is the zinc-finger protein at the end of the sex-determination pathway which is believed to control sex-specific transcription 2 . We mutated these binding sites in a full-length srd-1::GFP fusion kindly provided by Cori Bargmann. Mutation of the TRA-1A binding site at -2600 resulted in the expression of GFP in the ASI and ADF amphid neurons in hermaphrodites, suggesting that TRA-1A might bind directly to the promoter of srd-1 . We are testing this hypothesis by gel-shift assays. We are also investigating the function of srd-1 . We have isolated in collaboration with the Sanger Center a deletion at the srd-1 locus. The deletion covers -583 to +1017 eliminating the first two exons and parts of the third exon thus representing a likely null mutant. The mutant srd-1 is healthy and shows no morphological abnormalities in males or hermaphrodites. It forms dauers and stays on or near the bacterial lawn suggesting it is not impaired in any gross way for chemotaxis and males can mate. We are performing quantitative dauer formation, chemotaxis and mating assays. srd-2 is a close homolog of srd-1 with 48% identity. We will perform RNAi with srd-2 in a srd-1 mutant and test for behavioral alteration. 1.Troemel, E.R., Chou, J.H., Dwyer, N.D., Colbert, H.A. & Bargmann, C.I. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, 207-218 (1995). 2.. Hodgkin, J. A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev 1, 731-745 (1987).

Holmes, A et al. (2013) International Worm Meeting "Excluding membrane proteins from the cilia: A role for UNC-101 & LRK-1."

Doll, M, Gartner, A, Holmes, A

[International Worm Meeting, 2013]

Neurodegenerative diseases represent an increasing practical and financial burden on developed societies. Parkinson's Disease, affecting dopaminergic neurons, is one such disease, affecting ~1% of people over 60 in the UK. While most PD is idiopathic, ~15% is attributable to genetic causes. Of this, a significant proportion is linked to mutations in LRRK2, a Leucine-Rich Repeat Kinase; implicated in diverse cellular processes, from membrane traffic to mitochondria. The highly conserved C. elegans orthologue of LRRK2, lrk-1, interacts with unc-101, encoding a component of the AP-1 complex - important in post-Golgi transport, to regulate the localisation of synaptic vesicle proteins (SV) (Sakaguchi-Nakashima et al., 2007). We show here that these genes also interact to affect transport to the cilia of dopaminergic neurons. Perturbing unc-101, clathrin and rab-8 misroutes ODR-10, normally localized to the cilia, to all membrane compartments and that there is a requirement for the critical endocytosis proteins, clathrin, dyn-1 and the AP-2 complex in regulating cilium membrane volume (Dwyer et al., 2001, Kaplan et al., 2010, 2012). Using the dopamine transporter, DAT-1, fused to YFP, we show that it is excluded from the cilia of dopaminergic neurons, contrary to ODR-10, in wild type worms. In unc-101, and unc-16, a scaffold protein, mutants DAT-1 is misrouted to the cilia. Mutations in AP-2, dyn-1, & rab-8 did not result in DAT-1 misrouting, suggesting a novel role for the AP-1 complex in sorting cargoes away from the cilia or in establishing a membrane diffusion barrier at the base of the cilium. A lrk-1 mutation enhances the above DAT-1 ciliary misrouting phenotype, in contrast to the latter's rescuing effect on SV proteins. This suggests that lrk-1 may be a cargo dependent regulator of unc-101. Using a LRK-1::GFP we show that it is expressed in dopaminergic neurons and hope to study its localisation and protein interactions to further elucidate function. Additionally, we have performed mutagenesis screens for DAT-1 cilia misrouting phenotypes and have isolated several independent mutations. We hope that these will offer new insight into cilia establishment and maintenance.

Young-Kyung Bae et al. (2003) International Worm Meeting "Localization of C. elegans PKD-2"

Maureen M Barr, Young-Kyung Bae

[International Worm Meeting, 2003]

