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

Abstract

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 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-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) 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 Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

Figure 1.

Sequence alignment of human and C. elegans STIM1 homologues. Yellow and green shading indicates sequence identity and conserved amino acid substitutions, respectively. Conserved domains are shown in black boxes. Red boxes show location of N-linked glycosylation sites in human STIM1 (Williams et al., 2002). Light blue shading shows N-terminal cysteine residues conserved in human and Drosophila STIM homologues. Percent similarity and identity of signal peptide, EF-hand, SAM, transmembrane, and coiled-coil domains are 50.0% and 16.7%, 58.3% and 50.0%, 50.7% and 37.7%, 27.8% and 5.6%, and 57.4% and 21.3%, respectively. Alignment was performed using Vector NTI software (InforMax). Protein domains were identified by SMART (http://smart.embl-heidelberg.de/smart/change_mode.pl).

Figure 2.

STIM-1∷GFP expression pattern. (A) Low magnification confocal image of a whole worm. STIM-1∷GFP expression is present throughout the intestine, but is more pronounced in the anterior (AI) and posterior intestine (PI). Expression is also present in the spermatheca (Sp) and gonadal sheath cells (SC). Bar, 100 μm. (B) Expression of STIM-1∷GFP in head neurons. Arrows and arrowheads point to cell bodies and dendrites, respectively. Bar, 20 μm. (C) Series of confocal Z-sections through the anterior intestine. STIM-1∷GFP appears to be localized to a membrane or submembrane region as well as punctate and presumably intracellular domains. Lu, intestinal lumen. Bar, 20 μm. (D) Series of confocal Z-sections through the posterior intestine. STIM-1∷GFP appears to be localized to a membrane and/or submembrane region and a prominent intracellular reticular structure that resembles the intestinal endoplasmic reticulum (Rolls et al., 2002). Bar, 10 μm. (E) Series of confocal Z-sections through the spermatheca (Sp) and gonadal sheath cells (SC). Punctate localization pattern of STIM-1∷GFP can be seen in sheath cells in the second and third panels. Bar, 10 μm.

Figure 3.

Effect of stim-1 RNAi on fertility. Brood size is defined as the total number of progeny produced over 4 d. rrf-1 encodes an RNA-directed RNA polymerase homologue required for RNAi in somatic but not germ cells. pk1417 is a predicted rrf-1 null allele (Sijen et al., 2001). Values are means ± SEM (n = 5–8).

Figure 4.

Sheath cell and spermatheca function in control (GFP RNAi) and stim-1(RNAi) worms. (A) Rates of sheath contraction during a single ovulatory cycle. Time 0 is defined as the time at which ovulation was completed in control worms. stim-1(RNAi) worms never ovulated (see Results). Therefore in these animals, time 0 is defined as the first time point after peak sheath contraction rate was observed. Values are means ± SEM (n = 9–10). (B) Sheath cell displacement under basal conditions and during ovulation. Basal and ovulatory displacement were measured between −15 and −5 min and between −2 and 0 min, respectively. Values are means ± SEM (n = 6–9). *, P < 0.0001 compared with control ovulatory displacement. (C) Differential interference contrast micrographs of control and stim-1 RNAi worms. Spermatheca fails to open during ovulation in stim-1(RNAi) worms and oocytes are trapped in proximal gonad where they undergo endomitosis. Em, embryo; Emo, endomitotic oocytes; Oo, oocytes; Sp, spermatheca; Ut, uterus. Note that the uterus in the stim-1(RNAi) worm is empty, whereas the uterus in the control animal contains three developing embryos.

Figure 5.

Fertility and morphology defects in transgenic worms expressing the STIM-1 EF-hand mutant D55A;D57A. (A) Brood size in wild type and STIM-1 transgenic worms. Brood size is defined as the total number of progeny produced over 4 d. Wild type;gfp(RNAi), wild-type worms fed GFP dsRNA–producing bacteria. STIM-1∷GFP, transgenic worms expressing STIM-1 GFP fusion protein. D55A;D57A∷GFP, transgenic worms expressing STIM-1 EF-hand mutant GFP fusion protein. D55A;D57A∷GFP;gfp(RNAi), transgenic worms expressing STIM-1 EF-hand mutant GFP fusion protein fed GFP dsRNA–producing bacteria. Values are means ± SEM (n = 6–11). *, P < 0.001 compared with Wild type;gfp(RNAi), STIM-1∷GFP and D55A;D57A∷GFP;gfp(RNAi) worms. Brood size of D55A;D57A∷GFP;gfp(RNAi) worms was not significantly (P > 0.05) different from Wild type:gfp(RNAi) animals. (B–E) Differential interference contrast micrographs of transgenic worms expressing STIM-1(D55A;D57A)∷GFP. Gonads of STIM-1(D55A;D57A)∷GFP worms appeared to be stunted and contained endomitotic oocytes (B). Fluid accumulation was observed in the pseudocoel (C, arrowheads) and large vacuoles were present in the uterus (D). Smaller vacuoles were observed next to the pharynx (E, arrow) in a location similar to that of STIM-1∷GFP-expressing neurons (see Fig. 2 B). DG, distal gonad; Emo, endomitotic oocytes; Oo, oocytes; Phx, pharynx; Ut, uterus.

