Merrill, Sean et al. (2015) International Worm Meeting "Locating synaptic calcium channels."

Cherry, Alex, Merrill, Sean, Jorgensen, Erik, Watanabe, Shigeki

[International Worm Meeting, 2015]

Neurotransmission occurs when calcium triggers exocytosis of synaptic vesicles primed at release sites. The number, position and activity of nearby calcium channels determine the perdurance of free calcium at a release site. To understand the synapse we must identify the location of calcium channels in relation to synaptic vesicles, fusion proteins, and subcellular structures such as the dense projection. In the simplest model, calcium enters through a single source at the center of the synapse. However, calcium entry through multiple sources within a synapse has been studied only indirectly. In C. elegans unc-68 (RyR), egl-19 (L-type), and unc-2 (N-type) channels are each encoded by a single gene and contribute the calcium for synaptic vesicle exocytosis at neuromuscular junctions. We are determining the colocalization of calcium channels with other synaptic proteins at nanometer resolution. CRISPr and miniMos create single-copy transgenes that attach to each channel an enzymatic tag that covalently binds organic fluorophores suitable for imaging by biplane 3D super-resolution fluorescence microscopy. Transgenic animals are crossed with mutations that test the contributions of the vesicle priming proteins unc-13, unc-10 (RIM), and rimb-1 (RIM-binding protein) in localizing each synaptic calcium channel and its adjoining vesicles.

Sarfarazhussain Farooqui et al. (2006) European Worm Meeting "A GFP Reporter Based Screen for the Notch Target Genes during Vulval Development"

Sarfarazhussain Farooqui, ALEX HAJNAL, Ivo Rimann

[European Worm Meeting, 2006]

Sarfarazhussain Farooqui, Ivo Rimann and Alex Hajnal. During hermaphrodite vulval development, lateral signaling from the primary vulval precursor cell (VPC) P6.p activates the LIN-12 Notch pathway in the neighbouring secondary VPCs P5.p and P7.p. The LIN-12 Notch signal inhibits primary fate specification and promotes the secondary vulval fate by activating transcription factors of the CSL family, which turn on the expression of specific target genes.. LIN-12 Notch targets usually contain clusters of CSL binding sites (RTGGGAA) in their promoter/enhancer regions. To identify new target genes of the LIN-12 Notch pathway in a systematic way, we searched the C. elegans genome sequence for genes containing clusters of three or more CSL sites within 2 kbp upstream of the start codon. We further refined our list of candidate Notch targets by taking into consideration only those genes in which the putative CSL sites are conserved in the C. briggsae orthologs. Promoter-GFP fusion constructs for the 23 strongest candidate genes were made by fusion PCR and injected into wild-type worms to create transgenic lines. Details on the expression patterns of the reporter constructs generated so far will be presented at the meeting.

Tauffenberger, Arnaud et al. (2009) International Worm Meeting "Age-dependent modification of expanded CAG toxicity in simple C. elegans models."

Parker, Alex, Tauffenberger, Arnaud, Bel-Hadj, Samar

[International Worm Meeting, 2009]

Arnaud Tauffenberger1,2,3, Samar Bel-Hadj1, Alex Parker1,2, 3 1CRCHUM, 2Centre of Excellence in Neuromics, 3Department de pathologie et biologie cellulaire, Universite da Montreal, Montreal, Quebec, Canada The expansion of CAG trinucleotides coding for glutamine is the cause of at least nine late-onset neurodegenerative diseases. The expanded CAG (expCAG) diseases may have some pathogenic mechanisms in common as the disease threshold for all of these disorders is around 40 CAG repeats and they display misfolded intracellular protein aggregation phenotypes. We have turned to the model organism C. elegans to explore two areas of interest: 1. Age-dependent toxicity: Although the expCAG diseases show anticipation, the size of CAG repeat only partially correlates with the variation in age-of-onset. Evidence suggests that even for these single trait neurodegenerative diseases, disease onset and progression may have additional environmental and genetic components. Using C. elegans transgenic worms expressing expCAG in neurons we are exploring the links between aging and late-onset neurodegeneration. We hypothesize that genes regulating longevity, stress response, their associated signaling pathways and downstream effectors may encode new therapeutic targets for age-dependent proteotoxicity in humans. 2. Mechanisms of expCAG toxicity: Long homopolymeric CAG nucleotide stretches are unstable and may be prone to translation errors. Mistranslation of expCAG to alternate reading frames may produce hybrid proteins with additional toxicity. We have created transgenic strains to investigate this phenomenon. A summary of our data and recent findings will be presented.

