Nathalie Strutz et al. (2001) International C. elegans Meeting "Identification of functional ion channel domains of C. elegans glutamate receptor subunits"

Nathalie Strutz, David Madsen, Michael Hollmann, Andres V Maricq

[International C. elegans Meeting, 2001]

Glutamate receptors are not only abundant in the nervous system of vertebrates but also of invertebrates. In Caenorhabditis elegans , 10 putative ionotropic glutamate receptor subunits have been identified so far (Brockie et al. 2001, J. Neurosci. 21, 1510). Two of these, GLR-1 and GLR-2, have been expressed in Xenopus oocytes and failed to show ligand-activated currents. Therefore, we set out to investigate if the pore-forming domains of these two subunits are intrinsically functional ion conducting domains. We have previously shown that domain transplantation between different subtypes of glutamate receptors can generate functional chimeras that allow the characterization of those domains. To test for pore function, we transplanted the pore-forming region of GLR-1 or GLR-2 into either rat GluR1, as a representative of the AMPA receptor family, or rat GluR6, as a representative of the KA receptor family. The resulting chimeras GluR1-PGLR1, GluR1-PGLR2, GluR6-PGLR1, and GluR6-PGLR2, as well as the respective wildtypes GluR1, GluR6, GLR-1, and GLR-2 were expressed and characterized in Xenopus oocytes. Surprisingly, GluR1-PGLR1, GluR1-PGLR2, and GluR6-PGLR2 produced ligand-activated currents, despite the GLR-1 and GLR-2 wildtype receptor's apparent lack of ion channel function. The maximal current amplitudes of GluR6-PGLR2 were comparable to wildtype GluR6. GluR1-PGLR1 and GluR1-PGLR2 showed reduced current amplitudes in comparison to wildtype GluR1. The ion channel properties of the chimeras classify GLR-1 and GLR-2 into the group of non-NMDA receptor subunits, consistent with conclusions derived from sequence homology data.

Wang, Jennifer T. et al. (2013) International Worm Meeting "Asymmetric segregation of P granules requires granule remodeling by two novel serine-rich proteins."

Seydoux, Geraldine, Wang, Jennifer T.

[International Worm Meeting, 2013]

P granules are RNA-rich granules that are found exclusively in the germline. In adult germ cells, P granules are mostly perinuclear and stable. In contrast, in early embryos, P granules are cytoplasmic and highly dynamic. Activation of P granule dynamics in embryos is required to segregate P granules asymmetrically to the nascent germline [1,2]. We have found that activation of P granule dynamics requires P granule remodeling by two serine-rich proteins. We used a GFP::PGL-1 fusion to analyze P granule dynamics during the oocyte-to-embryo transition. Oocyte P granules are disassembled during ovulation and reappear during meiosis throughout the cytoplasm of the newly fertilized zygote. A similar disassembly/reassembly cycle is repeated in the zygote at each mitotic division, except that reassembly occurs preferentially in the cytoplasm destined for the germline blastomere. We have found that depletion of GEI-12 and its paralog C36C9.1 blocks the disassembly/reassembly cycle. In gei-12+C36C9.1(RNAi), oocyte P granules persist through ovulation and the first mitotic division, and do not segregate asymmetrically. Formation of new P granules is also blocked. GEI-12 and C36C9.1 are 70% identical serine-rich proteins with no recognizable motifs. Sequence analyses indicate that these proteins have low-complexity sequence (LCS) throughout their length. LCS domains have recently been proposed to form dynamic fibers that hold RNA granules together [3,4]. Consistent with functioning as a P granule scaffold, GEI-12 associates with P granules after reassembly in embryos. Deconvolution confocal microscopy suggests that, in the reassembled P granules, GEI-12 localizes to a core that joins together several small PGL-1 granules. We conclude that 1) GEI-12 and C36C9.1 remodel P granules during the oocyte-to-embryo transition and 2) P granule remodeling is essential for the asymmetric segregation of P granules in embryos. 1. Brangwynne CP et al (2009). Science 324, 1729 2. Gallo C et al (2010). Science 330(6011) 1685 3. Kato M et al (2012). Cell 149(4), 753-767 4. Han TW et al (2012). Cell 149(4), 768-779.

