Regulated Disruption of Inositol 1,4,5-Trisphosphate Signaling inCaenorhabditis elegansReveals New Functions in Feeding and Embryogenesis

Abstract

Inositol 1,4,5-trisphosphate (IP 3 ) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP 3 is to regulate release of Ca 2ϩ from intracellular stores through IP 3 receptors (IP 3 Rs). We describe a system for the transient disruption of IP 3 signaling in the model organism Caenorhabditis elegans. The IP 3 binding domain of the C. elegans IP 3 R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP 3 binding domain acting as an IP 3 "sponge." Disruption of IP 3 signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP 3 -mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP 3 signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP 3 R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP 3 signaling through the IP 3 R and thus determine that IP 3 -mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation. Inositol 1,4, ) is an important and widely used second messenger in animal cells. IP 3 is produced by the hydrolysis of phosphatidylinositol 4,5-bisphosphate after the activation of phospholipase C (PLC). The best understood routes of PLC activation are through the stimulation of G protein-coupled receptors or tyrosine kinase-linked receptors at the cell surface, although the presence of additional isoforms of PLC suggests other possible routes (Rhee and Bae, 1997;Katan, 1998). The only known action of IP 3 is to induce Ca 2ϩ release from intracellular stores through activation of IP 3 receptors (IP 3 Rs) (Berridge, 1993). This pathway and the Ca 2ϩ signals derived from it are central to a wide range of cellular responses (Berridge, 1993(Berridge, , 1997Clapham 1995). INTRODUCTIONTwo challenges in the study of this signaling network are to unravel the different functions played by IP 3 signaling in animals and to develop methods for manipulating these processes and thus advance our understanding of how the specificity of IP 3 signaling is achieved. Recent genetic approaches have provided new insights into IP 3 function in animals. The central role played by IP 3 Rs in a wide range of cellular responses is reflected by the high level of lethality resulting from gene knockout observed in both Drosophila (Acharya et al., 1997;Venkatesh and Hasan, 1997) and mice (Matsumoto et al., 1996). Nevertheless, careful analysis has enabled, for example, the functions of IP 3 Rs in cerebellum in mice (Inoue et al., 1998) and phototransduction in Drosophila to be studied (Acharya et al., 1997;Raghu et al., 2000). Caenorhabditis elegans is also proving to be a powerful system for understanding IP 3 signaling function. IP 3 Rs i...

Inositol 1,4,5-Trisphosphate Signaling Regulates Mating Behavior inCaenorhabditis elegansMales

“…For RNAi of itr-1 we used pNG030 (Walker et al, 2002), which carries a 1-kb region of the itr-1 cDNA. For RNAi of PLC genes we constructed derivatives of pPD129.36, carrying ϳ1 kb of the cDNA of each PLC gene; these are pHAB301 (egl-8), pHAB302 (plc-1) pHAB303 (plc-3) pHAB304 (plc-4), pHAB305, (plc-2), and pHAB306 (pll-1).…”

“…To this end, we investigated whether itr-1 (sy290), a gain-of-function allele (Clandinin et al, 1998), could rescue the sterility resulting from egl-8 RNAi. itr-1 (sy290) has a mutation, R582Q, in the IP 3 binding site (Clandinin et al, 1998), which results in a twofold increase in IP 3 binding affinity (Walker et al, 2002). In the strain PS2582 (Clandinin et al, 1998) the itr-1 (sy290) mutation is quite closely linked to an unc-24 (e138) mutation, resulting in a locomotion (weak kinker) phenotype, which would presumably itself interfere with mating efficiency.…”

“…Analysis of itr-1 loss-of-function mutants led to the identification of a central role in the regulation of the defecation motor program (Dal Santo et al, 1999). We have demonstrated, using itr-1 mutants, RNA-mediated interference (RNAi) and an inducible dominant-negative construct (IP 3 sponge), that IP 3 R and IP 3 signaling are involved in the up-regulation of pharyngeal pumping in response to food and in multiple stages of embryogenesis (Walker et al, 2002). Ca 2ϩ release is a key integratory intracellular signaling event and is central to a wide range of cellular responses (Berridge, 1993(Berridge, , 1997Clapham, 1995).…”

ANK Repeat-Domain of SHN-1 Is Indispensable for In Vivo SHN-1 Function in C. elegans

“…shn-1 regulates pharyngeal pumping with itr-1 itr-1 has been also involved in pharyngeal pumping in C. elegans (Walker et al, 2002) and shn-1 is known to be expressed in pharynx . Thus, we analyzed whether shn-1 is also involved in regulation of pharyngeal pumping with itr-1.…”

Regulation of maternal phospholipid composition and IP3-dependent embryonic membrane dynamics by a specific fatty acid metabolic event in C. elegans

“…Although a link between the IP 3 pathway and embryonic exocytosis has not been thoroughly investigated in C. elegans, an observation of egg-filling multinucleated embryos resembling acs-1(RNAi) C17ISO embryos was reported in mutants with decreased IP 3 signaling (Walker et al 2002). In addition, disrupting the function of RAB-11 GTPase (a major regulator of Ca 2+ -induced exocytosis located on the membrane of excretory vesicles) or SYN-4 (calcium-sensing target SNARE located on plasma membranes) was shown to mimic the blockage of chondroitin biosynthesis that also results in the egg-filling phenotype, failed cytokinesis, and multinucleated blastomeres (Jantsch-Plunger and Glotzer 1999; Sato et al 2008).…”