Shi, Zhen et al. (2011) International Worm Meeting "The mevalonate pathway has a role in microRNA repression of target genes."

Ruvkun, Gary, Shi, Zhen

[International Worm Meeting, 2011]

The mevalonate pathway is present in all higher eukaryotes and many bacteria and mediates the production of isoprenoids. The isoprenoids feed into a wide range of biosynthetic pathway: sterols, dolichol, ubiquinone, heme A, and the lipid moiety for protein prenylation. C. elegans possesses a functional mevalonate pathway but lacks enzymes for the cholesterol synthesis, suggesting that mevalonate generates precursors for the other biosynthetic pathways. Inactivation of genes function in the synthetic steps in the mevalonate pathway as well as drugs that inhibit this pathway (the statins) strongly enhance the retarded phenotypes of the hypomorphic mutation in the let-7 miRNA. These phenotypes are suppressed by the inactivation of let-7 targets, suggesting that mevalonate is needed for normal let-7 repression of target genes. We found that the non-cholesterol biosynthetic outputs of the mevalonate pathway are required for the repression of these let-7 targets. In addition, inactivation of the mevalonate pathway also results in the de-silencing of lin-14, the target of lin-4 miRNA, compared to stage-matched control worms. To address which downstream branch(s) of the mevalonate pathway regulate(s) the developmental timing, we are performing a candidate-based RNAi screen for gene inactivations that cause retarded developmental timing. The cherry-picked RNAi library we are screening includes genes that function in protein prenylation, dolichol, ubiquinone and heme A biosynthesis.

Sylvia E J Fischer et al. (2005) International Worm Meeting "Identification of factors required for down-regulation of lin-14 through its 3UTR using a reporter transgene."

Gary Ruvkun, Sylvia E J Fischer, Qi Pan

[International Worm Meeting, 2005]

To undergo correct timing of subsequent developmental events, LIN-14 expression needs to be down regulated during L1. The microRNA lin-4 is pivotal in this process and is thought to block translation of lin-14 mRNA by binding to complementary sites in the lin-14 3UTR. Although several factors that are required for microRNA biogenesis are known, such as the RNaseIII enzyme DCR-1, knowledge of downstream factors that may contribute to the translational block by microRNAs is very limited. In order to identify factors that contribute to lin-4 mediated regulation of gene expression, we are using a lin-14 reporter transgene in an RNAi screen. The transgene contains the rol-6(su1006) coding region, fused to the 3UTR of lin-14. Whereas the strain does not show a Rol phenotype upon L4440 control (RNAi), upon dcr-1 (RNAi) it shows a small percentage of rolling animals, presumably because of a reduction in mature lin-4 microRNA levels. We carried out a screen of a cherry picked RNAi library that contains 612 RNAi clones for genes encoding RNA binding proteins, helicases and nucleases, asking for expression of the transgene. 20 RNAi clones were identified that in feeding RNAi experiments, cause expression of the transgene. We are further analyzing the effect of RNAi of these genes on additional reporter transgenes, as well as their effect on lin-4 microRNA levels and transgene mRNA levels.

Villalobos, Tatiana et al. (2021) International Worm Meeting "Tubular lysosome induction links starvation to animal longevity"

Das, Suman, Ramos, Cara, Butsch, Tyler, Villalobos, Tatiana, Bohnert, Adam, Alam, Sanaa, Johnson, Alyssa, Ghosh, Bhaswati, Eymard, Eric

[International Worm Meeting, 2021]

