Werner, Kristen et al. (2013) International Worm Meeting "Small Molecule Communication: C. elegans and bacterial chemical signals."

Perez, Lark, Werner, Kristen, Semmelhack, Martin, Bassler, Bonnie

[International Worm Meeting, 2013]

Bacterial group behaviors are governed by a process called quorum sensing, in which bacteria produce, secrete, and detect extracellular signal molecules called autoinducers (AIs). Vibrios produce multiple AIs, some enable intra-species communication and others that promote inter-species communication. Vibrio cholerae produces an intra-species AI called CAI-1 that is a 13 carbon long fatty acyl molecule and the interspecies signal called AI-2 that is a boron-containing furanone. The information contained in the AIs is funneled into a shared phosphorelay signaling cascade that controls virulence, biofilm formation, and other traits. The bacteriovorous nematode, Caenorhabditis elegans, also uses small molecules to interpret its environment. A class of C. elegans-derived molecules called ascarosides influence nematode behaviors including attraction, repulsion, and mating. The presence of bacteria stimulates chemotaxis, egg-laying, and feeding in C. elegans, however, the bacteria-produced molecules that the nematode detects to control these phenotypes are largely unknown. We demonstrate that in addition to playing a vital role in quorum-sensing-regulated behaviors in V. cholerae, CAI-1 also influences behavior in C. elegans. C. elegans is more strongly attracted to V. cholerae than to its food source E. coli HB101 and C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant for CAI-1 production. Consistent with this finding, robust chemoattraction occurs to synthetic CAI-1. CAI-1 is detected by the sensory neuron AWCON. Laser ablation of this cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. To define which moieties of CAI-1 are crucial for recognition by C. elegans, we synthesized CAI-1 analogs and tested whether they promote chemoattraction. The fatty-acid chain length as and the precise position of the CAI-1 ketone group are the key features required for mediating CAI-1-directed nematode behavior. Together, these analyses define a bacteria-produced signal and the nematode detection apparatus that permit interkingdom communication.

Chih-Chun Hsu et al. (2010) East Asia Worm Meeting "Visualization of neuronal precursor interaction complex in C. elegans by the BiFC assay"

Oliver I. Wagner, Juan David Moncaleano, Chih-Chun Hsu

[East Asia Worm Meeting, 2010]

The study of protein interactions in the physiological context is a valuable tool for the investigation of biological regulatory mechanisms. Here, we are interested in the interactions between molecular motors and small adaptor proteins that might have a regulatory function. For example, our previous study shows a regulatory function of an active zone protein liprin-alpha (SYD-2) on UNC-104 (KIF1A). However, factors that control UNC-104/SYD-2 interactions are not known. Similarly, it is known that UNC-16 (JIP3) acts as a scaffold for kinesin-1 (KLC-2) regulating the transport of synaptic vesicles; while it is unknown which factors regulate the UNC-16/KLC-2 interaction. To investigate factors that activate or suppress the interactions between these kinesins and their adaptor proteins, we use a novel method BiFC (Bimolecular fluorescence complementation) in combination with forward and reverse mutagenesis. Here, we fuse fluorescent protein complementary fragments (hybrids of fluorophores) to each protein in the complex. In detail, we express the N-terminal half of a YFP fused to one protein and at the same time the C-terminal half fused to another protein in the complex. As it is known that YFP-N can complement with YFP-C to make a functional YFP, this method enables us to investigate physical interactions between two proteins in the living worm. Specifically, we use a native, pan-neuronal promoter pUnc104 to drive the expression of the following proteins in the nervous system of C. elegans: YN::SYD-2/UNC-104::YC, UNC-16::YN/KLC-2::YC as well as UNC-104::YN/UNC-104::YC. As a positive control we use bJUN::YN and bFOS::YC that are known to express and strongly interact in the nucleus. The investigation of UNC-104/UNC-104 interaction is of special interest as in the literature it is highly discussed whether this kinesin-3 exists as a monomer or dimer when activated or deactivated. The next important step in this research will be to use forward (EMS) and reverse (genome-wide RNAi screen) mutagenesis to identify genes that might disrupt the interaction between the BiFC pairs. Therefore we would be able to investigate novel regulators in the kinesin/adaptor protein complexes.

