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 C. elegans is unsegmented and weakly acidic (pH ~ 4.4), with ultradian fluctuations to pH > 6 every 45 to 50 seconds. 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 seconds every 45 to 50 seconds. 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.

Anilinomethylrhodamines: pH Sensitive Probes with Tunable Photophysical Properties by Substituent Effect

“…in the detection of nerve agents, 15 phosgene, 16 and biologically relevant aldehydes. 17 Furthermore, our work on the AMR scaffold, and more recently the work of Peterson et al ., 18 has shown that the pH range in which these probes respond can be tuned by introducing different functional groups to change the basicity of the amine moiety. We are interested in exploring functionalities that can be used to tune the pH dependent properties of the AMR scaffold.…”

TheC. elegansCHP1 homolog,pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen

“…Previously we developed a pH-responsive dye, KR35, and used it to monitor pH changes in the intestine of C. elegans [18]. KR35 is activated by acid in a range that is physiologically relevant to the C. elegans intestine, which maintains a pH gradient from anterior to posterior of ~5.5-3.5 [19].…”

“…The 50 second oscillations and the MAT are part of a process called the defecation motor program (DMP), whereby protons are transported across the basolateral membrane of the intestine by the Na + /H + exchanger PBO-4, where they act as neurotransmitters that trigger muscle contractions required for elimination of waste [16,17]. Using KR35 we previously showed that animals with mutations in pbo-4 fail to produce the MAT during pH oscillations, although other aspects of the acidic wave were grossly normal such as the acidification of the posterior intestine prior to the MAT [18].…”

Cycloalkyl-AminoMethylRhodamines: pH Dependent Photophysical Properties Tuned by Cycloalkane Ring Size

“…Our group and others have developed a structurally analogous scaffold, the aminomethylrhodamines or rhodaminedeoxylactams as fluorescent probes for pH, which have a pK a value that is strongly dependent on the electronic properties affecting the amino group [12–15]. This approach has allowed us to rationally design rhodamine pH probes where the photophysical response to pH is easily predicted by correlation to the amine's pK a .…”