Naoki Hisamoto et al. (2003) International Worm Meeting "Deletion mutants in components of MAPK cascades in C. elegans."

Naoki Hisamoto, Miho Kato, Kimika Maki, Kunihiro Matsumoto

[International Worm Meeting, 2003]

MAP kinase (MAPK) signal transduction pathways are evolutionarily conserved in eukaryotic cells and transduce signals in response to a variety of extracellular stimuli. Each pathway is composed of three classes of protein kinases: MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK). Scaffolding proteins such as JIPs serve to bring together these components, facilitating their interaction and regulation. MAPK phosphatase (MKP) negatively regulates MAPK pathways by dephosphorylating MAPKs. To understand the physiological roles of MAPK pathways in the normal development and function of organisms, we have isolated C. elegans deletion mutants in components involved in the MAPK cascades. They include;MAPKKK:dlk-1 (DLK/MUK homolog), mlk-1 (MLK homolog), mtk-1 (MTK1/MEKK4 homolog), zak-1 (ZAK/MRK homolog), wnk-1 (WNK homolog), lrk-1MAPKK: mkk-4 (MKK4 homolog)MAPK: kgb-1 (JNK-like),kgb-2 (JNK-like)Scaffold protein: jip-1 (JIP1/2 homolog)MKP: vhp-1We will discuss the phenotypes of these deletion mutants.

JJ Ewbank et al. (1999) International C. elegans Meeting "We do it, bees do it, even educated fleas do it, (plants as well), but how do worms fight infections?"

JJ Ewbank, P Bulet, JA Hoffmann, D Ferrandon

[International C. elegans Meeting, 1999]

Over the last few years, insights into the antimicrobial defenses of the fruit fly Drosophila melanogaster have revealed remarkable parallels between insect and mammalian innate immunity (1). Relatively little is known about the immune responses of C. elegans . In Drosophila , an infection provokes the rapid sysnthesis of powerful antimicrobial peptides and opsonising proteins (2). While seven distinct anti-microbial peptides have been identified in flies, thus far, only two have been characterised from C. elegans , encoded by abf-1 and abf-2 on C50F2 (3; see also 4). Searching for conserved peptide motifs has yielded two further family members, abf-3 and abf-4 , (on F54B8 and Y38H6C, respectively) the study of which we are undertaking. Further, we are using MALDI-TOF mass spectrometry to investigate whether other antimicrobial peptides are induced upon infection of C. elegans . In Drosophila , the expression of anti-fungal peptides is controlled by the Toll pathway (5). A search of the genome reveals that C. elegans possesses structural homologues of most of the components of this signalling cascade (for Toll, pelle, tube, cactus, on cosmids C07F11, K09B11, F45G2 and C04F12, respectively). As a first step, we are using Northern analysis to determine whether this putative pathway is implicated in worm defense mechanisms. The results of these investigations will be presented. 1. Medzhitov and Janeway, 1998, Curr Opin Immunol, 10, 12-5 2. Hoffmann and Reichhart, 1997, Trends in Cell Biology, 7, 309-316 3. Kato, 1997, International Worm Meeting abstract 296 4. Kato and Komatsu, 1996, J. Biol. Chem., 271, 30493-30498 5. Lemaitre et al., 1996, Cell, 86, 973-983

Kuramochi, Masahiro et al. (2015) International Worm Meeting "Calcium imaging and computational simulation for the spatio-temporal dynamics of the neural coding in a C. elegans chemotaxis circuit."

Kuramochi, Masahiro, Doi, Motomichi

[International Worm Meeting, 2015]

To understand the neural information processing navigating animal behaviors, integration of the in-vivo observation of neuronal activity and the computational modeling using observation data is quite useful. This strategy gives us important information to characterize the spatio-temporal dynamics of the activity in each single neuron and their roles on information processing through synaptic integration. Recent studies have succeeded to apply the integrated analysis into C. elegans sensory neurons [Kato et al., 2014]. We have focused on a C. elegans chemosensory circuit and are conducting an integrational analysis to understand the neural information processing during salt-chemotaxis behavior. By using calcium-sensitive fluorescent proteins, in this study, we monitored the activities of the ASE salt-sensing neurons and their postsynaptic interneurons, and using these data, we have developed a novel simulation model to estimate their activities in-silico.The calcium-sensitive fluorescent proteins GECO were expressed in either ASE neurons or their postsynaptic neurons such as AIY, and the transgenic animals were stimulated by the decrease or increase of NaCl concentration. To monitor and collect the patterns of neuronal activities close to worm's living conditions, we applied various types of stimuli to the transgenic animals, such as long or short duration of stimulation, tiny or large concentration change, and the rapidly-flickering concentration change. These imaging analyses imply several characteristics about one of the salt-sensing ASE neurons ASER. First, the ASER neuronal activity is described by a leaky integrator which includes an exponential decay occurred after initial activation process. Second, the activity of the ASER neuron has a nonlinear relationship with the concentration change of NaCl. Thirdly, the ASER can response reliably to rapidly-flickering stimuli as the temporal dynamics observed in the AWC neuron [Kato et al., 2014]. For the computational modeling of the ASER neuronal activity, we have developed a kind of 'leaky integrate-and-fire model', in which neuronal cells are described as multi-compartments. We confirmed that this model reproduces the neuronal activity of the ASER neuron in agreement well with imaging data. Our integrated strategy will help to understand how neurons are responded and how these responses do contribute to the spatio-temporal dynamics of the postsynaptic neuronal activity, as worms sense various types of stimulation.