Puri, Dharmendra et al. (2017) International Worm Meeting "Regulation of neuronal microtubule cytoskeleton."

Jin, Yishi, Puri, Dharmendra, Chisholm, Andrew, Ghosh-Roy, Anindya, Thyagarajan, Pankajam

[International Worm Meeting, 2017]

In the nervous system, directional flow of information depends on the polarized architecture of neuron. Neuron has a unique structure - many dendrites and one axon. Microtubule polymers are the building blocks of the polarized architecture of neuronal cell. Although MT protofilaments give stability in axon, they themselves are dynamic - undergoing polymerization and depolymerization. The dynamics of the microtubule network needs to be regulated in a context-dependent manner during polarization, maintenance or other remodeling processes. Loss of kinesin-13 family microtubule depolymerizing enzyme klp-7 (kinesin-like protein) leads to excess stabilization of microtubules, leading to a multipolar neuronal phenotype in C. elegans mechanosensory (1) and other neurons. We found that this phenotype in klp-7(lf) can be reversed by a microtubule-destabilizing drug Colchicine or in backgrounds lacking either alpha or beta tubulin. We hypothesized that a genetic screen for the suppressors of klp-7(lf) will help us identify regulators of microtubule cytoskeleton. We conducted a genetic screen saturating the mutagenized genome. By combining meiotic recombination mapping and whole genome sequencing, we have identified candidates such as tubulin genes, post-translation modification factors and other novel factors. We mapped one of the suppressors to a gene, muscleblind-1/mbl-1. We found that mbl-1 is necessary and sufficient for touch neuron axon extension in a cell-autonomous manner. Gentle touch response in posterior and anterior side is compromised in mbl-1 mutant. Mbl-1 function is mostly known in muscles as a splicing regulator. Recent study in worm suggested that MBL-1 is required for synapse stabilization in DA9 motor neuron (2). We hypothesize that MBL-1 is regulating splicing of genes required for microtubule stability. References: 1. Ghosh-Roy A, Goncharov A, Jin Y, & Chisholm AD (2012) Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration. Dev Cell 23(4):716-728. 2. Spilker KA, Wang GJ, Tugizova MS, & Shen K (2012) Caenorhabditis elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse formation. Neural Dev 7:7.regulation

Andersen*, Kristine et al. (2015) International Worm Meeting "An Investigation into the Affect of Neuronal Activity on Proper Neural Connectivity in C. elegans."

Andersen*, Kristine, Varshney, Aruna, Duong, Alex, Miller, Kristine, Park, Joori, Pyle, Jacqueline, Barsi-Rhyne*, Benjamin, VanHoven, Miri, Vargas, Christopher, Holdrich, Emma, Tsujimoto, Bryan, Tran, Alan

[International Worm Meeting, 2015]