Redemann, Stefanie et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Codon adaptation-based control of protein expression levels in C. elegans and its application to GPR-1 allows control of microtubule-based pulling force"

Redemann, Stefanie, Ernst, Susanne, Ayloo, Swathi, Bringmann, Henrik, Schloissnig, Siegfried, Pozniakowski, Andrej, Hyman, Anthony A

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The variation of the expression level of a protein could provide a powerful tool to study protein function. However, there is no method that allows the precise control of protein levels under a native promotor in eukaryotes. We developed a method, which enables us to fine tune the protein expression levels in C. elegans by using synthetic genes with adapted codons. By modifying the codon usage of a gene, the Codon adaptation index (CAI) can be changed and the level of protein expression can be controlled. We used this method to regulate the expression of the G-protein regulator GPR-1/2, which is involved in force generation during spindle positioning in the first asymmetric cell division in C. elegans. By gradually increasing the amount of GPR-1/2, we found that the amount of force acting on the spindle in C. elegans embryos is directly related to the amount of the G protein regulator GPR1/2 in the cell. In C. elegans GPR-1/2 is found in a complex, the force-generating complex,which is thought to consist of at least three proteins: GPR-1/2, LIN-5 and a G-alpha protein. Since increasing the amount of GPR1/2 is sufficient to increase the force, this suggests that the other proteins are there in excess and that GPR-1/2 is the limiting component. The modification of the CAI is a good example of how the ability to over-express proteins is essential for identifying components that are limiting as opposed to permissive for force generation. This method provides the first way to control the level of protein expression levels in C. elegans, and the first method for overexpression of proteins in the C. elegans germline. With this method the protein levels of a protein of interest can be varied, while maintaining all the wild type genetic regulation and the wild type protein sequence.

Tsukada, Y. et al. (2013) International Worm Meeting "System identification for thermosensory neuron encoding thermal environment."

Mori, I., Murase, A., Shimowada, T., Kuhara, A., Tsukada, Y., Ishii, S., Honda, N., Noriyuki, O.

[International Worm Meeting, 2013]

A central goal in neuroscience is the full characterization of the information processing of neural circuits. Despite the knowledge of the complete connectivity and the molecules important for the specific mechanisms of neural circuits, it remains understood about the mechanism of information processing in the neural circuit of Caenorhabditis elegans. Here, we focus on the response of thermosensory neuron AFD during thermotaxis and its change in different conditions. We used automated tracking system to capture freely moving single animals on thermal gradient (about 0.5 deg C/cm). Combining the tracking system with a calcium imaging system, we monitored behavior and activity of AFD thermosensory neuron during thermotaxis with genetically encoded calcium indicator, cameleon YC 3.60. The time course of temperature for the tracked worm was estimated by recorded xy-coordinates and thermography. Thus we quantified exact temperature input, AFD activity, and behavioral state of freely moving animal during thermotaxis. As we previously reported, wild-type N2 animals on an agar plate with thermal gradient showed thermotaxis behavior: they migrate to the cultivated temperature region when we keep the worms in a constant temperature during cultivation. Then we estimated response functions of neural activity of AFD for input of temperature. The estimated response functions indicate differential detection of temperature by AFD. We also found that the response functions of the worms cultivated in different temperatures are different forms, but not depending on food conditions. After the quantitative measurements of AFD response functions, we reconstructed AFD responses with temperature inputs and the estimated response functions. Then we compared the reconstructed responses with real data. The results showed reproductive high correlation, and thus we conclude that estimated response functions express dynamic property of AFD thermosensory neuron. We suggest that such quantitative approach has potential to understand non-linear characteristic of neural circuits.

