(A) Confocal images of transgenic animals expressing the pHluorin::PBO-4 fusion protein. DIC (top), confocal slice (middle) and confocal projection (bottom) of an adult hermaphrodite expressing the GFP fusion protein. Fluorescence was observed on the basolateral surface of the posterior intestine cells only. Animals carrying this fusion protein were rescued for posterior body contractions (pbo-4(ok583); oxEx584), demonstrating that the fusion protein is functional and properly localized. The dashed lines indicate the intestinal boundary in the DIC image. Scale bar is 50μM. (B) Top, cross-sectional electron micrograph of the posterior end of a wild-type C. elegans adult hermaphrodite. The intestine (green), muscle (red) and epidermis (blue) are pseudo-colored and the region of interest is denoted by a white box. Scale bar is 1μm. Bottom, closeup of the region of interest. Note the muscle extends a process to form a long muscle-intestinal interface (arrowhead). Scale bar is 0.5μm.

(A) – (C) Extracellular pH changes were measured using pHlourin expressed on the basolateral surface of the posterior intestine (pHlourin::PBO-4). (A) Acidification transients were observed in a wild-type strain (EG3326 pbo-4(ok583); oxEx584[pHlourin::PBO-4(+)]). The arrowhead denotes distinct accordion-like posterior body contractions (pBoc) observed following a transient drop in fluorescence. (B) Acidification transients were reduced in a pbo-4(−) strain (EG4176 pbo-4(n2658); oxEx782[pHluorin::PBO-4(ΔC)]). The carboxy terminus of PBO-4 is deleted in the PBO-4(ΔC) construct and it does not rescue posterior body contractions in the mutant strain. (C) No posterior body contractions were detected in a mutant lacking the acid receptor subunit PBO-5 (EG4178 pbo-5(ox4); oxEx784[pHlourin::PBO-4(+)]). Acid transients produced by the pHluorin::PBO-4 transgenic protein were observed with similar kinetics and frequency as in (A). (D) Image sequence of the first acid transient corresponding to the first event in (A), expanded on the right. The white dashed line indicates the initial location of the posterior intestine at the start of the pH decrease. The orange line indicates the anterior displacement and eventual posterior relaxation of the posterior body contraction. Fluorescence intensity is pseudo-colored to highlight differences. Scale bar (0.02mm), grayscale pixel intensity values in arbitrary units. Traces in A-C are mean pixel intensity recordings from individual animals; regions of interest were defined by hand drawn areas slightly larger than the fluorescent region of the posterior intestine. (E) The number of transients in a six minute recording were significantly fewer in the pHluorin::PBO-4(ΔC) strain (3.2 ± 0.8, n = 14) compared to pbo-4(−); pHluorin::PBO-4 (13.7 ± 1.6, n = 18, P<0.001) and pbo-5(−);pHluorin::PBO-4 (12.3 ± 2.4, n = 10,P<0.01). (F) Average transient amplitude per individual was significantly smaller in the pbo-4(−) pHluorin::PBO-4(ΔC) strain (1.0 ± 0.2% ΔF/F, n = 14) compared to pbo-4(−) pHluorin::PBO-4(+) (5.0 ± 0.7% ΔF/F , n = 18, P<0.0001) and pbo-5(−) pHluorin::PBO-4(+) (3.1 ± 0.5% ΔF/F, n = 10, P=0.0012). No transients were detected in 5/14 pbo-4(−)pHluorin::PBO-4(ΔC) individuals. (G) Average transient rise times were significantly slower in the pbo-4(−) pHluorin::PBO-4(ΔC) strain (2.7 ± 0.3 s, n = 9) compared to pbo-4(−) pHluorin::PBO-4(+) (1.9 ± 0.09 s , n = 18, P=0.01) and pbo-5(−) pHluorin::PBO-4(+) (1.7 ± 0.2 s, n = 10, P=0.01). (H) Average transient decay times were significantly slower in the pbo-4(−) pHluorin::PBO-4(ΔC) strain (9.6 ± 1.0 s, n = 9) compared to pbo-4(−) pHluorin::PBO-4(+) (2.9 ± 0.3 s , n = 18, P<0.001) and pbo-5(−) pHluorin::PBO-4(+) (4.3 ± 0.4 s, n = 10, P<0.001). No statistical significance in any of these parameters were found between pbo-4(−) pHluorin::PBO-4(+) and pbo-5(−) pHluorin::PBO-4(+) strains. Asterisks indicate statistical significance between pbo-4(−) pHluorin::PBO-4(ΔC) and pbo-4(−) pHluorin::PBO-4(+) strains. N is the number of six minute recordings made from individual animals. Bars indicate mean ± SEM. The Mann-Whitney statistical test was used for B and C, t-tests were used for A and D.

