Oscillatory Ca2+ Signaling in the IsolatedCaenorhabditis elegansIntestine

Abstract

Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45–50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca²⁺ oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca²⁺ signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca²⁺ oscillations with a period of ∼50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca²⁺ wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP₃) receptor ITR-1 reduce pBoc and Ca²⁺ oscillation frequency and intercellular Ca²⁺ wave velocity. In contrast, gain-of-function mutations in the IP₃ binding and regulatory domains of ITR-1 have no effect on pBoc or Ca²⁺ oscillation frequency but dramatically increase the speed of the intercellular Ca²⁺ wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)–encoding genes demonstrated that pBoc and Ca²⁺ oscillations require the combined function of PLC-γ and PLC-β homologues. Disruption of PLC-γ and PLC-β activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca²⁺ oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-γ functions primarily to generate IP₃ that controls ITR-1 activity. In contrast, IP₃ generated by PLC-β appears to play little or no direct role in ITR-1 regulation. PLC-β may function instead to control PIP₂ levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca²⁺ signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca²⁺ oscillations and intercellular Ca²⁺ waves in nonexcitable cells.