Analysis of diverse signal transduction pathways using the genetic model system Caenorhabditis elegans

Signal transduction allows cells to respond to signals from their environment and is therefore important for most biological processes. The binding of an extracellular signalling molecule to a cell-surface receptor is the first step in most signal transduction pathways. Cell-surface receptors transduce the extracellular signals by generating a cascade of intracellular signals that alter the behaviour of the cell. The proteins involved in signal transduction include, besides the receptors, GTP-binding proteins, protein kinases and ion channels, and these proteins are conserved between multicellular organisms. In this study, we used the nematode Caenorhabditis elegans, a simple and well-described organism that is very well suited to perform genetic experiments, to analyse conserved signal transduction pathways. Chapter two of this thesis describes the genetic analysis of an important signaltransducing molecule, the adenylyl cyclase SGS-1. Adenylyl cyclases act downstream of certain heterotrimeric G proteins and convert ATP into the second messenger cAMP. In C. elegans, SGS-1 can be activated by the homologue of mammalian Gas, GSA-1, since mutations in sgs-1 suppress the neuronal degeneration induced by expression of constitutively active GSA-1 from its own or heat-shock promoter. We show that SGS-1 is essential for viability and that it is involved in behaviours as diverse as pharyngeal pumping and locomotion. To regulate this latter behaviour SGS-1 needs to be activated by GSA-1. In chapter three, nxf-1, another suppressor of the neuronal degeneration induced by expression of constitutively active GSA-1 from a heat-shock promoter, is characterized. nxf-1 encodes a nuclear export factor that is involved in transport of mRNA out of the nucleus. Mutations in nxf-1 also suppress other heat-shock promoter-induced phenotypes, indicating that this locus is not a specific downstream target of Gas, but rather a general suppressor of heat-shock promoter-induced phenotypes. We postulate that the mutations in nxf-1 make the protein inactive during heat-shock, resulting in reduced transport of the activated Gas and other mRNAs out of the nucleus and thus the absence of the activated Gas phenotype after heat-shock. Chapter four reports the analysis of the C. elegans homologue of Ga12/13, GPA- 12. Loss of GPA-12 does not result in any obvious defect in development or behaviour. However, overexpression of constitutively active GPA-12 from its own or heat-shock promoter results in a developmental growth arrest that is caused by a feeding defect. Mutations in tpa-1, which encodes two PKC isoforms, suppress the developmental growth arrest induced by activated GPA-12, indicating that TPA-1 acts downstream of GPA-12. Activation of TPA-1 by the tumour-promoting phorbol ester TPA (or PMA) results in an identical developmental growth arrest, showing that activated GPA-12 and PMA use the same PKC signalling pathway. In chapter five of this thesis, the DYRK family of protein kinases is described. A human DYRK kinase is implicated in the cognitive defects caused by trisomy of chromosome 21, and we show that in Caenorhabditis elegans overexpression of a DYRK family member, mbk-1, results in signal transduction defects in olfactory neurons. mbk-1 knockout animals do not show any obvious defects. Loss of hpk-1, another member of the DYRK family in C. elegans, does also not result in any obvious phenotype. However, a third member, mbk-2, is essential for viability

Hyperactivation of the G12-Mediated Signaling Pathway in Caenorhabditis elegans Induces a Developmental Growth Arrest via Protein Kinase C

AbstractThe G12 type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells [1–5]. However, little is known about the molecular nature of the components that act in the G12-signaling pathway in an organism. We characterized a C. elegans Gα subunit gene, gpa-12, which is a homolog of mammalian G12/G13α, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G12QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G12 participates, we screened for suppressors of the G12QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCθ/δ. TPA-1 mediates the action of the tumor promoter PMA [6]. Expression of G12QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G12 signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G12 proteins

Comparison of Different RNAi Experiments of Chromosome I Using Wild-Type Bristol N2 and <i>rrf-3</i>

<p>Differences between different laboratories or investigators and between experiments done within the same laboratory and by the same investigators are observed. Ovals represent the amount of bacterial clones that gave an RNAi phenotype in an experiment. Areas that overlap represent clones for which in both experiments an RNAi phenotype was detected. Differences and overlap between an RNAi experiment done with the <i>rrf-3</i> mutant strain and the data obtained by <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000012#pbio-0000012-Fraser1" target="_blank">Fraser et al. (2000</a>) done with the standard laboratory strain, Bristol N2 (A); N2 and <i>rrf-3</i> tested at the same time within our laboratory (B); experiments done with N2 in two different laboratories: this study (‘NL') and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000012#pbio-0000012-Fraser1" target="_blank">Fraser et al. (2000</a>) (C); two experiments done with the same strain, <i>rrf-3</i>, within our laboratory (D).</p