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[
J Biol Chem,
2014]
In a process known as quorum sensing, bacteria use chemicals called autoinducers for cell-cell communication. Population-wide detection of autoinducers enables bacteria to orchestrate collective behaviors. In the animal kingdom detection of chemicals is vital for success in locating food, finding hosts, and avoiding predators. This behavior, termed chemotaxis, is especially well studied in the nematode Caenorhabditis elegans. Here we demonstrate that the Vibrio cholerae autoinducer (S)-3-hydroxytridecan-4-one, termed CAI-1, influences chemotaxis in C. elegans. C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant defective for CAI-1 production. The position of the CAI-1 ketone moiety is the key feature driving CAI-1-directed nematode behavior. CAI-1 is detected by the C. elegans amphid sensory neuron AWC(ON). Laser ablation of the AWC(ON) cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. These analyses define the structural features of a bacterial-produced signal and the nematode chemosensory neuron that permit cross-kingdom interaction.
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[
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.
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[
EMBO J,
2013]
A key finding of modern ageing research is that our limitation in lifespan is more than the result of accumulated organismal decay. Lifespan is regulated by genetically defined chemosensory and endocrine pathways, which integrate signals that reflect the internal and external status of the animal. New findings by Liu and Cai unravel a role for the environmental gases oxygen and carbon dioxide in the regulation of lifespan homeostasis and thus a novel function of oxygen-chemosensory neurons in C. elegans.
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[
International Worm Meeting,
2017]
Cilia are ubiquitous microtubule-based organelles found at surface of metazoan cells. Non-motile (sensory/primary) cilia act to receive signals from the environment, and motile cilia either move cells or the surrounding fluid. Cilia are built and maintained using a cargo-trafficking intraflagellar transport (IFT) machinery powered by kinesin (anterograde) and dynein (retrograde) molecular motors. How IFT-dynein contributes mechanistically to ciliary processes in metazoans remains poorly studied, in part because null mutants exhibit a severe terminal phenotype with IFT protein accumulations in the bulbous tips of highly truncated cilia. We carried out a screen for retrograde IFT transport defects in C. elegans, and identified the first temperature-sensitive IFT mutant in a metazoan. Using this strain, we show that the IFT-dynein (CHE-3) heavy chain is essential for cilium maintenance and correct formation of the transition zone (TZ) ciliary gate. Cilia resorb upon shift to restrictive temperature (inactive IFT-dynein), and are restored upon return to permissive temperature (active IFT-dynein). Importantly, this resorption and regrowth is observed for primary cilia in terminally differentiated, sensory neurons. We next investigated how IFT-dynein enables processive IFT, in contrast to the bidirectional tug-of-war mechanism of transport used by kinesin and dynein elsewhere in the cell. We find that the IFT-dynein heavy, intermediate and two light chains are trafficked by the IFT-subcomplex B, unlike the light intermediate chain, which is transported independent of the canonical IFT complex. This differential transport provides an effective mechanism by which the retrograde transport machinery is maintained in an inactive state during anterograde transport. Together, our findings reveal that IFT-dynein is required for ciliary maintenance and TZ formation, and regulates the processivity of anterograde IFT by spatial separation and thus inactivation of its components.
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[
MicroPubl Biol,
2018]
Disrupting the function of sensory neurons of C. elegans can increase their lifespan (Apeld and Kenyon 1999). This effect is not limited to large-scale disruption, as ablation of single pairs of neurons have been shown to modify lifespan (Alcedo and Kenyon 2004; Lee and Kenyon 2009; Liu and Cai 2013). We tested whether silencing the neuron pair ASI with the tetanus toxin light chain (Tetx), as opposed to ablating it, could increase lifespan. Tetanus toxin disrupts neurotransmission by blocking the release of both small clear-core vesicles and large dense-core vesicles, but should not affect communication via gap junctions (Schiavo et al. 1992; McMahon et al. 1992). We expressed GFP::Tetx using the ASI-specific promoter
pgpa-4 (Figure Panel A) and conducted lifespan assays comparing animals with high fluorescence and undetectable fluorescence. Tetx in ASI extended lifespan in otherwise wild-type animals (Figure Panel B, Table 1, 14.9% average median lifespan increase across 5 replicates).
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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.
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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.