Friedman LC et al. (1996) Midwest Worm Meeting "mog-3 ETC."

Friedman LC, Kimble JE

[Midwest Worm Meeting, 1996]

The fem-3 gene is regulated post-transcriptionally to effect the switch from spermatogenesis to oogenesis (Barton et al., 1987; Ahringer and Kimble, 1991). The mog genes are excellent candidates for encoding trans-acting regulators of fem-3 to achieve this switch (Graham and Kimble, 1993; Graham et al., 1993; Gallegos and Kimble, unpublished). We will describe our initial efforts to clone mog-3 and to characterize it genetically.

Zhou, Y et al. (2017) International Worm Meeting "Genetic and chemical modulation of C. elegans intestinal antiviral innate immunity."

Zhou, Y, Tao, Y, Bedian, L, Zhong, W

[International Worm Meeting, 2017]

The RNA virus Orsay is the only known natural viral pathogen infecting the intestine cells of the nematode C. elegans. The C. elegans intestine cells resemble human intestinal epithelial cells not only in morphological features such as microvilli, but also in molecular mechanisms such as antibacterial innate immune response. As Orsay was discovered only six years ago, the C. elegans antiviral innate immune system remains largely unknown. Only two pathways, RNAi and ubiquitin-mediated protein degradation, have been discovered to be involved in the C. elegans antiviral innate immunity. To explore the genetic landscape of the C. elegans antiviral innate immunity, we conducted a genome-wide RNAi screen for genes whose inactivation sensitizes worms to Orsay infection. 110 genes were identified to be required for C. elegans antiviral innate immunity. 72 of these genes have human orthologs. Tissue-specific RNAi experiments showed that the majority of these genes function in the intestine cells to modulate antiviral innate immunity. In addition to RNAi and ubiquitin-mediated protein degradation, these genes encompass pathways in autophagy, mitochondrial unfolded protein responses, collagens, cytoskeletal organization, RNA processing, and transcription. To identify the gene products that are druggable, we screened 2000 chemicals for drugs that can alleviate Orsay infection symptoms, and discovered four innate immunity enhancing drugs, resorcinol monoacetate (RM), berberine (BBR), bismuth subsalicylate, 3,3'- diindolymethane (DIM). BBR and bismuth subsalicylate are known antidiarrhea drugs, with possible antiviral functions against human gastrointestinal viruses. Chemical-genetic experiments revealed that these drugs have different mechanism of actions: RM strengthens the collagen barrier for viral entry; BBR and DIM enhances the ubiquitin-mediated protein degradation pathway. Together, these data revealed a multifaceted antiviral innate immune system in the C. elegans intestine. Aspects of genetic and chemical modulation of this system may be conserved in human innate immunity against gastrointestinal viruses.

A Fire et al. (1999) International C. elegans Meeting "Structural requirements for the trigger in RNA-mediated interference"

J Fleenor, S Parrish, A Fire

[International C. elegans Meeting, 1999]

This poster will describe in vivo interference activities for a series of modified double-stranded RNAs that are based on segments of the genes unc-22 and gfp . Double stranded RNAs have been shown to act as potent inducers of gene-specific molecular silencing in C. elegans [a]. This process apparently reflects a well conserved control mechanism: recent reports have confirmed the effectiveness of dsRNA-triggered interference mechanisms in a variety of additional species including plant, insect, and protozoan systems [b-e]. By characterizing structural requirements (on the triggering side) for these two well defined segments in C. elegans , we hope to illuminate general features of the interference mechanism. Our experiments involve a variety of manipulations to produce dsRNAs with sequence or chemical alterations, followed by injection into C. elegans and assays for genetic interference. The manipulations are designed to address the following questions: 1. How precise and extensive are requirements for homology with the target gene? 2. What features distinguish the incoming RNA as "foreign"? 3. What chemical groups on the incoming RNA participate in interference? 4. Do the incoming sense and antisense strands have distinct roles in triggering interference? a. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 b. Waterhouse, Graham, Wang, PNAS 95, 13959 c. Ngô, Tschudi, Gull, Ullu, PNAS 95, 14687 d. Kennerdell and Carthew, Cell 95, 1017 e. Misquitta and Paterson, PNAS 96, 1451

Jason M McEwen et al. (2005) International Worm Meeting "UNC-18/nSec1 is required for proper trafficking of UNC-64/Syntaxin-1 in C. elegans neurons"