Scholz, Monika et al. (2019) International Worm Meeting "Predicting locomotion from whole-brain neural dynamics in freely moving animals."

Randi, Francesco, Leifer, Andrew, Yu, Xinwei, Shaevitz, Joshua, Linder, Ashley, Scholz, Monika, Sharma, Anuj

[International Worm Meeting, 2019]

How do patterns of neural activity across the brain represent an animal's behavior? Recent techniques for recording from large populations of neurons are providing new insights into how locomotion is encoded in population-level neural activity. Studies from mammalian systems suggest that behavioral information may be more prevalent throughout the brain and may account for a larger fraction of neural dynamics than previously thought. In C. elegans, pioneering studies revealed that the worm's neural dynamics during immobilization exhibit striking stereotyped low-dimensional patterns of neural activity that dominate brain-wide dynamics (Kato et al., 2015). These dynamics are hypothesized to map onto a motor sequence consisting of forward, backward and turning locomotion. One interpretation is that the majority of the worm brain's activity may be involved in encoding these locomotory behaviors. Here we seek to directly measure how patterns of neural activity represent locomotion by recording brain-wide calcium activity in freely-moving animals. We record calcium activity simultaneously from the majority of head neurons in C. elegans during unrestrained spontaneous locomotory behavior (Scholz et al., 2018). We find that a subset of neurons distributed throughout the head encode locomotion. By taking a linear combination of these neurons' activity, we predict the animal's velocity and body curvature and further infer the animal's posture from neural activity alone. The collective activity of these neurons outperforms single neurons at predicting velocity or body curvature. We further attempt to estimate the identity of neurons involved in the prediction. Among neurons important for the prediction are well-known locomotory neurons, as well as neurons not traditionally associated with locomotion. We compare the neural activity of the same animal during unrestrained movement and during immobilization and observe large differences in their neural dynamics. Intriguingly, during unrestrained movement we estimate that only a small fraction of the brain's overall neural dynamics are encoding velocity and body curvature. We speculate that the rest of the brain's neural dynamics may be involved in encoding other behaviors, processing sensory information or maintaining internal brain states. Kato, S., Kaplan, H.S., Schrodel, T., Skora, S., Lindsay, T.H., Yemini, E., Lockery, S., and Zimmer, M. (2015). Global brain dynamics embed the motor command sequence of Caenorhabditis elegans. Cell 163, 656-669. Scholz, M., Linder, A.N., Randi, F., Sharma, A.K., Yu, X., Shaevitz, J.W., and Leifer, A. (2018). Predicting natural behavior from whole-brain neural dynamics. BioRxiv 445643.

Jennifer L. Tenor et al. (2007) International Worm Meeting "A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity."

Jennifer L. Tenor, Alejandro Aballay

[International Worm Meeting, 2007]

Pathogen recognition through Toll-like receptors (TLRs) is the first step required to mount an appropriate immune response against invading pathogens. The only toll-like receptor, tol-1, in Caenorhadbitis elegans was shown to be required for behavioral avoidance to a particular Gram-negative bacterium, Serratia marcescens (1). Although we did not observe significant differences in behavioral avoidance between wild type and the tol-1(nr2033) mutant to various other Gram-negative pathogens, the tol-1 mutant was hypersusceptible to these pathogens suggesting that TOL-1 may provide protection through other mechanisms. Based on survival assays and microscopy, we found that TOL-1 is essential for protecting the pharynx against the enteropathogenic bacterium, Salmonella enterica serovar Typhimurium. Loss of tol-1 not only led to rapid death of C. elegans but also resulted in rapid accumulation of S. enterica in the terminal bulb of the pharynx. We showed that TOL-1 is required for the proper expression of ABF-2 which is a defensin-like molecule expressed in the pharynx (2) and HSP-16.41 which is also expressed in the pharynx and is part of a family of heat shock proteins required for C. elegans immunity (3). These results indicate that TOL-1 plays a direct role in innate immunity. (1) Pujol et al., 2001; (2) Kato, 2002; (3) Singh and Aballay, 2006.