(A) Representative traces of PBO-6, PBO-5 and PBO-5/6 injected oocyte responses to a pH 6.0 test pulse for 5 seconds (black bar). PBO-6 injected eggs never exhibited a functional response to pH 6.0 application. PBO-5 injected oocytes exhibited a slight response with a mean current amplitude = −0.18 ± 0.06 μA. PBO-5/6 co-injected oocytes had robust response to pH 6.0 with a mean current amplitude =−3.9 ± 0.35 μA. 18 oocytes total from 6 different batches were tested for each condition. (B) Representative traces of PBO-5/6 dose-response experiments. pH and duration of applied solution is shown above each trace. (C) PBO-5/6 pH dose-response curve. PBO-5/6 expressing oocytes were voltage-clamped at −60mV and a series of test pH applications (7.2−6.0) were bath applied for 5 seconds. Each point represents the mean current value normalized to the maximum and minimum values. A pH₅₀ of 6.83 ± 0.01 and a Hill coefficient of 5.0 were determined for PBO-5/PBO-6 receptors (n = 18). Error bars represent standard error of the mean. (D) H⁺ ion are sufficient to activate PBO-5/6 receptors. A representative co-activation experiment is shown. All points are in response to a pulse of pH 6.8 with or without the addition of 1mM neurotransmitter. Cells were voltage clamped at −60mV.

(A) Schematic diagram of the defecation motor program. The defecation motor program occurs every 50 seconds. First, the posterior body muscles contract in a posterior-to-anterior wave, three seconds later the anterior body muscles contract, followed by contraction of the enteric muscles, which expels the contents of the intestine. (B) Behavioral characterization of pbo-4 mutants: pbo-4 mutants lack the posterior body contraction. Eleven defecation cycles in day 1 adult animals were scored for the presence or absence of each muscle contraction (n ≥ 6 for each genotype). The pbo-4 alleles, n2658, ok583, ox10, sa300, exhibited a complete loss of posterior body contractions. In addition, enteric muscle contractions were significantly reduced for pbo-4(n2658) and pbo-4(ok583) compared to the wild type (t-test, P < 0.05). The strain ok583; oxEx582[PBO-4] carries an extrachromosomal array containing the genomic pbo-4 rescuing plasmid (pPD58). The strain ok583; oxEx584[pHluorin::PBO-4] contains an array expressing the full-length pHluorin::PBO-4 fusion protein (pAB16). Error bars represent s.e.m. (C) The pbo-5 alleles exhibited a loss of posterior body contractions; other motor steps and cycle times were largely normal (wild type 43.0±1.4”, n2303 40.0±3.9”, ox4 41.2 ±6.1”, ox36 38.7±7.1”, rescue: 42.8±5.6). The strain ox4; oxEx599 is pbo-5(ox4) carrying an extrachromosomal array containing the pbo-5 mini-gene rescuing plasmid (pAB18). Error bars represent standard error of the mean.

pbo-4(n2658) animals have no or very weak posterior body contractions. When the wild-type pHluorin::PBO-4 transgene is expressed under the vit-2 promoter it rescues the pbo-4 phenotype in young adults (pbo-4(n2658); oxEx770[Pvit-2:pHluorin::PBO-4]). The vit-2 promoter is expressed only in the intestinal cells in the adult stage. Animals were scored for as having no posterior body movement (no pBoc), weak movement of the tail, a contraction that only sweeps the length of the posterior intestinal cell, or a full posterior body contraction that sweeps from the tip of the tail to almost the vulva.

Confocal images of adult animals expressing GFP fusion genes. The animals are oriented with anterior to the left, and posterior to the right. Left panels, an animal expressing the pbo-5 transcriptional GFP fusion protein. Right panels, an animal expressing the pbo-6 transcriptional GFP fusion protein. Note that pbo-5 and pbo-6 have overlapping expression in the posterior body muscles. In addition, pbo-5 is expressed in the head neurons RIFL, RIFR and RIS (data not shown). Anterior fluorescence in the top pbo-6 image is autofluorescence. The intestine of the animals is noted by a dashed white line. Arrows mark the posterior body muscle in the DIC image. Top, DIC, Middle, Epifluorescence, Bottom, merged image. Scale bar is 50μM.

(A) Flash uncaging of H⁺ ions can activate the posterior body contraction in vivo. Left panels, the wild type: Top, the tail of an adult animal before uncaging. Middle, the same animal after a flash of UV light was applied for 1−3 seconds. Bottom, the same animal three seconds after the flash. The posterior body muscles have relaxed after the flash stimulus. Right panels, pbo-4(sa300): Top, the tail of a pbo-4 mutant before the flash stimulus. Middle, a flash-induced posterior body contraction is observed in mutant animals, demonstrating that H⁺ ions can bypass the pbo-4 defect to activate the posterior body contraction. Bottom, pbo-4(n2658) mutants relax the posterior body muscles after the flash-induced contraction. All animals were injected with 0.1mg/ml of NPE caged-H⁺ ions. Scale bar is 50μM. (B) Quantification of flash-induced posterior body contractions. Controls included uninjected wild types, FITC-injected wild types, and uninjected pbo-4(n2658) animals (not shown). Control animals fail to activate a posterior body contraction upon flash uncaging of H⁺ ions. Wild-type and pbo-4(n2658) animals injected with caged protons exhibited a mean contractile distance of 45.4 ± 8.86 μm (n=11) and 52.82 ± 11.45 μm (n=7), respectively. pbo-5(ox4) lacks a subunit of the PBO proton receptor that is required for posterior body contractions. Uncaging H⁺ ions was unable to stimulate muscle contraction in this strain. Error bars represent standard error of the mean.