Neuronal activity has been implicated in the establishment and maintenance of appropriate synaptic connections in vertebrate and invertebrate systems. However, the molecular mechanisms by which neuronal activity affects connectivity are poorly understood. To understand this process, we have focused our studies on the PHB phasmid sensory neurons. We have begun to elucidate the pathway by which sodium dodecyl sulfate (SDS) is sensed by the phasmid neurons using a high throughput assay adapted from the method developed by Hilliard and colleagues (Current Biology, 2002). Briefly, SDS is sensed by the PHB neurons, which terminate backward movement via their connections with AVA interneurons. Using this assay, we find that odr-3/Galphaolf and tax-2/CNG-channel beta subunit, in addition to previously discovered tax-4/CNG-channel alpha subunit (Hilliard et al., Current Biology, 2002), are required for SDS chemosensation. To determine if defects in sensory signaling affect sensory synapses, we utilized the split-GFP-based trans-synaptic marker NLG-1 GRASP (Neuroligin-1 GFP Reconstitution Across Synaptic Partners) to visualize synapses between PHBs and AVA interneurons in live animals. Interestingly, we find that neuronal activity is required for maintaining these sensory synapses. Time course experiments indicate that odr-3/Galphaolf mutant L1s have normal synapses, but synapses are significantly reduced in later larval stages, although phasmid neuron morphology appears to be unaffected. Cell-specific rescue experiments indicate that odr-3/Galphaolf likely functions in PHB neurons. Subcellular localization of odr-3/Galphaolf shows localization to the PHB cilia, consistent with a role in sensory signaling being required to maintain synaptic integrity. These results indicate that C. elegans may be a powerful model organism for elucidating the molecular mechanisms by which sensory activity affects synaptic connectivity. Our future goal is to further characterize the mechanism by which sensory activity maintains synapses using molecular genetic and physiological approaches. Funded by NIH (1R01NS087544 to MV at SJSU and NL at UCSF, 5T34GM008253 MARC undergraduate fellowship to CV, 2R25GM071381 RISE undergraduate fellowships to CV and JP), HHMI (SCRIBE 52006312 undergraduate fellowship to BB and KM), and NSF (RUMBA REU 1004350 fellowship to KA and EH).

Rybarska, Agata et al. (2015) International Worm Meeting "Multiple mechanisms of germ granule condensation and distribution are essential for soma-germline distinction in the C. elegans embryo."

Eckmann, Christian, Rybarska, Agata

[International Worm Meeting, 2015]

Organization of the cytoplasm into dynamic assemblages, such as RNA/protein (RNP) granules, reflects physiological control of gene expression programs. However, the pathways of RNP granule formation and their function during development remain largely unclear. Moreover, in animals with preformed germ cell specification, such as C. elegans, maternal germplasm components asymmetrically condense into RNP (germ) granules that partition exclusively to the germline precursor (P) lineage, lending to the hypothesis that germ granule condensation is a physiochemical property of germ cells that is linked to the embryonic germ line establishment. However, recent findings challenged this view as failed asymmetric segregation of the canonical markers of P granules, PGL and GLH proteins, have no obvious role in germline fate specification [Gallo et al. 2010]. We set out to clarify this enigma by carefully studying the localization behavior and function of other known P granule-associated components. In particular, we focused on maternal RNA regulators that have-unlike PGL/GLH proteins-also an obvious non-granular phase. This includes GLD-2 and GLD-3, which form together a cytoplasmic poly(A) polymerase (cytoPAP). Using a suit of new antibodies, we found that both cytoPAP components are asymmetrically organized in dividing germline precursors. Their cytosolic phase is present at the "somatic" and "germline" poles; however, at the "germline" pole they condense into granules that partition exclusively to germline precursors. Strikingly, these germplasm granules segregate independently of phosphatase PPTR-1, which regulates asymmetric condensation of PGL/GLH-positive P granules. Instead, cytoPAP components follow a MEX-5-dependent dissolution pathway that acts in a later stage of mitosis. In contrast to PGL/GLH, condensation of cytoPAP-containing germ granules strongly correlates with nuclear accumulation of PIE-1, an essential germline fate determinant. Moreover, we uncovered an important functional interdependency between the non-granular form of both cytoPAP components and cortical polarity establishment, suggesting that RNA modifiers act in germline precursors to break cellular symmetry. Taken together, we identified two types of germ granules that depend on different molecular pathways for their asymmetric condensation and distribution. These differing mechanisms of intracellular phase separation appear to be essential for soma-germ line distinction. Thus, our work provides a paradigm for the intricate relation between position-dependent cytoplasm organization and cell fate establishment.