Zhu X et al. (1997) International C. elegans Meeting "MIG-15 is a C. elegans homolog of the murine protein NIK that activate the SAPK cascade"

Hedgecock EM, Zhu X

[International C. elegans Meeting, 1997]

mig-15 (rh148) mutants have pleiotropic defects in hypodermal development, Q neuroblast migrations and muscle arm targeting. Cosmid ZC504 rescues mig-15 mutant phenotypes. Subcloning revealed that mig-15 encodes a Ste20/p65PAK related ser/thr protein kinase that is most homologous to NIK, a newly identified murine protein (YC. Su et al., EMBO journal, 16, 6 1997 In press). MIG-15 and NIK share over 80% identity in the N-terminal kinase domain, and over 70% identity in the C-terminal regulatory domain. NIK was shown to specifically activate the SAPK pathway, and the activation can be blocked by a dominant-negative MEKK1.We have isolated a full length cDNA clone by RT-PCR technique. Sequence comparison of the full-length clone and two partial cDNA clones (from Y. Kohara) revealed two splice variant sites. Both sites are outside of the conserved N-terminal kinase domain and the C-terminal regulatory domain. The functional significance of these splice variant forms is currently unknown. As observed in a transgenic line expressing the MIG-15::GFP fusion protein from an extra chromosomal array, MIG-15 is expressed at a low level throughout the body. However, some neurons and muscle cells are brighter during particular developmental stages. The adhesion junctions between the seam cells and the hypodermal syncitium are visible as two parallel lines. We are investigating the role of mig-15 in the MAPK cascade in C. elegans as well as its role in the Q neuroblast migrations. We speculate that MIG-15 has dual functions in turning on the MAPK cascade and maintaining the activated state of the wingless signaling pathway, which is thought to be involved in the regulation of Q neuroblast migration (C.Guo and E. Hedgecock, 1996 East Coast C. elegans Meeting).

Martijn Dekkers et al. (2003) International Worm Meeting "Genetic analysis and molecular imaging of olfactory signalling in C. elegans"

Martijn Dekkers, Gert Jansen, Gerard Borst

[International Worm Meeting, 2003]

G-protein signal transduction is one of the most widely used mechanisms for cells to communicate with their environment. In a single cell several different G-protein signalling cascades are present, and recent evidence suggests extensive crosstalk between these individual cascades. How this is organised, while maintaining specificity in signalling is thus far poorly understood. To address this problem we study chemosensory behaviours in C. elegans. The animal is capable of detecting at least 60 odorants and to discriminate between many of them using only two pairs of cells, AWA and AWC. Interestingly, in these cells a specific subset of 6 G subunits is expressed (Jansen ea. 1999). To study the functions of the individual G subunits we use behavioural assays including an odorant discrimination assay (Bargmann ea 1993). Our preliminary data shows that the G subunit ODR-3 is sufficient to discriminate between at least some odorants. Many downstream effectors of G-proteins are known and are being tested for their function in these pathways. However, many of these genes are expressed ubiquitously and some have severe locomotory defects. This renders behavioural assays useless to study the effects of these proteins in signalling. Therefore we are developing imaging tools to visualise responses of individual cells to olfactory or gustatory stimuli. We will use two constructs, the Yellow Cameleon (YC) (Kerr ea. 2000) and the Voltage Sensitive Fluorescent Protein (VSFP) (Sakai ea. 2001). Both constructs utilise the Fluorescence Resonance Energy Transfer (FRET) principle to measure either the calcium fluxes in the cell or changes in the membrane potential, respectively. We will drive expression using cell-specific promotors, which allows us to elucidate the signal processing on a cellular level. To study effects in olfaction we will express the constructs in AWA using the promoter of the odr-10 gene and in AWC with the gpa-13 promoter, for gustation we will use ASE (flp-5 promoter) and ASI (gpa-4 promoter). Together with the data from behavioural assays, this will provide insight in the G-protein signalling pathways in olfaction in C. elegans.

Lao, R.X. et al. (2019) International Worm Meeting "Understanding Argonaute/Small RNA-based intercellular communication."

Buck, A.H., Claycomb, J.M., Wadi, L., Lao, R.X., Seroussi, U., Maity, T., Blaxter, M., Abreu-Goodger, C.

