Novel acid-activated fluorophores reveal a dynamic wave of protons in the intestine of Caenorhabditis elegans

Abstract

Unlike the digestive systems of vertebrate animals, the lumen of the alimentary canal of Caenorhabditis elegans is unsegmented and weakly acidic (pH ~4.4), with ultradian fluctuations to pH > 6 every 45-50 s. To probe the dynamics of this acidity, we synthesized novel acid-activated fluorophores termed Kansas Reds. These dicationic derivatives of rhodamine B become concentrated in the lumen of the intestine of living C. elegans and exhibit tunable pKa values (2.3-5.4), controlled by the extent of fluorination of an alkylamine substituent, that allow imaging of a range of acidic fluids in vivo. Fluorescence video microscopy of animals freely feeding on these fluorophores revealed that acidity in the C. elegans intestine is discontinuous; the posterior intestine contains a large acidic segment flanked by a smaller region of higher pH at the posterior-most end. Remarkably, during the defecation motor program, this hot spot of acidity rapidly moves from the posterior intestine to the anterior-most intestine where it becomes localized for up to 7 s every 45-50 s. Studies of pH-insensitive and base-activated fluorophores as well as mutant and transgenic animals revealed that this dynamic wave of acidity requires the proton exchanger PBO-4, does not involve substantial movement of fluid, and likely involves the sequential activation of proton transporters on the apical surface of intestinal cells. Lacking a specific organ that sequesters low pH, C. elegans compartmentalizes acidity by producing of a dynamic hot spot of protons that rhythmically migrates from the posterior to anterior intestine.

Figure 1

Panel A: Structures of the Kansas Red (KR) fluorophores. Panel B: Synthesis of the KR fluorophores. Panels C–D: Structures of control compounds used as mechanistic probes.

Figure 2

Spectral properties of the Kansas Red fluorophores and control compounds. Panels A–G: Absorbance spectra of compounds (10 μM) acquired in simulated gastric fluid comprising aqueous buffer containing BSA (1%), Triton X-100 (1%), and DMSO (1%) at the pH values shown. Fluorescence emission spectra (Em.), obtained in phosphate (10 mM) buffers (pH 1.5 for KR23 and pH 2.5 for KR35-KR54 fluorophores) containing Triton X-100 (1%), normalized to 100% of the abs. λ_max peak. Panel H: Quantification of pKa values from absorbance measurements. OG40: Oregon Green 488 fluorophore with pKa = 4.0 in simulated gastric fluid. Relative quantum yields of acid-activated fluorophores were determined in ethanol containing 1% TFA. Data used to calculate extinction coefficients and quantum yields is provided in the supporting information.

Figure 3

DIC and confocal micrographs of the intestine of mechanically-immobilized transgenic C. elegans expressing PEPT-1GFP on the apical face of intestinal cells after feeding on KR35 (10 μM). Scale bar = 20 microns.

Figure 4

Fluorescence micrographs of unrestrained C. elegans after feeding on KR35 for 30 min (10 μM). Time-dependent images during the DMP extracted from video microscopy are shown. Fluorescence is rendered as a spectrum heat-map, with red representing the most intense fluorescence (highest acidity) and black the least intense fluorescence (lowest acidity). The head of the animal is on the left side of each image. Scale bar = 250 microns.

Figure 5

Panels A–E: Three-dimensional plots of changes in fluorescence in the intestine of unrestrained C. elegans during the DMP. Single frames isolated from video microscopy were normalized and analyzed after animals fed on media containing KR23, KR35, KR41, KR52, or KR54 (10 μM, 0.1% DMSO) for 30 min. Maximum anterior fluorescence was observed at t ~ 7.2 s. Each plot represents analysis of a single animal. Panel F, left: Fluorescence standard curve constructed by imaging of KR54 in glass microneedles, pulled to an internal diameter of 15 microns, filled with this fluorophore in aqueous solution (pH 2.5) containing BSA (1%), Triton X-100 (1%), and DMSO (10%). This elevated concentration of DMSO (10%) facilitated solubility of the fluorophore at high (e.g. mM) concentration in needles. Panel F, right: Quantification of the concentration of KR54 in the lumen of the posterior intestine, between DMP cycles, assuming an intestinal pH of 4.4. Each data point represents analysis of a single animal (mean ± SEM shown).

Figure 6

Fluorescence micrographs of unrestrained C. elegans during the DMP after co-feeding on a mixture of the green fluorescent probe Oregon Green dextran (25 μM) and either the red fluorescent probes RhB amide (10 μM, panel A) or KR35 (10 μM, panel B) for 2 h. Time-dependent images during the DMP extracted from video microscopy are shown. Fluorescence is rendered as a spectrum heat-map, with red representing the most intense fluorescence and black the least intense fluorescence. The head of the animal is on the left side of each image. Independent imaging of these probes confirmed spectral orthogonality with the filter sets employed. A major shift of fluorescence from the posterior intestine to the anterior-most intestine during the DMP was only observed with the acid activated KR35 fluorophore (panel B, t = 6.8 s). Scale bar = 100 microns.

Figure 7

Panels A–B: Fluorescence ratios in unrestrained wild type (N2) and mutant C. elegans (N ≥ 6) after feeding on KR35 (30 min, 10 μM). The data shown in panel A was collected between DMP cycles whereas the data shown in panel B was obtained during the DMP. Anterior to posterior ratios were calculated from fluorescence values quantified with 10 micron circular ROIs placed in the intestine at 5% (anterior) and 75% (posterior) as measured from the grinder to the anus (mean ± SEM shown). Panels C–D: Confocal micrographs of mechanically immobilized C. elegans expressing PBO-4::GFP (lhEx290) in the intestine and colocalization with KR35 after feeding on 10 μM for 30 min (panel D). The images show PBO-4::GFP expressed almost exclusively in the four posterior pairs of intestinal cells (cell pairs 6–9). Nuclei of cells 6–8 are marked with arrows (Int. 6L, 7L, 8L). PBO-4 is more abundant on the apical membrane of the more anterior cells. The head of the animal is on the left side in each image. Scale bars = 10 microns.