Lysosomes are digestive organelles that govern cellular metabolism and homeostasis. Despite their importance to animal health and disease, the current model of lysosome structure and function is quite simplistic; autophagic lysosomes are thought to exist mainly as discrete vesicles with uniform activity. Here, we report that vesicular lysosomes in the C. elegans gut transform into an extensive, dynamic, tubular network in response to animal starvation. These tubular lysosomes (TLs) are acidic, serve as preferential sites of starvation-triggered autophagy, and are stimulated via inhibition of the mTOR pathway. Consistent with our past studies in flies, we identify spin-1, a C. elegans Spinster ortholog, as a critical TL gene. Using an endogenously-tagged SPIN-1::Cherry transgenic line, we find that genetic models of caloric restriction show atypically high spin-1 expression levels, which correlates with the constitutive presence of TLs throughout the gut. TLs also appear to contribute to the transgenerational effects of starvation; well-fed descendants of starved worms can show gut TLs for several generations. Notably, the presence of gut TLs in well-fed progeny is predictive of enhanced adult lifespan. Further, we find that experimental expression of the Drosophila TL stimulator SVIP in C. elegans is sufficient to induce gut TLs in well-fed worms and improve C. elegans health during aging. These findings highlight a new class of degradative lysosomes that act at the center of the cellular response to starvation, potentially coordinating its positive effects on animal health and longevity.

Milica Margeta et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "From Building a Neuron to Building a Circuit: Polarity and Synaptic Connectivity in the RIA Interneuron Pair"

Kang Shen, Milica Margeta

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

RIAL and RIAR are major integrating interneurons located in the head of the worm. They have been implicated in the behavior thermotaxis, since laser ablation of RIA neurons leads to an athermotactic phenotype (Mori et al., 1995). RIAL/R receive inputs from a number of other neurons, including thermophilic AIY interneurons and cryophilic AIZ interneurons, and provide output to head motor neurons (RMDs and SMDs). Notably, these connections are made in the cluttered nerve ring neighborhood, implying that there exists a high degree of specificity ensuring that these connections are properly formed. Anatomically, RIA neurons are highly polarized: the proximal region of their processes is exclusively postsynaptic, while the distal region is largely presynaptic as based on the ultrastructural EM pattern (White et al., 1986). We used a RIA-specific promoter, Pglr-3, to drive the expression of fluorescently tagged pre- and postsynaptic proteins and visualize these discrete domains in vivo. RIA neurons have been labeled with a synaptic vesicle marker Pglr-3::Cherry::RAB-3 and a glutamate receptor marker Pglr-3::GLR-1::GFP; these tagged proteins selectively localize to pre- and postsynaptic regions of RIA, respectively. We are interested in using this system to address two main questions: 1) how is the intrinsic polarity of RIA neurons established? and 2) how is correct connectivity to appropriate synaptic partners (in particular, AIY) generated? We are currently performing a number of candidate crosses and genetic screens in order to address these questions.

Ivo Rimann et al. (2006) European Worm Meeting "The Transcription Factor EGL-43 is Necessary for Anchor Cell Invasion during Vulval Development"

Ivo Rimann, ALEX HAJNAL

[European Worm Meeting, 2006]

Ivo Rimann and Alex Hajnal. For proper egg-laying by the hermaphrodite, the uterus has to become firmly connected to the vulva during late larval development. The anchor cell (AC), a specialized cell in the somatic gonad, coordinates vulval and uterus development and initiates the formation of the connection between these two tissues.. During the L3 larval stage, the AC induces six neighboring ventral uterine (VU) cells to adopt the Pi cell fate. Around the same time of development, the basal laminas separating the uterus from the vulva begin to dissolve and the AC invades the vulval tissue by extending a process between the primary cells (Sherwood et al., 2003). In this way, the AC connects the vulval and uterine tissues.. In an RNAi-based screen for genes regulating vulval morphogenesis, we have found that knock-down of egl-43 causes a penetrant protruding vulva (Pvl) phenotype because the connection between the uterus and vulva is not formed correctly. egl-43 encodes a zinc finger transcription factor homologous to the human EVI1 proto-oncogene. Closer examination showed that in egl-43 RNAi animals the basal lamina under the AC is not removed, which prevents the AC from invading the vulval tissue. In addition, egl-43 RNAi animals show defects in the specification of the Pi cell fate. Similar defects in AC invasion have been reported for fos-1 mutants (Sherwood et al., 2005), and like fos-1, a transcriptional egl-43p::gfp reporter is expressed at high levels in the AC and at lower levels in the adjacent VU cells. Moreover, egl-43p::gfp expression in the AC is 5-fold reduced in a fos-1(0) background, indicating that FOS-1 promotes AC invasion at least in part by up-regulating egl-43 expression in the AC.

