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Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma.

Espelt MV, Estevez AY, Yin X, Strange K - J. Gen. Physiol. (2005)

Bottom Line: The function of the two enzymes is additive.In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation.PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

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+) 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 approximately 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 (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) 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+) 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-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta 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-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta 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.

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Isolated intestine preparation. (A) Schematic diagrams of worm digestive tract and isolated intestine. (B) Differential interference contrast (DIC) and fluorescence micrographs of an isolated intestine loaded with fluo-4 AM. Bar, 20 μm. (C) DIC and fluorescence confocal micrographs (panels a and b) of an isolated intestine loaded with fluo-4 AM. Panel c is a reconstruction of a series of cross sections. Location of the cross sections is shown by the white vertical line in panel b. Bars in panels b and c are 20 μm and 2 μm, respectively.
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fig1: Isolated intestine preparation. (A) Schematic diagrams of worm digestive tract and isolated intestine. (B) Differential interference contrast (DIC) and fluorescence micrographs of an isolated intestine loaded with fluo-4 AM. Bar, 20 μm. (C) DIC and fluorescence confocal micrographs (panels a and b) of an isolated intestine loaded with fluo-4 AM. Panel c is a reconstruction of a series of cross sections. Location of the cross sections is shown by the white vertical line in panel b. Bars in panels b and c are 20 μm and 2 μm, respectively.

Mentions: The C. elegans digestive tract consists of a pharynx, intestine, and rectum (Fig. 1 A). The pharynx is a muscular organ that pumps food into the pharyngeal lumen, grinds it up, and then moves it into the intestine. 20 epithelial cells with extensive apical microvilli form the main body of the intestine, which is ∼750 μm long in a full-grown adult worm. Intestinal epithelial cells secrete digestive enzymes, absorb nutrients, and store lipids, proteins, and carbohydrates (White, 1988; Leung et al., 1999; Ashrafi et al., 2003).


Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma.

Espelt MV, Estevez AY, Yin X, Strange K - J. Gen. Physiol. (2005)

Isolated intestine preparation. (A) Schematic diagrams of worm digestive tract and isolated intestine. (B) Differential interference contrast (DIC) and fluorescence micrographs of an isolated intestine loaded with fluo-4 AM. Bar, 20 μm. (C) DIC and fluorescence confocal micrographs (panels a and b) of an isolated intestine loaded with fluo-4 AM. Panel c is a reconstruction of a series of cross sections. Location of the cross sections is shown by the white vertical line in panel b. Bars in panels b and c are 20 μm and 2 μm, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266627&req=5

fig1: Isolated intestine preparation. (A) Schematic diagrams of worm digestive tract and isolated intestine. (B) Differential interference contrast (DIC) and fluorescence micrographs of an isolated intestine loaded with fluo-4 AM. Bar, 20 μm. (C) DIC and fluorescence confocal micrographs (panels a and b) of an isolated intestine loaded with fluo-4 AM. Panel c is a reconstruction of a series of cross sections. Location of the cross sections is shown by the white vertical line in panel b. Bars in panels b and c are 20 μm and 2 μm, respectively.
Mentions: The C. elegans digestive tract consists of a pharynx, intestine, and rectum (Fig. 1 A). The pharynx is a muscular organ that pumps food into the pharyngeal lumen, grinds it up, and then moves it into the intestine. 20 epithelial cells with extensive apical microvilli form the main body of the intestine, which is ∼750 μm long in a full-grown adult worm. Intestinal epithelial cells secrete digestive enzymes, absorb nutrients, and store lipids, proteins, and carbohydrates (White, 1988; Leung et al., 1999; Ashrafi et al., 2003).

Bottom Line: The function of the two enzymes is additive.In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation.PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

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+) 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 approximately 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 (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) 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+) 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-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta 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-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta 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.

Show MeSH
Related in: MedlinePlus