Ana Y. Estevez

A Primary Culture System for Functional Analysis of C. elegans Neurons and Muscle Cells

C. elegans has provided important insights into neuromuscular system function and development. However, the animals small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.

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

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 Ca2+ oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca2+ signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca2+ 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 Ca2+ wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP3) receptor ITR-1 reduce pBoc and Ca2+ oscillation frequency and intercellular Ca2+ wave velocity. In contrast, gain-of-function mutations in the IP3 binding and regulatory domains of ITR-1 have no effect on pBoc or Ca2+ oscillation frequency but dramatically increase the speed of the intercellular Ca2+ wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)–encoding genes demonstrated that pBoc and Ca2+ oscillations require the combined function of PLC-γ and PLC-β homologues. Disruption of PLC-γ and PLC-β activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca2+ oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-γ functions primarily to generate IP3 that controls ITR-1 activity. In contrast, IP3 generated by PLC-β appears to play little or no direct role in ITR-1 regulation. PLC-β may function instead to control PIP2 levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca2+ 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 Ca2+ oscillations and intercellular Ca2+ waves in nonexcitable cells.

Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells.

The nematode Caenorhabditis elegans offers significant experimental advantages for defining the genetic basis of diverse biological processes. Genetic and physiological analyses have demonstrated that inositol-1,4,5-trisphosphate (IP3)–dependent Ca2+ oscillations in intestinal epithelial cells play a central role in regulating the nematode defecation cycle, an ultradian rhythm with a periodicity of 45–50 s. Patch clamp studies combined with behavioral assays and forward and reverse genetic screening would provide a powerful approach for defining the molecular details of oscillatory Ca2+ signaling. However, electrophysiological characterization of the intestinal epithelium has not been possible because of its relative inaccessibility. We developed primary intestinal epithelial cell cultures that circumvent this problem. Intestinal cells express two highly Ca2+-selective, voltage-independent conductances. One conductance, IORCa, is constitutively active, exhibits strong outward rectification, is 60–70-fold more selective for Ca2+ than Na+, is inhibited by intracellular Mg2+ with a K1/2 of 692 μM, and is insensitive to Ca2+ store depletion. Inhibition of IORCa with high intracellular Mg2+ concentrations revealed the presence of a small amplitude conductance that was activated by passive depletion of intracellular Ca2+ stores. Active depletion of Ca2+ stores with IP3 or ionomycin increased the rate of current activation ∼8- and ∼22-fold compared with passive store depletion. The store-operated conductance, ISOC, exhibits strong inward rectification, and the channel is highly selective for Ca2+ over monovalent cations with a divalent cation selectivity sequence of Ca2+ > Ba2+ ≈ Sr2+. Reversal potentials for ISOC could not be detected accurately between 0 and +80 mV, suggesting that PCa/PNa of the channel may exceed 1,000:1. Lanthanum, SKF 96365, and 2-APB inhibit both IORCa and ISOC reversibly. Our studies provide the first detailed electrophysiological characterization of voltage-independent Ca2+ conductances in C. elegans and form the foundation for ongoing genetic and molecular studies aimed at identifying the genes that encode the intestinal cell channels, for defining mechanisms of channel regulation and for defining their roles in oscillatory Ca2+ signaling.

Function of a STIM1 Homologue in C. elegans: Evidence that Store-operated Ca2+ Entry Is Not Essential for Oscillatory Ca2+ Signaling and ER Ca2+ Homeostasis

1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca2+ entry (SOCE) is activated during endoplasmic reticulum (ER) Ca2+ store depletion and is believed to be an essential and ubiquitous component of Ca2+ signaling pathways. SOCE is thought to function to refill Ca2+ stores and modulate Ca2+ signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca2+ sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530–amino acid protein with ∼21% sequence identity to human STIM1. Green fluorescent protein (GFP)–tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1∷GFP expression, suppresses the EF-hand mutation–induced pBoc arrhythmia, and inhibits intestinal store-operated Ca2+ (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca2+ signaling, in wild type and IP3 signaling mutant worms, and has no effect on intestinal Ca2+ oscillations and waves. Depletion of intestinal Ca2+ stores by RNAi knockdown of the ER Ca2+ pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca2+ signaling processes and for maintenance of store Ca2+ levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

Highly Ca2+-selective TRPM channels regulate IP3-dependent oscillatory Ca2+ signaling in the C. elegans intestine.

Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45–50 s and mediates defecation. pBoc is controlled by inositol-1,4,5-trisphosphate (IP3)–dependent Ca2+ oscillations in the intestine. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express an outwardly rectifying Ca2+ (ORCa) current with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL-l and GON-2 function inhibits IORCa ∼70% and ∼90%, respectively. IORCa is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating IORCa are interdependent. IORCa, IGON-2, and IGTL-1 have nearly identical biophysical properties. Importantly, all three channels are at least 60-fold more permeable to Ca2+ than Na+. Epistasis analysis suggests that GON-2 and GTL-1 function in the IP3 signaling pathway to regulate intestinal Ca2+ oscillations. We postulate that GON-2 and GTL-1 form heteromeric ORCa channels that mediate selective Ca2+ influx and function to regulate IP3 receptor activity and possibly to refill ER Ca2+ stores.

Calcium feedback mechanisms regulate oscillatory activity of a TRP‐like Ca2+ conductance in C. elegans intestinal cells

Inositol‐1,4,5‐trisphosphate (IP3)‐dependent Ca2+ oscillations in Caenorhabditis elegans intestinal epithelial cells regulate the nematode defecation cycle. The role of plasma membrane ion channels in intestinal cell oscillatory Ca2+ signalling is unknown. We have shown previously that cultured intestinal cells express a Ca2+‐selective conductance, IORCa, that is biophysically similar to TRPM7 currents. IORCa activates slowly and stabilizes when cells are patch clamped with pipette solutions containing 10 mm BAPTA and free Ca2+ concentrations of ∼17 nm. However, when BAPTA concentration is lowered to 1 mm, IORCa oscillates. Oscillations in channel activity induced simultaneous oscillations in cytoplasmic Ca2+ levels. Removal of extracellular Ca2+ inhibited IORCa oscillations, whereas readdition of Ca2+ to the bath caused a rapid and transient reactivation of the current. Experimental manoeuvres that elevated intracellular Ca2+ blocked current oscillations. Elevation of intracellular Ca2+ in the presence of 10 mm BAPTA to block IORCa oscillations led to a dose‐dependent increase in the rate of current activation. At intracellular Ca2+ concentrations of 250 nm, current activation was transient. Patch pipette solutions buffered with 1–4 mm of either BAPTA or EGTA gave rise to similar patterns of IORCa oscillations. We conclude that changes in Ca2+ concentration close to the intracellular opening of the channel pore regulate channel activity. Low concentrations of Ca2+ activate the channel. As Ca2+ enters and accumulates near the pore mouth, channel activity is inhibited. Oscillating plasma membrane Ca2+ entry may play a role in generating intracellular Ca2+ oscillations that regulate the C. elegans defecation rhythm.

Highly Ca2+-selective TRPM Channels Regulate IP3-dependent Oscillatory Ca2+ Signaling in the C. elegans Intestine

Xing et al. 2008. J. Gen. Physiol. doi:10.1085/jgp.200709914nn[OpenUrl][1][Abstract/FREE Full Text][2]nn [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1085%252Fjgp.200709914%26rft_id%253Dinfo%253Apmid%252F18299395%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%