Xiong, R. et al. (2019) International Worm Meeting "Cellular and molecular mechanisms of mok-1 function in left-right neuronal asymmetry."

Xiong, R., Chuang, C.-F., Wang, X.

[International Worm Meeting, 2019]

The developing nervous system generates a large diversity of cell types with distinct patterns of gene expression and functions. One way to establish neuronal diversity is to specify neuronal subtypes across the left-right axis. The C. elegansleft and right AWC olfactory neurons communicate to specify asymmetric subtypes, AWCOFF and AWCON. The default AWCOFF is specified by a Ca2+-regulated kinase cascade that is activated by influx of Ca2+ through the voltage-gated Ca2+ channel UNC-2/UNC-36. Intercellular communication between the two AWC neurons and other neurons through the NSY-5/innexin gap junction network antagonizes unc-2/unc-36 Ca2+ signaling in the induced AWCON cell. Our recent data suggest that voltage- and calcium-activated SLO BK potassium channels slo-1and slo-2acts redundantly downstream of nsy-5 to inhibit unc-2/unc-36 Ca2+ signaling in the specification of AWCON. To identify the genes required for slo-1function in inhibiting unc-2/unc-36 Ca2+ signaling for promoting AWCON, we performed a non-biased forward genetic screen to isolate mok (modifier of K+ channel) mutants that suppress the slo-1(gf) 2AWCON phenotype. mok-1(vy11) is one of the mutants identified from this screen. The 2AWCOFF phenotype of mok-1(vy11) mutants is suppressed by loss-of-function mutations in the Ca2+ channel gene unc-36, suggesting that mok-1 acts upstream of the unc-2/unc-36 Ca2+ signaling pathway. Together, our results suggest a model in which nsy-5 inhibits unc-2/unc-36 Ca2+ signaling through slo-1 and mok-1. We identified the mutation responsible for the vy11 phenotype using whole genome sequencing with the help of Alex Boyanov in the Oliver Hobert lab. Further genetic analysis and behavior analysis of the vy11 mutant, as well as molecular characterization of mok-1 will be presented in the meeting.

Claudia Walser et al. (2006) European Worm Meeting "Distinct Roles of the Pumilio and FBF Translational Repressors during C. elegans Vulval Development"

Gopal Battu, ALEX HAJNAL, Claudia Walser, Erika Frohli Hoier

[European Worm Meeting, 2006]

Claudia Walser, Gopal Battu, Erika Frhli Hoier and Alex Hajnal. Members of the conserved PUF (Pumilio and FBF repeat) protein family regulate various aspects of germ line development by selectively binding to the 3 untranslated region of their target mRNAs and repressing translation. FBF-1 and FBF-2 regulate the sperm/oocyte switch and mitosis versus meiosis decision by repressing fem-3 and gld-1 translation, respectively (Zhang et al., 1997; Crittenden et al., 2002). We found that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulval development.. Loss-of-function mutations in puf-8 cause excess vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway, and they suppress the Vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signaling. A PUF-8::GFP translational reporter is initially expressed in all six vulval precursor cells (the VPCs P3.p through P8.p) but gets restricted to the distal, uninduced (3) VPCs after vulval fate specification. Moreover, the fusion of the distal VPCs to the hypodermal syncytium is retarded in puf-8(lf) mutants. Thus, PUF-8 acts cell-autonomously to limit the temporal competence of the vulval precursor cells to respond to the extrinsic patterning signals. fbf-1 and fbf-2, on the other hand, function as redundant inhibitors of vulval cell fate specification in a distinct pathway that involves the repression of gld-1 translation. fbf-1 fbf-2 double mutants show ectopic 1 cell fate marker expression in adjacent VPCs. We conclude that the FBFs ensure that only one VPC (P6.p) is selected for the 1 cell fate.. Therefore, the PUF family translational repressors regulate various aspects of cell fate specification in the soma, and they may play a conserved role in modulating signal transduction during animal development.