Vidal-Gadea, Andres G et al. (2011) International Worm Meeting "C. elegans selects distinct crawling and swimming gaits via dopamine and serotonin."

Elbel, Erin, Axelrod, Abram, Erbguth, Karen, Topper, Stephen, Brauner, Martin, Vidal-Gadea, Andres G, Crisp, Ashley, Pierce-Shimomura, Jonathan, Young, Layla, Siegel, Dionicio, Maples, Thomas, Kressin, Leah, Gottschalk, Alexander

[International Worm Meeting, 2011]

The ability to select and switch between alternate locomotory gaits is shared among many animals that are required to transition between different environments. Failure to achieve motor transitions is often devastating to the organism as exemplified by patients with advanced Parkinson's disease. The nematode C. elegans is well adapted for locomotion on land and in water and is likely required to transition between these sub-niches in nature. Despite recent advances, it remains controversial if swimming and crawling are distinct gaits in C. elegans, and if so, how transitions between them are accomplished (1-4). Answering these questions could enable the use of this powerful model system to study the mechanisms underlying locomotory transitions. We used a multifaceted approach spanning in depth behavioral assays, mechanic and optogenetic stimulation, laser ablations, mutant analysis, and in vivo photolysis of caged amines to test whether the worm uses distinct gaits and to determine how they transition between them. We show through a series of behavioral assays that in C. elegans crawling and swimming are distinct gaits. Dopamine released by ADE and PDE neurons is necessary and sufficient to initiate and maintain crawling by a pathway activating D1-like receptors. Conversely, serotonin is necessary and sufficient to initiate and maintain transition from crawling to swimming and to inhibit a set of crawl-specific behaviors. We found these transitions to be modulated by the balance between dopamine and serotonin. These amines have been found to play crucial roles in transition between alternate locomotory forms in diverse species stressing the importance locomotor transitions have for survival. Further study of locomotory switching in C. elegans and its dependence on dopamine may provide insight into comparable motor deficits observed in Parkinson's disease. 1) Boyle H et al. N. Front. Behav. Neurosci. 2011 5:10 doi: 10.3389/fnbeh.2011.00010. 2) Mesce KA, Pierce-Shimomura JT. Front. Behav. Neurosci. 2010 4:49 doi: 10.3389/fnbeh.2010.0004 3) Vidal-Gadea AG et al. 2009 Neuroscience 366:17 Calabrese R: Faculty of 1000 Biology, 11 November, 2009. Conference 2009 October 16-21. Available at: http://f1000biology.com/ article/id/1182956/evaluation 4) Fang-Yen C et al. 2010 PNAS 107, 20323-20328.

Simske JS et al. (2000) Midwest Worm Meeting "jc5: A Role for Inositol 1, 4, 5-Triphosphate Receptor in Ventral Enclosure?"

Simske JS, Hardin JD, Thomas CL

[Midwest Worm Meeting, 2000]