Josh Kaplan, Jason M McEwen

[International Worm Meeting, 2005]

UNC-18/nSec1 appears to have many roles in regulating synaptic vesicle release. Studies in Drosophila and cell culture have shown that UNC-18 may act as an inhibitor of synaptic transmission 2. In C. elegans, unc-18 mutant animals show a decrease in neurotransmitter secretion at the neuromuscular junction and in both worms and mice there is a decrease in the number of docked vesicles 1,4,5. One contributing factor in how UNC-18 positively regulates synaptic release may be the availability of UNC-64/Syntaxin-1 for forming SNARE complexes. If UNC-18/nSec1 is functioning to maintain levels of UNC-64/Syntaxin at the plasma membrane, this may explain some of the synaptic transmission defects seen in unc-18 mutant worms. Studies using non-neuronal vertebrate cell types have shown that UNC-18/nSec1 is required for UNC-64/Syntaxin-1 to be properly trafficked to the plasma membrane 3.To address the role of UNC-18 in regulating anterograde transport in neurons, we looked to see if UNC-64 is properly localized in unc-18 mutant worms. Using both antibodies against UNC-64 and an UNC-64a::GFP fusion protein we showed that a larger fraction of UNC-64 is retained in the cell bodies of neurons in unc-18 animals when compared to wild type. Trafficking of other synaptic proteins such as SNB-1/Synaptobrevin, RIC-4/SNAP-25, UNC-10/RIM-1 and SYD-2 is unaffected in unc-18 mutants, suggesting that this is not a general defect in intracellular transport. Current experiments are testing whether UNC-18 regulates ER or Golgi exit of UNC-64.1. Gengyo-Ando K et al. Neuron 19932. Schulze KL et al. Neuron 19943. Rowe J, et al. J Cell Sci 20014. Voets T, et al. Neuron 20015. Weimer RM, et al. Nature Neuroscience 2003

Michelle Po et al. (2007) International Worm Meeting "An unc-7 suppressor screen to identify novel active zone regulators."

Taizo Kawano, Michelle Po, Mei ZHEN, Magali BOUHOURS

[International Worm Meeting, 2007]

Synapses are asymmetric structures that facilitate the transmission of signals between neurons and their targets. The mature synapse consists of a presynaptic terminal in direct apposition to a postsynaptic terminal. Neurotransmission occurs when neurotransmitter-filled presynaptic vesicles dock and fuse with the plasma membrane. The neurotransmitter contents are released into the synaptic cleft and subsequently bind to receptors at the postsynapse. The active zone is an electron-dense region of the synapse that is required for the docking and fusion events of synaptic transmission. Our lab is interested in identifying genes that regulate the formation of active zones. In our study we are using the GABAergic nervous system of C. elegans as our model system. The active zones in this nervous system are visualized using a unique active zone specific marker SYD-2::GFP that was developed in our laboratory (Yeh et al., 2006). A forward genetic screen isolated an unc-7 loss-of-function mutant that shows a significant decrease in number of SYD-2::GFP puncta. We have determined that the innexin UNC-7 localizes to perisynaptic regions in addition to gap junctions. Subsequently, we showed that loss-of-function unc-9 mutants, which encodes an innexin protein that is 56% identical to UNC-7, shows the same synaptic defects as unc-7 mutants (Yeh and Ng, unpublished), suggesting a novel regulatory role for innexins at chemical synapses. We further investigated the role of innexins at the synapse by performing an unc-7 suppressor screen. We identified 10 dominant alleles of unc-1, a gene that encodes a stomatin homologue, that suppress the kinker behavior as well as the synaptic phenotype of unc-7 loss-of-function mutants. We found that UNC-7 and UNC-1 co-localize at peri-synaptic regions, indicating that the two proteins may functionally interact. We are investigating the physical properties of the potential UNC-7, UNC-9, and UNC-1 channel complexes. Current progress will be presented at the meeting. Yeh E, Kawano T, Weimer RM, Bessereau JL, Zhen M. 2005. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J Neurosci. 25(15):3833-41.

Edward Yeh et al. (2003) International Worm Meeting "A screen to identify factors involved in active zone formation."

