Salt taste
Salt taste is essential for salt and water homeostasis in all animals and salts are probably cues for food. Defects in salt taste can lead to high blood pressure and malnutrition. Despite its importance, relatively little is known about the molecular mechanisms of salt taste. Salts are thought to be primarily sensed by epithelial Na+ channels (ENaC) and perhaps transient receptor potential (TRP) channels. Not much is known about possible downstream signalling molecules. We aim to unravel the molecular mechanisms of salt taste. A better understanding of these mechanisms would allow the development of new drugs, such as taste enhancers or alternative tastants, to help patients with taste deficits.

We use genetic approaches to identify additional genes that function in NaCl perception in the nematode C. elegans. Previous studies have identified the most important sensory neurons of C. elegans involved in the detection of NaCl. Also several forward genetic screens have been performed to identify genes involved in this process. However, these screens identified only few genes.

Using a candidate gene approach, we have recently identified seven new genes that play a role in NaCl detection. In addition, we found that C. elegans uses two pathways to detect NaCl. This latter finding probably explains that previous screens did not identify many NaCl detection molecules, since mutants that have lost only one of the two NaCl detection pathways still respond to NaCl. Only when both pathways are inactivated the animals lose NaCl taste. We use this knowledge to perform synthetic genetic screens to identify proteins involved in NaCl chemotaxis. The functions of newly identified genes are subsequently studied using gain- and loss-of-function mutants and analysis of expression patterns. Mutant phenotypes are characterized using a variety of behavioural assays, cell specific rescue, in vivo Ca2+ imaging and genetic epistasis analysis.

Movie of the assay we use to quantify the response of C. elegans to NaCl. Adult animals are put in the middle of an assay plate. The assay plate is divided in 4 quadrants by plastic spacers. Two quadrants are filled with buffered agar containing a certain NaCl concentration (in this case 25 mM), the other two quadrants only contain buffered agar. Just before the assay the ridges between the quadrants are covered with a thin layer of buffered agar to allow the worms to move across the plate. These wild type animals rapidly move to the quadrants that contain NaCl (upper right and lower left).

We expect that our studies will increase our understanding of the molecular mechanisms of salt taste. Since many processes have been conserved in evolution and most results obtained in the worm can be extrapolated to mammals, we expect that this knowledge will also be instrumental to further our understanding of salt taste in mammals.

Gustatory plasticity
Wild type C. elegans are attracted to low salt concentrations (0.1 - 200 mM NaCl), but avoid higher NaCl concentrations. The balance between NaCl attraction and avoidance is not fixed, but depends on experience: after prolonged exposure to 100 mM NaCl, in the absence of food, or in the presence of a repellent, the behaviour of the animals changes drastically, resulting in avoidance of all NaCl concentrations. In this response, called gustatory plasticity, the animals associate the presence of NaCl with the absence of food. We use this rather simple assay to unravel the cellular circuitry and the molecular mechanisms that regulate this form of behavioural plasticity in C. elegans.

In a candidate gene approach we have identified 78 genes involved in gustatory plasticity, including G protein signalling genes that function in sensory neurons, cGMP signalling genes in neurons exposed to the body fluid, genes involved in serotonin, dopamine and glutamate neurotransmission and genes that probably function in the integration of the different signals. In addition, we identified four pairs of sensory neurons that play a role in gustatory plasticity, the ASE, ADF, ASH and ASI neurons.

Previous studies have shown that the ASE and ASH neurons primarily mediate attraction and avoidance, respectively. Using behavioural analyses, genetics and in vivo Ca2+ imaging we found that in naïve animals, the ASE neurons respond to NaCl concentrations from 1 mM up to 1 M, while the ASH neurons respond to NaCl concentrations above 100 mM. This suggests that in the naïve animal the response to NaCl is determined by a balance between attraction (via ASE) and avoidance (via ASH). It is unclear where these signals are integrated and what mechanism sets the balance.

Movie of the imaging approach to visualize the Ca2+ response of individual neurons in C. elegans. A transgenic animal, expressing a cameleon construct in a specific sensory neuron is fixed in a fluorescence microscope and exposed to a defined sensory cue. When the Ca2+ concentration in the cell increases as a result of the detection of the cue, the fluorescence emission of the cameleon construct will change from CFP to YFP (in the movie from blue to green). Focus on the small cell in the middle of the movie, the fluorescence at the bottom is GFP emission of the transgene marker.

However, after pre-exposure to NaCl in the absence of food the response of the ASE neurons to low NaCl concentrations is decreased, while the response in the ASH neurons is increased. Thus, prolonged exposure in the absence of food results in desensitisation of the ASE neurons, and sensitisation of the ASH neurons. We are currently testing various mutants to find out which signals are required for these (de)sensitization events.

Since this is a relatively simple behaviour we expect that our multidisciplinary approach using classical genetics, behavioural assays, cell specific rescue, laser ablation and in vivo Ca2+ imaging, will ultimately allow us to explain the behaviour of the animals on the molecular and cellular level.