[International Worm Meeting, 2019]

Cells have many ways to regulate gene expression, one of which is by using small RNAs (sRNAs) and sRNA pathway effectors, called Argonautes (AGOs). Studies in plants and worms point to sRNA mediated communication between cells in a single organism (e.g., systemic RNAi; Winston et al., Science 2002). Perhaps even more exciting is the prospect that sRNA/AGO complexes could be used as a means of communication between organisms (Buck et al., Nat Comm. 2014, Cai et al., Science 2018). The discovery that sRNAs exist extracellularly in conjunction with AGOs is particularly important for host/parasite or pathogen relationships. For example, a parasitic nematode of mouse, H. polygyrus bakeri (Hb), secretes extracellular vesicles, which possess sRNAs and an extracellular AGO protein, exWAGO (extracellular Worm AGO). However, Hb is not genetically tractable, hence we have turned to C. elegans to study the functions of exWAGO. Although exWAGO is not present in the C. elegans genome, three homologs, SAGO-1, SAGO-2, and PPW-1 exist. We have engineered strains of C. elegans expressing exWAGO in various tissues to characterize its molecular function in sRNA pathways. In parallel, we engineered GFP-tagged strains of SAGO-1, -2, and PPW-1 using CRISPR-mediated gene editing. These three C. elegans AGOs share a common localization pattern to the apical gut membrane, where exWAGO is expressed in Hb. To further understand the roles of these intestinal secondary AGOs (iSAGOs), we immunoprecipitated the iSAGOs and performed sRNA sequencing and mass spectrometry analysis to identify sRNA and protein binding partners. Top hits from the IP/MS analysis included trafficking, secretory pathway, and membrane anchoring proteins, highlighting the potential for the iSAGOs to be secreted and act intercellularly in C. elegans. We are now in the midst of identifying the pathways that contribute to apical membrane anchoring of iSAGOs and determining what role the iSAGOs may play in systemic RNAi in C. elegans.

Tsukada, Yuki et al. (2011) International Worm Meeting "Long term calcium imaging of AFD thermosensory neuron revealed behavioral strategy for exploratory movements during thermotaxis."

Tsukada, Yuki, Mori, Ikue, Ohnishi, Noriyuki, Kuhara, Atsushi, Shimowada, Tomoyasu

[International Worm Meeting, 2011]

Despite the identification of neurons, their connectivity, and molecules related to the specific mechanisms of neural circuits, little is known about the mechanism of information processing in Caenorhabditis elegans. An essential obstacle is the lack of methods that can monitor time course of information flow during information processing tasks. To address this issue, we focus on thermotaxis behavior of C. elegans because the information flow is relatively simple: the essential neural circuit related to thermotaxis is thought to consist of only several neurons. We quantitatively monitored information input (temperature) and output (behavior) by using thermography and our live imaging/tracking system that pictures freely moving single animals; the time-lapse images of a single animal were recorded up to several hours at about 30 frames/sec video rate with migratory trajectory. We simultaneously monitored activity of AFD thermosensory neuron and behavior during thermotaxis by combining the tracking system with the calcium imaging system with genetically encoded calcium indicator, Cameleon YC 3.60. Thus we quantified exact temperature, AFD activity, and behavioral state of freely moving animal during thermotaxis. Wild-type N2 animals on an agar plate with thermal gradient showed thermotaxis behavior depending on their cultivated temperature or food availability. Long-term monitoring of AFD activity showed spike-like increments of calcium concentration despite the continuous, slow temperature change of about 0.1 deg C/min. By comparing AFD activity and the behavioral states, we found that the peak of AFD activity corresponded to the timing of turn, although the AFD activity and turning behavior was not one-to-one coupling and one activity peak seemed to correspond to a few turns. This relationship changed when the animals were conditioned with off-food plate, by experiencing the animal off-food for two hours before the thermotaxis assay. Off-food conditioned animals tended to show one AFD activity peak for one turn or AFD activity peaks without any turn. According to these results, we suggest that AFD thermosensory neurons recognize thermal environment as discrete system even in shallow continuous thermal environment and that the animals change behavioral strategy during thermotaxis by adjusting relationship between AFD activity input and turning output. These hypotheses brush up biased random walk model of exploratory behavior of C. elegans and shed light on a specific type of information processing systems which utilize random signal.