Oscillatory Ca2 Signaling in the Isolated Caenorhabditis elegans Intestine: Role of the Inositol-1,4,5-trisphosphate Receptor and Phospholipases C ß and γ

MARIA V. ESPELT, ANA Y. ESTEVEZ, XIAOYAN YIN, AND KEVIN STRANGE

JOURNAL OF GENERAL PHYSIOLOGY

ROCKEFELLER UNIV PRESS

Año: 2005 vol. 126 p. 379 - 392

0022-1295

Defecation in the nematodo 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 Ca 2+ oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca 2+ signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca 2+ 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 Ca 2+ wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP3) receptor ITR-1 reduce pBoc and Ca 2+ oscillation frequency and intercellular Ca 2+  wave velocity. In contrast, gain-of-function mutations in the IP3 binding and regulatory domains of ITR-1 have no effect on pBoc or Ca 2+  oscillation frequency but dramatically increase the speed of the intercellular Ca 2+  wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)–encoding genes demonstrated that pBoc and Ca 2+  oscillations require the combined function of PLC- ã and PLC- â homologues. Disruption of PLC- ã and PLC- â activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca 2+ oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC- ã functions  rimarily to generate IP 3 that controls ITR-1 activity. In contrast, IP 3 generated by PLC- _ appears to play little or no direct role in ITR-1 regulation. PLC- â may function instead to control PIP 2 levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca 2+  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 Ca 2+  oscillations and intercellular Ca 2+  waves in nonexcitable cells.