Autosomal dominant polycystic kidney disease (ADPKD) affects 1 in 500 to 1000 individuals. The primary cause of the disease is associated with the defective function of polycystin-1(PC1) and polycystin-2(PC2) (1). The C. elegans homologues of PC1 and PC2 are LOV-1 and PKD-2, respectively (3), and they are localized in cilia of male-specific sensory neurons in C. elegans and required for male-mating behavior. Recently, the mammalian PC1 and PC2 have been localized to kidney primary cilia and shown to act as a mechanosensitive channel in vitro (2). The ciliary localization of polycystins is thus evolutionarily conserved between vertebrates and C. elegans. As the presence of LOV-1 and PKD-2 in sensory cilia is essential, the proper localization of human PC1 and PC2 may also be essential for function. However, the molecular mechanism of PC localization to cilia is unknown. We will use C. elegans as a model to determine the mechanism of PC localization. We are determining whether vesicular transport or intraflagellar transport (IFT) is required for the localization. Vesicular transport plays an important role in protein sorting of sensory receptors including the ODR-10 GPCR and OSM-9 TRP channel (4). IFT is an evolutionarily conserved process required for ciliogenesis(5). To begin characterization of the mechanisms of PKD-2 localization, we have examined the subcellular localization of functional PKD-2:GFP in various transport mutant backgrounds. The C. elegans gene unc-101 encodes 1 adaptor clathrin mediating vesicular transport, and che-3 encodes a heavy chain of cytoplasmic dynein necessary for retrograde IFT. Under the unc-101 mutant background, PKD-2:GFP is mislocalized to dendrites of CEM neurons in adult males. The che-3 mutant does not affect the PKD-2:GFP localization. Thus, the proper localization of PKD-2:GFP requires the AP-1 clathrin protein in vesicular transport system. We are currently examining PKD-2:GFP in other transport mutant backgrounds. We are also performing time-lapse motility assays in PKD-2:GFP transgenic animals. (1) Torres, V. D. (1998) Curr. Opin. Nephrol. Hypertens. ,7 159-69 (2) Yoder, B. K. et al. (2002) J. Am. Soc. Nephrol. 13, 2508-16 (3) Barr, M. M. & Sternberg, P. W. (1999) Nature 401, 386-89 (4) Dwyer, N. D. et al. (2001) Neuron 31, 277-87 (5) Rosenbaum, J. L. & Witman, G. B. (2002) Nat. Rev. Mol. Cell. Biol. 3, 813-25

Bickle M et al. (2000) European Worm Meeting "Investigating the sex-specific expression and function of the putative chemosensor srd-1"

Hodgkin JA, Bickle M, Hughes D

[European Worm Meeting, 2000]

We are analysing the function and expression of the putative chemosensor encoded by srd-1. srd-1 was identified as a predicted seven transmembrane receptor by Troemel et al in 1995 1 . A fusion of 3750 bp of promoter region of srd-1 to GFP is expressed in the ASI amphid neurons in hermaphrodites whereas, in males, it is expressed in the ASI, ADF and the R8/9 ray neurons of the tail. We have identified five putative TRA-1A binding sites in the promoter of srd-1 at approximately -810, -910, -2020, -2600 and -3480. TRA-1A is the zinc-finger protein at the end of the sex-determination pathway which is believed to control sex-specific transcription 2 . We mutated these binding sites in a full-length srd-1::GFP fusion kindly provided by Cori Bargmann. Mutation of the TRA-1A binding site at -2600 resulted in the expression of GFP in the ASI and ADF amphid neurons in hermaphrodites, suggesting that TRA-1A might bind directly to the promoter of srd-1. We are testing this hypothesis by gel-shift assays. We are also investigating the function of srd-1. We have isolated in collaboration with the Sanger Center a deletion at the srd-1 locus: srd-1(eh1). The deletion covers -583 to +1017 eliminating the first exon and parts of the second exon thus representing a likely null mutant. srd-1(eh1) mutants are healthy and show no morphological abnormalities. srd-1(eh1) mutants are fertile, form dauers and stay on the bacterial lawn, indicating they have no gross defect in chemotaxis. srd-1(eh1) males mate normally. srd-2, a close homolog of srd-1 with 48% identity, is expressed solely in males, in the R9 neurons like srd-1. It is therefore possible that srd-1 and srd-2 are functionally redundant, which would account for the lack of phenotypes of srd-1(eh1). We have made a putative dominant negative mutation in srd-1: srd-1Y231C. Overexpression of srd-1Y231C in males inhibits mating, indicating that srd-1 might play a role in mating. We plan to perform RNAi with srd-2 in a srd-1 mutant and test for behavioral alteration. 1.Troemel, E.R., Chou, J.H., Dwyer, N.D., Colbert, H.A. & Bargmann, C.I. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, 207-218 (1995). 2.Hodgkin, J. A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev 1, -745 (1987).