Gert Jansen et al. (2002) European Worm Meeting "Cellular and genetic analysis of salt adaptation in C. elegans"

Gert Jansen, Suzanne Rademakers

[European Worm Meeting, 2002]

C. elegans shows strong chemotaxis to salts. However, prolonged exposure to salts abolishes chemoattraction to these same salts. This decrease in chemotaxis is time and concentration dependent, partially salt specific and reversible. Thus far we identified 3 loci that affect adaptation to salts: gpc-1, adp-1 and osm-9. gpc-1 encodes a G protein g subunit, specifically expressed in the amphid sensory neurons. Both gain- and loss-of-function mutations in this gene affect salt adaptation. We have not been able to find other defects in the gpc-1 mutant animals. It is striking that G proteins are involved in taste perception, since this process is presumed to be mediated by channels. The gene affected in adp-1 mutants has not been identified. This mutation also affects adaptation to odorants. The osm-9 gene encodes a putative channel protein with limited similarity to the Drosophila TRP phototransduction channel and is expressed in a subset of sensory neurons. Mutations in this gene also affect odorant adaptation. It is unclear which cells mediate salt adaptation. The ASE neurons have been found to be the most important cells for salt detection. However, gpc-1 is not expressed in these neurons. We are currently performing cell specific rescue and genetic cell ablation experiments to determine which cells are involved in salt detection and adaptation in our assay. To identify more molecules involved in salt adaptation we have performed a genetic screen for salt adaptation mutants. Thus far, we have identified 10 different mutants. Genetic mapping using SNP analysis is ongoing.

Gert Jansen et al. (2001) International C. elegans Meeting "The C. elegans G-protein gamma subunit gpc-1 is involved in adaptation to water-soluble attractants."

David Weinkove, Gert Jansen, Ronald H A Plasterk

[International C. elegans Meeting, 2001]

Heterotrimeric G proteins form a first intracellular step in many signal transduction cascades. C. elegans has 21 Galpha, 2 Gbeta and 2 Ggamma subunits. Based on functional analyses the Galpha subunits can be divided into 2 groups. The conserved alpha subunits are ubiquitously expressed and regulate muscle and neuron activity. At least 14 other Galpha's play a role in sensory transduction. A similar discrimination can be made between the 2 G-protein gamma subunits, gpc-1 and gpc-2 . This latter gamma subunit is quite ubiquitously expressed. By contrast, gpc-1 is specifically expressed in only 6 pairs of amphid neurons and 1 pair of phasmid neurons, suggesting a function in sensory signaling. However, gpc-1 is not essential for the detection of various environmental cues, such as odorants or salts. To test if GPC-1 is involved in other types of sensory plasticity we developed a water-soluble compound adaptation assay. The behavior of wild-type animals in this assay confirms the idea that prolonged exposure to water-soluble compounds can abolish chemo-attraction to these same water-soluble compounds. This decrease in chemotaxis is time and concentration dependent, salt specific and reversible. gpc-1 mutant animals show clear deficits in their ability to adapt to three water-soluble compounds, NaAc, NaCl and NH 4 Cl. Also adp-1 and osm-9 , two loci previously implicated in odorant adaptation, are involved in adaptation to these salts. Our findings clearly indicate that G proteins are involved in the perception of salts. It is still unclear where in the signaling cascade these molecules function.

Rademakers, Suzanne et al. (2021) International Worm Meeting "Identifying novel interactors of the guanylate cyclase GCY-22 involved in NaCl chemotaxis"

Rademakers, Suzanne, Jansen, Gert

[International Worm Meeting, 2021]