Figure 6.

Effect of stim-1 RNAi on pBoc period (A) and coefficient of variance (B), which is a measure of cycle rhythmicity in wild type and IP₃ signaling mutant worms. itr-1(sa73) is a loss-of-function allele and itr-1(sy290) and itr-1(sy327) are gain-of-function alleles of the C. elegans IP₃R. lfe-2(sy326) and ipp-5(sy605) are loss-of-function alleles of an IP₃ kinase and IP₃ phosphatase, respectively. Values are means ± SEM (n = 5–19).

Figure 7.

Examples of intestinal Ca²⁺ oscillations. (A and B) Calcium oscillation imaging in dissected intestines loaded with fluo-4. Intestines were isolated from wild-type worms fed either GFP (A) or stim-1 (B) dsRNA–producing bacteria. Fluorescence images were acquired during the last two oscillations shown in B at 1 Hz. All other images were acquired at 0.2 Hz. (C and D) In vivo Ca²⁺ oscillation imaging in intestines of freely moving L3 larvae expressing the FRET-based Ca²⁺ indicator protein cameleon. Worms were fed bacteria containing the empty RNAi feeding vector (C) or stim-1 dsRNA–producing bacteria (D) for three successive generations.

Figure 8.

Effect of stim-1 RNAi on STIM-1∷GFP expression. (A) Fluorescence micrographs of anterior and posterior intestines from wild type and STIM-1∷GFP worms and STIM-1∷GFP worms fed stim-1 dsRNA–producing bacteria. Images were obtained using a CCD camera and inverted microscope (see Materials and methods). Camera settings were the same for all three groups of worms. Weak fluorescence in wild-type worms is autofluorescence from intestinal granules. (B) Maximal mean pixel intensity in anterior and posterior intestines. Mean pixel intensity was measured in a region 28 μm wide by 18 μm high. The measuring region was positioned over the anterior or posterior intestine at a point where mean pixel intensity was maximal. Values are means ± SEM (n = 7–9). *, P < 0.002 compared with STIM-1∷GFP worms.

Figure 9.

Effect of stim-1 RNAi on cultured intestinal cell SOC current. Examples of whole cell currents induced by store depletion in a control cell (A) and a cell treated with stim-1 dsRNA (B). Store depletion was induced using a pipette solution containing 10 μM IP₃, 10 mM BAPTA, and 18 nM free Ca²⁺. Currents were elicited by ramping membrane voltage from −120 mV to +80 mV at 200 mV/s every 5 s. (C) Effect of stim-1 dsRNA on peak I_SOC measured 5 min after obtaining whole cell access. Solid lines are the mean currents for the cells shown.

Figure 10.

Effect of expression of stim-1 EF-hand mutation on pBoc period and rhythmicity. (A) pBoc cycles of individual STIM-1 (D55A;D57A)∷GFP worms fed bacteria containing the empty vector (top) or fed stim-1 dsRNA-producing bacteria (bottom). (B) Mean pBoc period and coefficients of variance for wild-type worms and worms shown in A. Wild type;gfp(RNAi), wild-type worms fed GFP dsRNA–producing bacteria. D55A;D57A∷GFP, transgenic worms expressing STIM-1 EF-hand mutant GFP fusion protein fed bacteria containing the empty feeding vector. D55A;D57A∷GFP;stim-1(RNAi), transgenic worms expressing STIM-1 EF-hand mutant GFP fusion protein fed stim-1 dsRNA–producing bacteria. Values are means ± SEM (n = 8–9). Wild-type data are reproduced from Fig. 6. *, P < 0.001 compared with wild-type worms. **, P < 0.001 compared with D55A;D57A∷GFP worms. pBoc period and coefficient of variance in D55A;D57A∷GFP;stim-1(RNAi) worms were not significantly (P > 0.05) different from wild type;gfp(RNAi) worms.

Figure 11.

Induction of the intestinal unfolded protein response. (A) Fluorescent micrographs of control hsp-4∷GFP worms fed bacteria containing the empty feeding vector, control hsp-4∷GFP worms treated with tunicamycin, and hsp-4∷GFP worms fed either sca-1 or stim-1 dsRNA–producing bacteria. Worms were imaged using a stereo dissecting microscope and CCD camera (see Materials and methods). Images were obtained with identical camera settings. (B) Changes in whole worm fluorescence induced by tunicamycin (Tun), sca-1 RNAi, or stim-1 RNAi. Values are means ± SEM (n = 275–732) and are plotted relative to fluorescence in control animals. *, P < 0.001 compared with control worms. (C) Effect of combined sca-1 and stim-1 RNAi on whole worm fluorescence. Values are means ± SEM (n = 242–756) and are plotted relative to fluorescence in control animals. *, P < 0.0001 compared with sca-1(RNAi) worms. Fluorescence levels in B and C were quantified using a COPAS BioSort and normalized to time-of-flight (i.e., GFP fluorescence/time-of-flight), which is a measure of worm size. Raw GFP fluorescence levels showed an identical pattern of change and statistical significance.