Publications

• Jansen, G., Weinkove, D. & Plasterk, R.H.A. (2002) The G-protein gamma subunit gpc-1 of the nematode C. elegans is involved in taste adaptation.  EMBO J. 21, 986-994.
• Hukema, R.K., Rademakers, S. & Jansen, G. (2008) Gustatory plasticity in C. elegans involves integration of negative cues and NaCl taste mediated by dopamine, serotonin, and glutamate. Learn. Mem. 15, 829-836.
• Hukema, R.K., Rademakers, S., Dekkers, M.P.J., Burghoorn, J. & Jansen, G. (2006) Antagonistic sensory cues generate gustatory plasticity in Caenorhabditis elegans. EMBO J. 25, 312-322.

Regulation of intraflagellar transport.
Almost all mammalian cells have cilia, small protrusions from the cell that are used for motility or to sense cues from the environment. It is becoming clear that cilia are essential for the function, differentiation or localisation of cells. This is most apparent from the fact that many human diseases are caused by defects in ciliary proteins, collectively called ciliopathies.

The development, maintenance and function of cilia require an evolutionarily conserved transport system, called intraflagellar transport (IFT). Several recent studies suggest that IFT is regulated, allowing modulation of cilia length and of signalling pathways in cilia. Results obtained in C. elegans suggest that this regulation may involve the use of different kinesin motors. The cilia of a subset of C. elegans’ sensory neurons consist of two segments, a middle segment and a distal segment. In the middle segments IFT is mediated by two kinesin-2 motor complexes together, heterotrimeric kinesin II and homodimeric OSM-3 kinesin, moving at 0.7 μm/s. In the distal segment transport is mediated by OSM-3 alone, at 1.2 μm/s. We have identified two genes that play a role in the coordination of transport by the two kinesins: the MAP kinase, dyf-5, and the sensory Gα protein gpa-3.

Four of C. elegans’ cilia visualized using GPA-15::GFP, expressed in the ADL, ASH and ASK neurons in the head.

dyf-5: dyf-5 loss-of-function (lf) animals have longer cilia, while in dyf-5 overexpressing animals the cilia are shorter. Genetic analyses and live imaging of IFT proteins have shown that in dyf-5(lf) animals kinesin II can enter the distal segments and that OSM-3 is not attached to IFT particles and moves at a reduced speed. Mammals have three DYF-5 homologues, MAK, MOK and MRK. We are characterizing the functions of these proteins in the cilia of cultured mammalian cells, using life imaging of GFP tagged IFT (motor) proteins and MAK, MOK and MRK knockdown or overexpression.

gpa-3: Animals that carry a dominant active gpa-3, gpa-3QL, show a defect in the uptake of dyes in ciliated sensory neurons. The cilia of gpa-3QL animals show severe morphological defects, and are shorter. gpa-3(lf) animals show no morphological abnormalities. Interestingly, both gpa-3(lf) and gpa-3QL affect the speed of the anterograde IFT motors: kinesin II speed is reduced and OSM-3 kinesin speed is increased in the middle segments of the cilia. However, the speeds of other IFT proteins (complex A and B) are not affected. This suggests that kinesin II and OSM-3 are partially uncoupled, but both motors transport complex A/B proteins. Previous studies have shown that GPA-3 functions in the developmental switch that regulates dauer formation. Exposure to dauer pheromone affects cilia length and affects OSM-3 and kinesin II speeds, similar to mutation of gpa-3. We propose that environmental cues mediated by GPA-3 regulate cilia length by regulating the constitution of IFT particles. Since both gpa-3(lf) and gpa-3QL affect the speeds of the IFT motors, but only gpa-3QL affects cilia length we speculate that these mutations have opposite effects on the constitution of IFT particles. To address this issue, we are trying to measure speeds of cargo molecules.

To identify additional genes that regulate IFT, we are performing genetic screens for suppressors of gpa-3QL and dyf-5 loss- or gain-of-function. We identified six gpa-3QL suppressor mutants, called sql (suppressor of gpa-3QL). sql-1 encodes the homologue of the mammalian Golgi protein GMAP-210. sql-1(lf) has a very similar effect on the speeds of OSM-3 and kinesin II as mutation of gpa-3. In addition, we will try to identify proteins present in a complex with C. elegans DYF-5 and/or its mammalian homologues using biotin pull down and mass spectrometry. The functions of newly identified proteins will be characterized in C. elegans and cultured mammalian kidney cells using reverse genetics, EM and life imaging of GFP tagged proteins.

Publications

• Burghoorn, J., Dekkers, M.P.J., Rademakers, S., de Jong, T., Willemsen, R. Swoboda, P., & Jansen, G. (2010) Environmental cues and G protein signalling modulate the coordination of intraflagellar transport kinesin motor proteins in C. elegans. J. Cell Sci. 123, 2077-2084.
• Burghoorn, J., Dekkers, M.P.J., Rademakers, S., de Jong, T., Willemsen, R. & Jansen, G. (2007) Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of C. elegans. Proc. Natl. Acad. Sci. USA 104, 7157-7162.