G protein signalling in sensory behaviour
One of the most widely used signal transduction pathways in eukaryotic cells employs G protein coupled receptors (GPCR) and heterotrimeric G proteins. We use the nematode Caenorhabditis elegans to analyse G protein mediated signal transduction in vivo. We focus our research on the signalling pathways and cellular circuits that govern the response of C. elegans to salts and on the pathways that allow structural plasticity of cilia, small cellular extensions that function as sensory organelles.

G protein signal transduction
Signalling through serpentine receptors and heterotrimeric G proteins is one of the main means of transducing extracellular signals in the cell. Binding of a ligand to a specific GPCR results in the activation of a G protein complex. In the inactive state the G protein complex consists of a Gα (GDP-bound), Gβ and Gγ subunit. Upon activation GDP is exchanged for GTP, which results in dissociation of the GTP bound α subunit and the βγ dimer. Both entities can activate effector molecules, such as adenylate or guanylate cyclases, cGMP phosphodiesterases, phospholipase C, phosphoinositide 3-kinases, ion channels selective for K+, Ca2+ and Na+, tyrosine kinases and MAP kinases. Gα subunits have an intrinsic GTPase activity, which in time results in hydrolysis of the GTP to GDP and reassociation of the inactive heterotrimeric complex. Accessory proteins, termed regulators of G protein signalling (RGS) proteins, regulate this GTPase activity and the association of the α- with the βγ-subunits. Despite the extensive characterisation of these signalling cascades, many questions remain, such as how different signals are integrated, segregated and insulated from another within a cell.

The nematode C. elegans
Previous studies have made it clear that many molecular and cellular processes are often conserved between humans and simple organisms such as worms. This enables us to use genetic analyses in the nematode Caenorhabditis elegans to analyse the fundamental mechanisms of G protein signal transduction. C. elegans has been a very successful model system for the in vivo analysis of many processes. Despite its small body size, simple body plan and short life cycle C. elegans still shows many behaviours that make life interesting, such as directed movements, feeding, responses to various environmental cues and sexual reproduction. 

The movie shows a typical view of the behaviour of C. elegans on a culture plate. We see adult animals and larvae crawling through their food, bacteria. Scientifically, the simplicity of C. elegans offers many advantages. Furthermore, the animals show hermaphroditic reproduction, are accessible to classical genetics, the complete cell lineage and neural wiring diagram have been determined and we can use many molecular techniques for our functional analyses (for further information on C. elegans see http://elegans.swmed.edu/, http://www.wormbase.org/, or http://www.wormbook.org/).

G proteins in C. elegans
The genome of C. elegans contains 21 Gα, 2 Gβ and 2 Gγ genes. Functionally, these proteins can be divided into 2 groups, G protein subunits that regulate muscle and neuron activity in general and sensory specific G proteins. We focus on the latter group of G proteins. C. elegans responds to a wide range of chemicals, including salts, amino acids, bitter and sweet compounds, water-soluble repellents and volatile chemicals. The nematode uses 11 bilateral symmetric pairs of chemosensory neurons, the amphid neurons, to detect compounds in its environment (see figure).

The function of each of these neurons has been determined. With just 11 pairs of chemosensory cells to detect at least 100 water-soluble attractants and repellents and at least 60 odorants, C. elegans seems significantly restricted in its possibilities to discriminate between chemical compounds. Still, the animal can discriminate between Na+ and Cl-, sensed by the ASE neurons, and it can discriminate between various odorants sensed by the AWC neurons. The functional analysis of all G protein genes in C. elegans showed that each amphid neuron expresses multiple Gα subunits, suggesting extensive modulation of signalling. The analysis of all olfactory Gα subunits confirmed this: We found that olfactory signalling is mediated by 1 main signal (ODR-3), which is modulated by 2 to 5 Gα subunits, with either stimulatory or inhibitory functions. These results have shown that sensory signalling in C. elegans offers an excellent model system to analyse G protein signal transduction and, particularly, determine how several different signalling pathways can function within one cell, enabling cross-talk and modulation while preserving specificity.

Our analysis of sensory G proteins signalling has lead us into the following fields (see also lines of investigation):

1. Salt taste. We identified several new genes that play a role in the detection of NaCl in C. elegans. The functions of these 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.

2. Behavioural plasticity. After prolonged exposure of C. elegans to normally attractive concentrations of NaCl in the absence of food, the animals learn to avoid NaCl. We call this response gustatory plasticity. Using a candidate gene approach we have identified many genes that play a role in gustatory plasticity, including sensory signalling molecules, neurotransmitter systems and genes that probably function in the integration of the different signals. We use this rather simple behavioural assay, molecular genetic tools and Ca2+ imaging to unravel the cellular circuitry and the molecular mechanisms that regulate this behaviour in C. elegans (Jansen et al., 2002, EMBO J; Hukema et al., 2006, EMBO J; Hukema et al., 2008, Learn Mem).

3. 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 the cilia perform very important functions that 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. The development, maintenance and function of the cilia depend on a specialised transport system, called intraflagellar transport (IFT). We have recently identified a novel mechanism that regulates transport in the cilia of C. elegans in response to environmental cues. In addition, we have identified a protein that regulates the length of the cilia of C. elegans. We use molecular genetic techniques and in vivo imaging of IFT to unravel the molecular mechanisms of these processes (Burghoorn et al., 2007, PNAS; Burghoorn et al., 2010, JCS).

4. Modulation of longevity by sensory signalling. C. elegans is an ideal model organism to identify genes that regulate longevity. Various cues that regulate longevity in C. elegans have been described, including sensory cues. We identified several Gα subunits that function in the sensory neurons and are involved in regulating longevity. We expect that these studies will ultimately lead to the identification of the sensory cues that modulate longevity in C. elegans (Lans et al., 2007, Dev Biol).

5. Specificity of signal transduction in the olfactory system. C. elegans uses only four pairs of sensory neurons to detect many attractive olfactory cues in its environment. To this end, each olfactory neuron expresses many different receptors and several Gα subunits. We found that olfactory signalling is mediated by at least three Gα subunits, the stimulatory Gα subunits ODR-3 and GPA-3 and the inhibitory Gα subunits GPA-2 or GPA-5. Using genetic analyses, we aim to identify additional molecules from these G protein signalling pathways and identify the signals that determine specificity of signal transduction (Lans et al., 2004 & 2006, Genetics).