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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: 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.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.

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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Effect of egl-8(n488) loss-of-function mutation and plc-3(RNAi) on pBoc rhythm. (A and B) Individual pBoc cycles in egl-8(n488) and plc-3(RNAi) worms. (C and D) pBoc cycles in individual egl-8(n488) worms fed plc-3 dsRNA.
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fig6: Effect of egl-8(n488) loss-of-function mutation and plc-3(RNAi) on pBoc rhythm. (A and B) Individual pBoc cycles in egl-8(n488) and plc-3(RNAi) worms. (C and D) pBoc cycles in individual egl-8(n488) worms fed plc-3 dsRNA.

Mentions: Loss-of-function mutations in egl-8 have been shown previously to disrupt the defecation cycle in C. elegans (Lackner et al., 1999). Fig. 6 shows the effect of the egl-8 loss-of-function allele, n488, and plc-3(RNAi) on pBoc cycles in individual worms. In addition to prolonging cycle time, loss of EGL-8 and PLC-3 activity induces striking pBoc arrhythmia. Mean ± SEM pBoc coefficients of variance for egl-8(n488) and plc-3(RNAi) worms were 38 ± 7% (n = 6) and 22 ± 3% (n = 9). These values were significantly (P < 0.01) different from the mean ± SEM pBoc CV of 6 ± 1% (n = 10) observed in control animals. Worms carrying the plc-3 deletion allele plc-3(tm1340) also exhibited a highly arrhythmic pBoc cycle (mean ± SEM CV = 53 ± 8%; n = 11).


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)

Effect of egl-8(n488) loss-of-function mutation and plc-3(RNAi) on pBoc rhythm. (A and B) Individual pBoc cycles in egl-8(n488) and plc-3(RNAi) worms. (C and D) pBoc cycles in individual egl-8(n488) worms fed plc-3 dsRNA.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Effect of egl-8(n488) loss-of-function mutation and plc-3(RNAi) on pBoc rhythm. (A and B) Individual pBoc cycles in egl-8(n488) and plc-3(RNAi) worms. (C and D) pBoc cycles in individual egl-8(n488) worms fed plc-3 dsRNA.
Mentions: Loss-of-function mutations in egl-8 have been shown previously to disrupt the defecation cycle in C. elegans (Lackner et al., 1999). Fig. 6 shows the effect of the egl-8 loss-of-function allele, n488, and plc-3(RNAi) on pBoc cycles in individual worms. In addition to prolonging cycle time, loss of EGL-8 and PLC-3 activity induces striking pBoc arrhythmia. Mean ± SEM pBoc coefficients of variance for egl-8(n488) and plc-3(RNAi) worms were 38 ± 7% (n = 6) and 22 ± 3% (n = 9). These values were significantly (P < 0.01) different from the mean ± SEM pBoc CV of 6 ± 1% (n = 10) observed in control animals. Worms carrying the plc-3 deletion allele plc-3(tm1340) also exhibited a highly arrhythmic pBoc cycle (mean ± SEM CV = 53 ± 8%; n = 11).

Bottom Line: 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.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.

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