C. elegans senses salts in their environment using the ASE neurons. Detection of salts occurs in sensory organelles, called primary cilia. cGMP signalling plays an important role in salt detection. The receptor-type guanylate cyclase (rGC) GCY-22 is involved in the response to NaCl in the environment. We generated a full-length GFP knock-in the gcy-22 gene. GCY-22::GFP shows unique localization to the ciliary tip and periciliary membrane compartment (PCMC) of one ciliated neuron, ASER. Our goal is to understand the molecular mechanisms that regulate its trafficking and unique localization. To identify proteins that physically interact with GCY-22, we performed mass spectrometry after immunoprecipitation to identify proteins bound to GFP-tagged GCY-22. Next, we study where the identified candidate interacting proteins are expressed and localized. Mutants are used to investigate their role in salt detection, GCY-22::GFP trafficking towards the cilium and localization to the ciliary tip. The most prominent candidate interacting protein is GCY-19. GCY-19::GFP is expressed in ASER and colocalized with GCY-22. Loss-of-function of gcy-19 resulted in lower levels of GCY-22::GFP at the ciliary tip. Similarly, gcy-22 loss-of-function animals showed lower levels of GCY-19::GFP at the tip of the ASER cilium . We also found GCY-4 and GCY-5 as possible interactors of GCY-22. As rGCs are thought to act as dimers, these findings suggests that GCY-22 might be a common subunit for heterodimeric complexes possibly to achieve ion-selectivity. In addition, we identified DAF-25 in our GCY-22::GFP IP-MS experiments. DAF-25 is the ortholog of the mammalian ankyrin repeat and Mynd domain containing protein Ankmy2. DAF-25 has been reported previously to be important for rGC transport. Mutants lacking DAF-25 show no ciliary tip localization of GCY-22::GFP and do not respond to NaCl. Other candidate genes are currently being investigated. This work will allow us to gain insight in the molecular mechanisms that regulate ciliary tip localization of GCY-22.

UMUERRI, OLUWATOROTI O. et al. (2011) International Worm Meeting "Molecular mechanism of salt taste."

UMUERRI, OLUWATOROTI O., Jansen, Gert

[International Worm Meeting, 2011]

NaCl is essential for salt and water homeostasis and it is important for physiological functions in many organisms. However, the detailed molecular mechanism of NaCl sensation is not well known. In mammals, the epithelial Na+ channel (ENaC) has been shown to be involved in NaCl detection but there is also an ENaC channel-independent salt taste mechanism. Several genes involved in NaCl chemoattraction in C. elegans have been identified. These include tax-2 and tax-4 (cyclic nucleotide gated (CNG) channel subunits), tax-6 and cnb-1 (calcineurin A and B subunits) and ncs-1 (neuronal calcium sensor). Analysis of these mutants in our assay, in which we exposed the animals to a very steep NaCl gradient, showed reduced chemotaxis to NaCl. However, these mutants still showed significant attraction to higher NaCl concentrations. By analyzing the behaviour of double mutants, we found that chemotaxis to NaCl involves two genetic pathways. The first pathway involves two mitogen activated protein (MAP) kinases, nsy-1 and sek-1, and three genes that have been previously characterized, tax-2, tax-4 and tax-6. The second pathway involves tax-2, another CNG channel subunit, cng-3, the Ga protein odr-3, the TRPV channel subunit, osm-9, and the guanylate cyclase, gcy-35. We use cell specific rescue of the mutant genes and neuronal calcium imaging to find out where in the neuronal circuit of C. elegans these genes function. The involvement of the main salt sensing neurons, ASE, has been confirmed. In addition, we found that the Ga protein, odr-3, functions in chemosensation in the ADF neurons. We have recently performed a forward genetic screen to identify additional genes that play a role in NaCl chemotaxis. We found 16 independent mutants among 20,000 haploid genomes screened. 6 mutants have completely lost chemosattraction to NaCl and 10 mutants showed highly reduced chemoattraction. One of the 6 identified mutants is a mutation in the che-1 gene (allele gj1010). We are currently using SNP-mapping and sequencing to identify the genes affected in two other mutants, gj1008 and gj1009 . gj1008 mapped to the left arm of chromosome I and gj1009 mapped to the middle of chromosome III. This will help in deciphering the signal transduction pathway that mediates NaCl sensation.

Sanders, Tom et al. (2013) International Worm Meeting "A circuit for decision making in C. elegans: a computational approach."

