C. elegans is capable of sensing various environmental stimuli and modifying its behavioral responses. We are particularly interested in molecularly dissecting temperature-evoked behavior (thermotaxis), because of its experience-dependent plasticity. In order to identify molecular components that regulate thermotaxis, we have isolated many thermotaxis-defective mutants (see abstract by Okumura et al. ). Of these,
ttx-4(
nj1,
nj3,
nj4) mutants showed thermophilic phenotype. In addition,
ttx-4 mutants were found to be defective in chemotaxis to ASE-sensed NaCl, AWA- and AWC-sensed odorants, and in ASH-mediated osmotic avoidance. We found that
ttx-4 gene encodes a novel Protein Kinase C (nPKC)-epsilon homologue. nPKCs consist of a regulatory domain in the amino terminus and a catalytic domain in the carboxyl terminus. In the regulatory domain, there are two conserved regions, C2-like region and C1 region. Unlike conventional PKCs (cPKCs), nPKC activation is Ca 2+ -independent, but is known to be DAG-dependent due to the presence of C1 region. However, in vivo substrates phosphorylated by nPKC are not well understood (see abstract by Kimura et al. ).
ttx-4(
nj1) and
ttx-4(
nj4) mutants have missense mutations in kinase domain, and the
ttx-4(
nj3) mutant has nonsense mutation in C1 domain.
ttx-4 mutants are all genetically recessive and
ttx-4(
nj1) mutant was rescued by a low concentration of wild type
ttx-4 transgene, suggesting that
ttx-4 mutations are loss of function mutations. The functional GFP-tagged TTX-4 protein was expressed in many sensory and interneurons, and some motorneurons. These neurons include AFD thermosensory neuron, AIY and AIZ interneurons, all of which are essential for thermotaxis. TTX-4 protein was also expressed in AWA, AWC and ASH neurons. Cell-specific expression of
ttx-4 cDNA in AFD or AWC of
ttx-4 mutants rescued abnormal thermotactic or chemotactic behavior, respectively, implicating that the function of TTX-4 nPKC in sensory neurons is sufficient for normal behavioral responses to sensory stimuli. Previous laser-killing experiments and genetic studies demonstrated that animals whose AFD neurons are inactive or barely active show cryophilic phenotype. As TTX-4 functions in AFD neurons and loss of function mutations of
ttx-4 cause thermophilic phenotype, it is likely that TTX-4 functions as negative regulator of temperature signaling in AFD neurons. This negative regulation resembles the proposed role of TAX-6 calcineurin in AFD (see abstract by Kuhara et al. ). To explore the functional relationship in temperature signal transduction between TTX-4 and TAX-6, if any, we are in the process of analyzing thermotactic phenotypes of
ttx-4(lf) ; Ex[TAX-6(gf)] and
tax-6(lf) ; Ex[TTX-4(gf)] animals.
tax-6 mutants show partial olfactory responses, which turned out to be caused by hyper-sensitivity to olfactory adaptation. By contrast,
ttx-4 mutants are completely defective in chemotaxis to odorants. To investigate whether these severe olfactory defects in
ttx-4 mutants are caused by hyper-sensitivity in olfactory adaptation as in
tax-6 mutants, we are analyzing primary olfactory responses to AWC-sensed odorants in
osm-9(
ky10);
ttx-4(
nj1) mutants, where
osm-9 mutation is expected to suppress olfactory adaptation to isoamyl alcohol and butanone.