Kato Y (1997) International C. elegans Meeting "IMMUNE DEFENSE OF WORMS: AN ANTIBACTERIAL PEPTIDE FAMILY IN A. SUUM AND C. ELEGANS"

Kato Y

[International C. elegans Meeting, 1997]

It is still largely unknown how C. elegans defends itself against microbes. Recent developments in the fields of vertebrate and D. melanogaster immunity indicate that some innate immune systems are very ancient in origin, i.e., common between insects and mammals. These results suggest that model organisms are useful to study innate immunity. The introduction of C. elegans into this research field may, therefore, provide novel tools for the analyses of innate immune systems. Antibacterial peptides have been identified as effector molecules in innate immune systems in various animal species. One of our strategies is (1) to identify antibacterial peptides in C.elegans, (2) to explore their regulatory mechanisms. Since the rate of molecular evolution of these antibacterial peptides is thought to be rapid, direct screening based on sequence data of known antibacterial peptides is difficult. Consequently, no peptide was found in the C. elegans genome data base using sequence homology. Since C. elegans is not suitable for biochemical analyses of tissue specific peptides because of its small body size, we purified antibacterial peptides from the body fluid of the pig round worm, Ascaris suum. These antibacterial peptides are basic peptides consisting of 61-71 residues and contain 4 intramolecular disulfide bridges, and are especially effective against gram positive bacteria, such as Staphylococcus aureus (Kato and Komatsu (1996) J. Biol. Chem. 271, 30493). They are designated as ASABF (Ascaris suum antibacterial factor) -alpha, beta, gamma, and delta, respectively. BLAST data base searches revealed two ASABF homologs in C. elegans. These putative peptides were designated ABF (antibacterial factor)-1 and 2. Genes encoding ABF-1 and ABF-2 were found in cosmid C50F2 and may be a part of a polycistronic mRNA. Although abf-2 lacks introns, abf-1 is interrupted by an intron whose position is identical to that of the A. suum asabf genes.

Hwang, W. et al. (2017) International Worm Meeting "Glutamine 5'-tRNA-drived small RNAs promote longevity via increasing mitochondrial activity through AMPK."

Shin, G.W., Nam, H.G., Seo, M., Lee, S.J.V., Kim, Y.E., Jung, G.Y., Hwang, W., Lee, Y., Ahn, G.O., Ha, C.M.

[International Worm Meeting, 2017]

Transfer RNA-derived small RNAs (tsRNAs) are abundant small non-coding RNAs discovered in various organisms, including C. elegans. Studies have begun to unravel the biological functions of tsRNAs, but their roles in aging processes remain elusive. Here we demonstrate the functional importance of specific tsRNAs for C. elegans longevity. We first determined age-dependent changes in tsRNA levels by analyzing available small RNA sequencing data (1) and confirmed the results by using Northern blot analysis. We found that the level of various tsRNAs gradually increased during aging. We overexpressed one of such tsRNAs, glutamine (Gln) 5'-tsRNA, by using an RNA polymerase III-responsive U6 (K09B11.12) promoter. Interestingly, we found that overexpression of the Gln 5'-tsRNA significantly extended lifespan. Our results suggest that the level of life-extending Gln 5'-tsRNA is increased during normal aging as a compensatory response. We then asked which longevity factors were required for the life extension. We showed that mutations in aak-2/the catalytic alpha subunit of AMP-dependent protein kinase (AMPK) fully suppressed the Gln 5'-tsRNA-mediated longevity. Through performing RNA seq. analysis, surprisingly, we found that the expression of all 12 mitochondrial DNA-encoded genes was highly increased by Gln 5'-tsRNA overexpression. We further showed that Gln 5'-tsRNA overexpression increased mitochondrial DNA contents in an AMPK-dependent manner and enhanced muscle-specific mitochondrial RFP intensity. Together, our data raised the possibility that age-dependent increases in Gln 5'-tsRNA levels delay aging and promote longevity by enhancing mitochondrial functions through AMPK. Currently we are testing whether our findings regarding the role of tsRNAs in mitochondrial function in C. elegans are conserved in mammals by using mice. Our study provides key information regarding anti-aging and mitochondria-enhancing functions of tsRNAs, which are universal small non-coding RNAs. (1) Kato et al., 2011, RNA

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.