Mei ZHEN, Edward YEH

[International Worm Meeting, 2003]

The active zone is a specialized presynaptic structure where vesicles containing neurotransmitters fuse with the plasma membrane, releasing the neurotransmitters to the post-synaptic membrane. At the EM level, electron dense active zones are surrounded by clusters of vesicles. A few components of the active zone have been identified such as UNC-10 (Koushika et al., 2001), UNC-13 (Richmond et al., 1999; Aravamudan et al., 1999) and SYD-2 (Zhen and Jin, 1999) but even fewer have been implicated in active zone establishment, differentiation or maintainance.syd-2 encodes a homolog of liprin- and has been shown to be a component of the active zone. Mutations in syd-2 give rise to larger active zones. At a previous meeting, we reported a SYD-2::GFP marker that could be used to visualize active zones in live animals. The SYD-2::GFP protein marker was placed under the promoter of unc-25 which expresses in GABAergic neurons. We have performed a SYD-2::GFP screen to identify a dditional factors involved in the formation of the active zone. An EMS mutagenesis screen was performed looking for mutations affecting the formation of the active zone. Approximately 2500 haploid genomes were screened and we are currently characterizing 5 isolated mutant alleles. hp77 and hp121 represent two loci on chromosome X, hp102 and hp198 represent two loci on chromosome IV and hp129 is on chromosome V. hp121 and hp102 have behavioural unc phenotypes, hp198 are slightly dumpy while hp77 and hp129 do not appear to have any behaviour or physical phenotypes. Initial characterization and phenotypic analysis will be presented on these mutants. It is our hope that the molecular characterization of these mutants will provide information on factors involved in the establishment, differentiation, and maintenance of active zones. Ultimately, this information will provide insight into the genetic pathways used to establish neural synapses in general.References: Aravamudan B, Fergestad T, Davis WS, Rodesch CK, and Broadie K. (1999) Nature Neuroscience. 2:965; Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM and Nonet M. (2001) Nature Neuroscience. 4:997-1005; Richmond JE, Davis WS and Jorgensen EM. (1999) Nature Neuroscience. 2:959-64; Zhen M and Jin Y. (1999) Nature. 401:371-5

Rebecca M Fox et al. (2005) International Worm Meeting "MAPCeL: Microarray Profiling C. elegans Cells"

Thomas Brodigan, Rebecca M Fox, Michael Krause, Kellen Olszewski, Susan J Barlow, Joan McDermott, W Clay Spencer, David M Miller, Stephen E Von Stetina

[International Worm Meeting, 2005]

Individual C. elegans cells can be easily visualized in vivo with GFP reporters but, owing to their small size, they are generally inaccessible for molecular analysis. We have now developed new methods for the isolation and gene expression profiling of GFP marked C. elegans cells (MAPCeL Microarray Profiling C. elegans Cells). In this strategy, GFP cells are isolated by FACS from in vitro cultures and RNA extracted for application to the C. elegans Affymetrix array. To demonstrate the utility of our approach, we have profiled cells in the embryonic motor circuit; we have identified genes that are differentially expressed in body muscle cells and in each of the three classes of embryonic ventral cord motor neurons (DA, DB, DD). Microarray data in each case were validated by strong correlation with genes known to be expressed in these cells as well as with GFP reporters that we constructed (~80%). For example, our MAPCeL data from inhibitory DD motor neurons confirms enrichment of transcripts required for the GABA phenotype (i.e. unc-25, unc-30, unc-47, snf-11). Conversely, the excitatory DA and DB motor neurons express cholinergic genes (unc-17, cha-1) as well as specific transcription factors that regulate cholinergic signaling (unc-3, unc-4). These data can now be used to identify novel genes with key roles in the locomotory circuit. For example, we have shown that DA motor neurons express a surprisingly diverse array of G-protein coupled receptors with the potential to modulate excitatory activity in response to neuropeptides as well as classical neurotransmitters. MAPCeL data from body muscle cells lead to the discovery that ACR-16 is a component of the levamisole-insensitive nicotinic ACh receptor (See Touroutine et al., this meeting). Finally, by identifying cohorts of genes with common expression patterns we can now search for cis-regulatory elements necessary for cell-specific expression. For example, we have identified enhancer element candidate motifs in the body wall muscle and DA motor neuron datasets (see Olszewski et al, this meeting). We conclude that MAPCeL can be used to generate reliable profiles of cell specific gene expression in the motor circuit and that these data will be valuable resources for correlating gene expression with function.1.Fox, RM, Von Stetina, SE, Barlow, SJ, Shaffer, C, Olszewski, KL, Moore, JH, Dupuy, D, Vidal, M, Miller, DM III. A gene expression fingerprint of C. elegans Embryonic motor neurons. BMC Genomics 2005, 6:42.