Jansen, Gert, Sanders, Tom, Cohen, Netta

[International Worm Meeting, 2013]

The head navigation circuit in the nerve ring of C. elegans performs a wide variety of computations with a limited number of neurons. Indeed, overlapping neurons have been implicated in navigation, sensory integration and decision making. Here, we create a computational model capable of reproducing a wide variety of behaviors, as observed in various assays performed on wild type, mutant and other defective animals. To this end, we developed a modular computational framework, in which neuronal, circuit and motor output functions and properties, as well as the assay specification can all be easily modified. In our model, individual worms are represented by position, direction pairs controlled by a reduced nervous system consisting of several sensory neuron pairs, an interneuron layer and an abstract motor system. We focus on a decision making assay, in which animals are placed on a dish with quadrants containing a particular concentration of NaCl or no NaCl. Before the assay, the animals are washed for 15 minutes with a low salt buffer to test their naive response, or a buffer containing 100 mM NaCl to test the response of animals that have associated NaCl with the absence of food (called gustatory plasticity). We show that the model is capable of reproducing the behavior of naive and conditioned animals in decision making. Model analysis suggests specific neuronal roles and circuit mechanisms. Specifically, our model distinguishes the roles of ASEL and ASER in decision making and suggests that ASH sensory neurons are recruited into the functional circuit, switching the preferred decision of the animals. We are currently testing the performance of our model in other navigation assays, for example a NaCl chemotaxis assay where animals are exposed to a shallow gradient of NaCl and a copper-diacetyl integration assay. The ability to validate the model by testing it in a variety of assays, and under a variety of conditions is a first step towards building more complete models of ever richer behaviors in C. elegans.

Umuerri, Oluwatoroti et al. (2013) International Worm Meeting "ASE and ASH neuron sensitivities determine NaCl attraction or avoidance behaviour."

Dekkers, Martijn, Jansen, Gert, Umuerri, Oluwatoroti

[International Worm Meeting, 2013]

Behavioral analyses in the quadrant assay has shown that C. elegans is attracted to 0.1 to 200 mM NaCl and avoids higher NaCl concentrations. In addition, C. elegans shows NaCl-taste related learning behavior: after 15 minutes exposure to 100 mM NaCl, in the absence of food, the animals avoid all concentrations of NaCl. We call this behavior gustatory plasticity. Previous analyses have shown that NaCl attraction is mainly mediated by the ASE neurons, while avoidance is mainly mediated by the ASH neurons. The ASE and ASH neurons are also essential for gustatory plasticity. We used Ca2+ imaging to visualize the responses of the ASE and ASH neurons both in naive animals and after pre-exposure to NaCl. Ca2+ imaging in naive animals confirmed that the left ASE neuron responds to an increase in NaCl (upstep), both at low and high NaCl concentrations. The ASER neuron has been reported to respond to a decrease in NaCl concentration (downstep). However, we found no downstep response of ASER after only 3 or 30 sec exposure. ASH neurons responded to NaCl concentrations above 100 mM. Interestingly, prolonged exposure to NaCl desensitized ASEL and completely abolished its responsiveness to NaCl after 5 or 10 minutes pre-exposure. In addition, prolonged exposure to NaCl sensitized ASER, resulting in robust downstep responses after 1 to 10 minutes exposure to NaCl. These processes seem mostly cell-autonomous, although serotonin and glutamate play a role. Prolonged exposure to NaCl also sensitized ASH. In agreement with the results of our behavioral assays sensitization of ASH required functional ASE neurons. In addition, we found that sensitization of ASH involves serotonin, dopamine, glutamate and neuropeptide signalling. Our results suggest that naive C. elegans are attracted to NaCl, predominantly mediated by ASE, and that at high NaCl concentrations this attraction is overruled by osmotic avoidance, mediated by ASH. Pre-exposure to 100 mM NaCl in the absence of food, sensed by ASE and other neurons, changes this circuit, resulting in desensitization of attraction and sensitization of avoidance.

Wesseling, Matthijs et al. (2019) International Worm Meeting "Expressing human epithelial Na channel (ENaC) subunits in C. elegans to model human salt taste."