During embryogenesis, epithelial cells migrate and change shape in order to generate three dimensional structure. How these cells move,and the signals that cause their movement is the focus of our research. During the process known as ventral enclosure epithelial cells that originate on the dorsal surface of the embryo migrate to the ventral midline, forming adhesive junctions. jc5 is a cold sensitive mutant that is defective in ventral enclosure and was identified in an EMS mutagenesis screen performed in the Hardin laboratory. Phenotypically, jc5 mutants show defects during varying stages of embryogenesis. Approximately 20% of embryos die because migration of epithelial cells from the dorsal side never occurs. Another 20% die during enclosure, predominantly displaying dorsal retraction or partial enclosure phenotypes. 40% of dead embryos that enclose properly, but develop severe body shape defects as they try to elongate into a worm. In addition, another 20% of embryos may display cytokinesis defects leading to too few hypodermal cells. These phenotypes have been observed using 4D Normarski microscopy, as well as with multiphoton microscopy with the Junction Associated Molecule-1 (JAM-1) GFP. We have mapped the gene to a small region of approximately 80KB on LG IV. One candidate gene in the region is an inositol 1,4,5-triphosphate receptor (ITR-1). In RNA interference experiments using itr-1, we have been able to phenocopy the jc5 phenotypes. Preliminary evidence with individual cosmids that break within itr-1 is consistent with the hypothesis that the jc5 mutation is caused by a lesion in the itr-1 gene. In order to test whether itr-1is jc5, we will try to rescue the jc5 phenotype by injection using a cosmid that spans the itr-1region. ITR-1 has already been found to act in behavioral rhythms in C. elegans larva and adults (Dal Santo, P. et al, 1999. Cell 98, 757), and can act as a Ras-independent effector of the LET-23 epidermal growth factor receptor (Clandinin, T. et al, 1998. Cell 92, 523). However, no one has characterized the role of this protein in embryonic development. It is known that ITR-1 controls intracellular calcium levels; recent papers in other systems have implicated calcium in the regulation of actin during cell migration (Gallo, G. and Letourneau, P.C., Current Biology 9, 490). This may indicate a role for ITR-1 in cytoskeletal rearrangement during ventral enclosure.

Christina L Thomas et al. (2001) International C. elegans Meeting "A Role for Inositol 1, 4, 5-Triphosphate Receptor During the Process of Ventral Enclosure in Caenorhabditis Elegans"

Jeff Hardin, Jeff S Simske, Christina L Thomas

[International C. elegans Meeting, 2001]

During embryogenesis, epithelial cells migrate and change shape in order to generate three dimensional structure. During the process known as ventral enclosure in C. elegans , epithelial cells that originate on the dorsal surface of the embryo migrate to the ventral midline, forming adhesive junctions. One mutant that is defective in ventral enclosure is jc5 . jc5 is a cold sensitive maternal effect mutant that displays defects during varying stages of embryogenesis. At least 20% of embryos arrest because migration of epithelial cells from the dorsal side never occurs. Approximately 20% die during enclosure. Often in these embryos, the epithelial sheet initiates migration towards the ventral midline, fails to seal, and eventually retracts back to the dorsal surface. Others display partial enclosure phenotypes. Approximately 40% of dead embryos enclose properly, but develop severe body shape defects during elongation. Using RNA interference, we have been able to phenocopy the jc5 phenotypes with RNA corresponding to the inositol 1, 4, 5-triphosphate receptor-1 gene, itr-1 . In addition , we have obtained rescue of the jc5 phenotype by injection of genomic DNA corresponding to the itr-1 gene. It is known that ITRs control intracellular calcium levels, and a recent paper has shown a role for calcium in the regulation of actin in growth cone turning in another system(Gallo et al, Current Biology 9, 490). From work done in our laboratory, we know that the ventral hypodermal cells extend actin-rich filopodia before they migrate (Williams-Masson et al, Development 124, 2889). We hypothesize that ITR-1 regulates cytoskeletal rearrangement during ventral enclosure through localized calcium release. To test this, we are planning to study calcium dynamics in itr-1(jc5) mutants using the calcium indicator fluo-4. Additional experiments must be done to explore how ITR-1 is functioning to regulate the cytoskeleton of hypodermal cells. To this end, we are analyzing alpha-catenin-GFP dynamics during migration in epithelial cells using multiphoton microscopy. Preliminary data suggest that in some cases filopodia are misdirected, failing to migrate to the proper contralateral partner cell. In addition, preliminary phalloidin staining suggests that actin is disorganized in epithelial cells that have halted migration in mutants. Currently, we are sequencing the lesion in the itr-1 , gene as well as eliminating itr-1 activity in epithelial cells using antisense expression to test for epithelial-specific defects. In order to find other proteins that may affect ITR-1 function either directly or indirectly, we have initiated a suppressor screen for rescue of the itr-1(jc5) phenotype. Thus far, we have screened 1100 genomes and have found 5 candidate suppressors.