EM Link et al. (1999) International C. elegans Meeting "C. elegans immunity? A reverse genetic analysis of components of the Toll signaling pathway"

J Hodgkin, G Hardiman, EM Link, LX Liu, L Nelson

[International C. elegans Meeting, 1999]

The Toll signaling pathway is involved in innate immunity in both flies and mammals. The Drosophila pathway, including the Toll receptor, Pelle kinase, and Dorsal transcription factor, regulates both dorsal-ventral polarity in the embryo and antimicrobial peptide production in adult flies. The human inflammatory cytokine IL-1 signals through the IL-1 receptor (whose cytoplasmic domain is homologous to that of Toll) and the Pelle homolog IRAK, ultimately activating NF k B, a rel protein homologous to Dorsal. Human Toll homologs also signal to NF k B(1). To investigate this pathway in C. elegans , we identified orthologs of four genes and generated deletion mutations for each: tol-1 = Tol l receptor, pik-1 = P elle/ I RAK k inase, trf-1 = TR A F , and ikb-1 = I k B . Worms with a deletion affecting all of tol-1 except the N-terminal leucine-rich repeats arrest as small, deformed larvae, a phenotype rescued by the tol-1 cosmid. However, a second tol-1 deletion in the intracellular domain seems wild-type. Deletion mutants of the other three genes show no obvious phenotype. To test whether these deletion alleles affect putative C. elegans host defenses, we exposed worms to an Aureobacterium strain that causes anal infection and local tissue swelling (2). The swelling phenotype was not altered in the four viable mutants. We also examined the expression of abf-1 and abf-2 , which encode homologs of Ascaris antibacterial peptides (3). abf-1 and abf-2 expression was not altered in the four mutants compared to wild type, following either usual culture or Aureobacterium infection. Orthologs for other downstream elements of the Toll pathway, including NIK, IKK and NF k B (1), are conspicuously absent from the complete worm genome, suggesting that C. elegans has evolved an alternative transcriptional output for tol-1 . Although our preliminary assays were negative, we cannot rule out a possible role for tol-1 in worm immunity, given the dearth of known microbial pathogens of C. elegans . (1) Kopp ER & Medzhitov R, Curr Opin Immunol 1999; 11: 13. (2) Hodgkin J & Corneliussen B, WBG 1997; 15 (1):73. (3) Kato Y, WBG 1996; 14 (4):37.

Burke, Samantha et al. (2011) International Worm Meeting "A function for mir-34 in gonad morphogenesis."

Hammell, Molly, Burke, Samantha, Ambros, Victor

[International Worm Meeting, 2011]

Mir-34 is a highly conserved microRNA whose function is not yet fully understood. Human cell culture and mouse model work has shown that mir-34 plays a role in cell cycle progression, senescence, and apoptosis [1,2], while work in C. elegans has implicated mir-34 in radiation-induced DNA damage response [3]. In transgenic C. elegans expressing GFP under the control of the mir-34 promoter, expression is seen broadly throughout neural and muscle cells and in the hypodermis. Levels of both mature mir-34 and the GFP reporter increase during adulthood. In starved L1 and dauer larvae mature mir-34 levels are elevated compared to the developing L1 and L2 stage larvae, respectively [4]. We examined mir-34 mutants for developmental phenotypes and determined that the mutants display incompletely penetrant gonad migration defect, including overextension of the gonad arms, improper contact between the arms and hypodermis, and extra turns. The incomplete penetrance of this phenotype suggested the presence of co-regulators. Therefore, we tested for genetic enhancement of the mir-34 phenotype by loss of other microRNAS that share predicted targets with mir-34. Mutation of one such microRNA, mir-83, also causes a slight gonad migration defect. In mir-34;mir-83 double mutant animals, the migration defect is significantly more pronounced, suggesting that mir-34 and mir-83 function redundantly in the regulation of targets involved in guiding the migration of the gonad during larval development. We are in the process of identifying transcripts co-targeted by mir-34 and mir-83 in order to better understand how the loss of both microRNAs can lead to such defects. References: [1] C. He et al., Biochemical and Biophysical Research Communications 388 (2009) 35-40. [2] M. Yamakuchi and C.J. Lowenstein, Cell Cycle 8 (2009) 712-715. [3] M. Kato et al., Oncogene 28 (2009) 2419-2424. [4] X. Karp et al., RNA 17 (2011) 639-651.