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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 600 |
Oscillatory
Ca2+ Signaling in the Isolated Caenorhabditis elegans Intestine: Role of the Inositol-1,4,5-trisphosphate
Receptor and
Phospholipases C
and |
Maria
V. Espelt, Ana Y. Estevez, Xiaoyan Yin, and Kevin Strange |
Caenorhabditis
elegans, posterior body wall muscle contraction (pBoc), oscillatory Ca2signaling, intercellular
Ca2+ waves |
Isolated intestines were transferred
by pipette to a 35-mm Petri dish with a 14-
mm microwell (MatTek Corp.).
|
Inverted
Microscope |
worm |
epithelial-like |
intestinal
epithelium |
| 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 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- and PLC- homologues. Disruption of PLC- and PLC-
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- 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 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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