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

C. elegans rrf-1 Mutations Maintain RNAi Efficiency in the Soma in Addition to the Germline

“…In addition, we found that RNAi of two endogenous targets, elt-2 and pept-1 , induced robust phenotypes in two different rrf-1 mutants. While elt-2 RNAi seemed to play an exclusively intestinal-specific role, pept-1 RNAi induced effects in both intestinally and hypodermally rescued rde-1 strains [12], [13]. This result may indicate that pept-1 functions as an oligopeptide transporter not only in the intestine, but also in the hypodermis.…”

A novel kinase regulates dietary restriction‐mediated longevity in Caenorhabditis elegans

“…Expression was also noticed in some neurons but is excluded from the intestine. To find tissues where knocking down of mekk-3 is sufficient to extend lifespan, we used tissue-specific RNAi systems (Espelt et al ., 2005; Qadota et al ., 2007). Interestingly, we found that mekk-3 RNAi did not extend lifespan when specifically knocked down in the intestine, the major metabolic tissue in the worm.…”

Intestinal Insulin Signaling Encodes Two Different Molecular Mechanisms for the Shortened Longevity Induced by Graphene Oxide in Caenorhabditis elegans

“…To further examine the tissue-specific activity of other genes in the insulin signaling pathway in regulating GO toxicity, we performed intestine-specific RNAi on the VP303 strain33. In nematodes, we found that intestine-specific RNAi of the daf-2, age-1, akt-1 , or akt-2 gene resulted in prolonged lifespan, whereas intestine-specific RNAi of the daf-16 or daf-18 gene led to reduced lifespan (Fig.…”