Hoorn, Ewout, Jansen, Gert, Wesseling, Matthijs

[International Worm Meeting, 2019]

Human salt taste is one of the main drivers of dietary salt intake and directly correlates with blood pressure. The average western diet contains a high amount of salt and this contributes to various pathologies including kidney diseases and cardiovascular problems. Salt sensitivity and preference vary among people, but how this is regulated molecularly is unclear. The main salt sensor in the human taste buds is the epithelial sodium channel (ENaC), a heterotrimeric channel consisting of an ?, ? and ? subunit. It is unclear how ENaC activity in the taste buds is regulated. In the kidney, the activity of ENaC is regulated by proteases. Cleavage of the ? subunit by furin and the ? subunit by furin and other 'channel activating' proteases such as prostasin, kallikrein and plasmin have been shown to increase the open-probability of the channel, whereas protease inhibitors reduce ENaC activity. It is unclear if this mechanism also applies to the regulation of ENaC in salt taste, where proteases and protease inhibitors could be supplied in saliva. We study the molecular mechanisms of salt taste in C. elegans. C. elegans is attracted to NaCl concentrations between 0.1 and 200 mM and avoids higher NaCl concentrations. Low NaCl concentrations are mainly sensed by the ASE sensory neurons and high concentrations by the ASH nociceptive neurons. Thus far, there are no indications that ENaC channels play a role. We aim to generate a humanized NaCl-taste worm model that expresses all three human ENaC subunits in the ASH cells. To achieve this, we will use a two-step approach using CRISPR/Cas9 induced homology directed repair for each subunit. First, we will introduce sra-6::gfp in one of three different loci on different chromosomes. These strains will allow us to confirm proper expression in the ASH neurons. Subsequently, we will replace GFP for an ENaC subunit. Expression of each ENaC subunit and localization within the cell will be analyzed using immunofluorescence experiments. We expect that, should the human ENaC channel be functional, the ENaC expressing animals will be less attracted or even repelled by salt. To reduce interference of the C. elegans NaCl-taste machinery, we will use che-1 mutant animals, that lack functional ASE neurons. This 'humanized' model system will subsequently be used to study the possible role that proteolytic processing of ENaC may have in salt detection.

van der Burght, Servaas et al. (2015) International Worm Meeting "Contribution of protein localization to variability in salt chemotaxis behavior."

van der Burght, Servaas, Jansen, Gert

[International Worm Meeting, 2015]

C. elegans perceives salt and shows attraction to NaCl concentrations between 0.1-200 mM NaCl but avoidance of higher concentrations. This response can be analyzed behaviorally with quadrant assays in which worms are presented with a choice between buffered agar with or without NaCl. When concentrations of 0 and 100 mM NaCl are used, WT worms move to the salt containing quadrants. However, a small fraction of (genetically identical) animals will end up on the quadrants without salt. We aim to explain the randomness in C. elegans behavior by examining stochasticity at a molecular and cellular level.Perception of NaCl probably occurs by receptors and channels in the sensory cilia of the ASE neurons. Stochasticity could be caused by variability in the localization of these proteins, the in- and export of proteins in the cilia, and by the intraflagellar transport (IFT) machinery. To study these processes we will use CRISPR/Cas9 technology to tag proteins of interest to visualize their in- and export dynamics and localization, and correlate this with the response to NaCl.The way in which a worm perceives salt, however, is not completely understood. Forward and reverse genetic approaches have identified several proteins involved, including the guanylate cyclase subunits gcy-22 and gcy-14 as possible receptors, the cyclic nucleotide-gated channel subunits tax-2, tax-4 and cng-3, the TRPV1 subunit osm-9, and the G protein alpha subunit odr-3. We are generating strains in which each of these proteins is tagged with GFP, specifically in the ASE neurons.In a forward genetic screen, we recently found that C. elegans chemotaxis to NaCl involves a degenerin-like/ENaC channel subunit. We are now in the process of generating knockout mutants to determine its contribution to NaCl perception. Additionally, we are creating animals in which the ENaC channel subunit is fused, in frame, to GFP. This GFP-fusion strain will be used to examine its expression pattern and the localization of the protein. Possible stochasticity in localization of the ENaC channel subunit could explain the randomness in C. elegans salt chemotaxis behavior.