Klosterman S M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Regulation of tomosyn (TOM-1) trafficking in C. elegans"

Klosterman S M, Richmond J E

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Regulated exocytosis of secretory vesicles is critically dependent on the assembly of SNARE complexes between the plasma membrane SNAREs, synatxin and SNAP-25 and vesicle-associated synaptobrevin, which render vesicles fusion competent. Tomosyn, a syntaxin-binding protein, forms a complex with syntaxin and SNAP-25 in competition with synaptobrevin predicted to limit fusogenic SNARE complex formation (Fujita et al, 1998). Consistent with these data, the highly conserved C. elegans tomosyn homolog, TOM-1, has been shown to negatively regulate both synaptic and dense-core vesicle release (Gracheva et al., 2006; Gracheva et al.,2007). TOM-1 appears to be associated with both synaptic vesicles (Takamori et al., 2006) and dense core vesicles (Gracheva et al., 2007) via an unknown mechanism. Consequently, the synaptic localization of TOM-1 is dependent on the anterograde vesicle kinesin motor (UNC-104) (McEwen et al., 2006). Given the importance of TOM-1 in the regulation of synaptic strength, we are interested in identifying the mechanism responsible for the synaptic localization of this protein. Neither syntaxin nor SNAP-25, the two established tomosyn-interacting proteins appear to be involved in TOM-1 transport to synapses (McEwen et al., 2006). In order to screen for TOM-1 trafficking defective mutants, we have generated an integrated transgene expressing TOM-1::GFP. We have verified that this protein is expressed at synapses and localizes in an UNC-104-dependent manner. We are currently screening known vesicle-associated candidate proteins by RNAi or by crossing the TOM-1::GFP transgene into reference alleles to examine their potential role in the regulation of tomosyn trafficking. Fujita Y, Shirataki H, Sakisaka T, Asakura T, Ohya T, Kotani H, YokoyamaS, Nishioka H, Matsuura Y, Mizoguchi A, Scheller RH, Takai Y (1998) Tomosyn: asyntaxin-1-binding protein that forms a novel complex in the neurotransmitterrelease process. Neuron 20:905-915. Gracheva EO, Burdina AO, Touroutine D, Berthelot-Grosjean M, Parekh H,Richmond JE (2007) Tomosyn negatively regulates CAPS-dependent peptide releaseat Caenorhabditis elegans synapses. J Neurosci 27:10176-10184. Gracheva EO, Burdina AO, Holgado AM, Berthelot-Grosjean M, Ackley BD,Hadwiger G, Nonet ML, Weimer RM, Richmond JE (2006) Tomosyn inhibits synapticvesicle priming in Caenorhabditis elegans. PLoS Biol 4:e261.McEwen JM, Madison JM, Dybbs M, Kaplan JM (2006) Antagonistic regulationof synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51:303-315. Takamori S et al. (2006) Molecular anatomy of a trafficking organelle.Cell 127:831-846.

Norman KR et al. (1998) West Coast Worm Meeting "let-268 ENCODES A COLLAGEN PROCESSING ENZYME AND IS NECESSARY FOR BASEMENT MEMBRANE STABILITY."

Norman KR, Moerman DG

[West Coast Worm Meeting, 1998]

In C. elegans the basement membrane overlying the bodywall muscle quadrants is a thin specialized extracellular matrix that separates the hypodermis from the muscle. The basement membrane has many functions, one of which is its role in myofilament assembly and stability. In a screen for embryonic lethals with defects in myofilament structure, we have identified an allele of let-268, ra414. let-268(ra414) mutants arrest around 470 minutes after fertilization and have disorganized muscle and basement membrane. Unlike earlier blocking severe pats, the muscle and basement membrane appear normal until contraction commences. The terminal phenotype of let-268(ra414) mutants is identical to that of mutants for the type IV collagen genes, emb-9 and let-2. Our immunofluorescence data and the late paralysis suggest the protein product of let-268 must act in or on a component(s) of the basement membrane overlying the muscle quadrants. In order to clone let-268 we used a combined PCR/deficiency mapping strategy. Three overlapping deficiencies that failed to complement let-268 were used to identify a single cosmid, F52H3. One of the predicted genes on this cosmid, a procollagen lysyl hydroxylase, seemed a likely candidate. We have sequenced this open reading frame in the three existing alleles and found the alterations, demonstrating the identity of LET-268 as a collagen modifying enzyme. Mutations in the human lysyl hydroxylase results in a connective tissue disorder known as Ehlers-Danlos syndrome type VI. Patients with this disease display signs of collagen pathology such as abnormal curvature of the spine, weak joints, loss of tonicity of the muscle and ocular fragility. To determine whether LET-268 acts on type IV collagen, let-268 mutants were stained with antibodies to the type IV collagen alpha 1 and alpha 2 chains (kindly provided by J. Kramer). These antibodies stain most of the basement membranes in the wild type embryo including the basement membrane overlying the body wall muscle, the pharynx and the intestine (Graham et al., 1997 JCB : 1171-1183; Gupta et al., 1997 JCB 137: 1185-1196). Antibody staining of let-268(ra414) with the type IV collagen sera indicates that type IV collagen is not secreted from the muscle cells or the head mesodermal cells. This staining appears perinuclear suggesting that the collagen is held up in an organelle near the nucleus, most likely the ER, the organelle where lysyl hydroxylase functions. This implies that let-268 is essential for excretion of type IV collagen as well as its function.

Huang C-c et al. (1998) East Coast Worm Meeting "ANALYSIS OF THE LAMININ SUBUNITS REVEALS TISSUE SPECIFIC ASSEMBLY AND MOLECULAR SPECIALIZATION OF BASEMENT MEMBRANES"

Kao G, Huang C-c, Vogel BE, Zhu X, Hutter H, Hedgecock EM, Wadsworth WG, Karantza V, Joh K

[East Coast Worm Meeting, 1998]

We are studying the developmental functions of laminin using the nematode C. elegans. Laminin is a heterotrimeric glycoprotein composed of a, b, and g subunits. The genome sequencing project reveals two a, one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin aA and laminin aB, are most similar to the vertebrate a1/a2 and a5 chains respectively, while the b and g resemble the b1/b2 and g1 chains. Thus there are two predicted laminin trimers, aAbg and aBbg. Mutations in the aB and b genes (designated epi-1 and lam-1 respectively) have been isolated and cause defects that are consistent with the developmental roles for basement membranes. These include miswiring of the nervous system, disorganization of internal tissues, malformation of male tail, and sterility. We are examining the expression patterns of laminin subunits by in situ hybridization, immuno-histochemistry, and GFP fusions. Endodermal and mesodermal cells express the genes throughout gastrulation. During later development, they are also expressed in many cells. Interestingly, while gene expression of b and g is detected in all these cells, the expression of aA and aB do not overlap. For example, aB is expressed in body wall muscles but aA is not, aA is expressed in pharyngeal and epidermal cells but aB is not. Immunostaining using affinity purified polyclonal antibody against the G2-3 domain of the laminin aB indicates cytoplasmic distribution during early gastrulation and, later extracellular accumulation between germ layers that marks early basement membrane formation. Together with the mutant phenotypes this suggests one of the major functions of laminin is to separate the different tissue types during gastrulation. The antibody recognizes all basement membranes (BM) at later stages, including the BM surrounding the epidermis, gonad, intestine, pharynx and all muscles. In the body wall muscle BM, laminin aB accumulates in an unusual pattern at and between dense body attachments. Because the myofilaments of skeletal muscle are disorganized in epi-1 mutants, this expression pattern indicates that laminin might play a role in myofilament stabilization or organization. During gonadogenesis, laminin aB is detected at high level around the migrating distal tip cells. While laminin aB is present in all BM, collagen IV is not, indicating that BMs have different molecular features (1). Although integrins are the only identified receptors for laminin, the published expression patterns of integrin subunits, pat-3 and ina-1(2,3), only have some overlap with the laminin distribution, implying that other laminin receptor(s) may also be used to help assemble BMs. We have shown that often the expression of a subunit gene is not detected in the cells where the protein will be assembled into a BM. Furthermore, the a mutant knockout phenotypes show the disruption of different tissues. Taken together, our results indicate that tissues selectively assemble BM at their surfaces and the assembled BMs have different protein compositions. 1.Graham, P.L. et al. JCB 137: 1171-1183, 1997 2.Gettner, S.N. et al. JCB 129:1127-1141, 1995 3.Baum, P.D. and Garriga, G. Neuron 19:51-62, 1997