van Vuuren, Laura et al. (2021) International Worm Meeting "Expressing human epithelial Na channel subunits in C. elegans to model human salt taste"

van Vuuren, Laura, Jansen, Gert, Hoorn, Ewout

[International Worm Meeting, 2021]

Human salt taste is one of the main drivers of dietary salt intake and directly correlates with blood pressure. Salt sensitivity and preference vary among people, but the underlying molecular mechanism of this variation is unknown. Many studies have shown that the epithelial sodium channel (ENaC) is the main salt sensor involved in salt taste in rodents and humans. In the kidney, the activity of ENaC is regulated by proteases. Recently it was shown that the salivary proteome is different in salt sensitive and salt insensitive tasters. We hypothesize that salivary proteases regulate ENaC open-probability and salt taste. We use C. elegans as model to study human salt taste. C. elegans is attracted to NaCl concentrations up to 200 mM and avoid higher NaCl concentrations. Low NaCl concentrations are mainly sensed by the ASE neurons and high concentrations by the ASH nociceptive neurons. Thus far, there are no indications that ENaC channels play a role. We will generate a humanized NaCl-taste worm model that expresses all three human ENaC subunits in the ASH cells. We use a two-step approach using CRISPR/Cas9 induced homology directed repair for each subunit. We first introduced a 3.7 kb sra-6::gfp construct in a save harbor locus on chromosome I. This strain showed proper GFP expression in the ASH neurons. Subsequently, we introduced the SCNN1A gene, encoding the ENaC alpha subunit, after the sra-6 promoter and fused in frame with GFP. In these animals we see weak GFP expression in the cell bodies of the ASH neurons. We are currently in the process of integrating sra-6::mScarlet on chromosome II, where we will subsequently introduce the human SCNN1B gene, and sra-6::tagBFP2 on chromosome IV, for subsequent introduction of the SCNN1C gene. We expect that the ENaC expressing animals will be less attracted or even repelled by salt. To reduce interference of the C. elegans NaCl-taste machinery, we will use che-1 mutant animals, that lack functional ASE neurons. This 'humanized' model system will subsequently be used to study the possible role that proteolytic processing of ENaC may have in salt detection.

Broekhuis, Joost et al. (2011) International Worm Meeting "Identification of novel proteins involved in the regulation of IFT."

Leong, Weng Yee, Broekhuis, Joost, Jansen, Gert

[International Worm Meeting, 2011]

The cilia of C. elegans' amphid channel neurons can be divided into a middle and distal segment. Anterograde intraflagellar transport (IFT) in these cilia is mediated by two kinesin-2 complexes, kinesin II and OSM-3. In the middle segment OSM-3 and kinesin II move together, and in the distal segments OSM-3 moves alone. The architecture of C. elegans' cilia suggests that cilia length and function can be dynamically regulated. We recently identified and characterized the C. elegans gene dyf-5. The dyf-5 gene encodes a conserved MAP kinase. Loss-of-function and overexpression studies showed an affect of DYF-5 on the length of the cilia, as well as morphology. In addition we found several effects on IFT, including uncoupling of the two kinesins. Both dyf-5 overexpression and loss-of-function result in a dye filling defect. To identify new proteins involved in the regulation of IFT we performed a screen for mutants that suppress the dyf-5(lf) or dyf-5XS Dyf phenotype. Thus far, we identified one mutant that suppresses dyf-5XS, but this inactivated the endogenous dyf-5 gene. The dyf-5(lf) suppressor screen is ongoing. To identify binding partners of DYF-5 we are performing pull-down/mass spec experiments with N- and C- terminally Bio-tagged DYF-5. We are currently awaiting the mass spec results. In addition, we are investigating the three mammalian homologues of dyf-5, MAK, MRK, and MOK. In our model system, cultured kidney epithelial cells (IMCD3), MRK and MOK are both expressed. In knockdown and overexpression experiments with MRK we see similar effects on cilia length as we did in C. elegans. However, we see no effects on the velocities of IFT particles in the IMCD3 cells after knockdown of MRK. Thus far we did not observe any effects of MOK on cilia length. We are currently performing pull-down/mass spec on MOK::Bio and MRK::Bio to identify binding partners.