The Dynamic Range of Acidity: Tracking Rules for the Unidirectional Penetration of Cellular Compartments

Abstract

Labeled ammonium cations with pK a ~7.4 accumulate in acidic organelles because they can be neutralized transiently to cross the membrane at cytosolic pH 7.2 but not at their internal pH < 5.5. Retention in early endosomes with less acidic internal pH was achieved recently using weaker acids of up to pK a 9.8. We report here that primary ammonium cations with higher pK a 10.6, label early endosomes more efficiently. This maximized early endosome tracking coincides with increasing labeling of Golgi networks with similarly weak internal acidity. Guanidinium cations with pK a 13.5 cannot cross the plasma membrane in monomeric form and label the plasma membrane with selectivity for vesicles embarking into endocytosis. Selfassembled into micelles, guanidinium cations enter cells like arginine-rich cell-penetrating peptides and, driven by their membrane potential, penetrate mitochondria unidirectionally despite their high inner pH. The resulting tracking rules with an approximated dynamic range of pK a change ~3.5 are expected to be generally valid, thus enabling the design of chemistry tools for biology research in the broadest sense. From a practical point of view, most relevant are two complementary fluorescent flipper probes that can be used to image the mechanics at the very beginning of endocytosis.

G‐Quartets, 4‐Way Junctions and Triple Helices but Not DNA Duplexes: Planarization of Twisted Push–Pull Flipper Probes by Surface Recognition Rather Than Physical Compression

“…Arguably the cleanest series of spectra was obtained for Flipper‐TR ® 1 at 13 n m ( Figure 5 ,A ). The response from the anionic but neutralizable [32] Flipper‐TR ® 1 was most convincing and consistent at all concentrations ( Figures 5 ,A , 5,D , S1 ). This result confirmed that detectable (planarizing) or undetectable (non‐planarizing) ion pairing of the cationic flippers 2 ( Figure 5, middle) and particularly the non‐neutralizable [32] 3 (right) with the non‐neutralizable [32] anionic phosphodiester backbone along DNA grooves was more disturbing than helpful.…”

“…The response from the anionic but neutralizable [32] Flipper‐TR ® 1 was most convincing and consistent at all concentrations ( Figures 5 ,A , 5,D , S1 ). This result confirmed that detectable (planarizing) or undetectable (non‐planarizing) ion pairing of the cationic flippers 2 ( Figure 5, middle) and particularly the non‐neutralizable [32] 3 (right) with the non‐neutralizable [32] anionic phosphodiester backbone along DNA grooves was more disturbing than helpful. Despite much precedence in the literature for cationic probes binding to DNA, [46,51] repulsion between negative charges is also understood as the basis of all DNA architectures.…”

“…[29,30] The planarization of flipper probes by physical compression from the surrounding has been explored extensively to image membrane order and tension in living cells. [18,27,31,32] Planarization of flipper probes by packing forces in single crystals has been reported as well. [28] In clear contrast, flipper planarization by noncovalent interactions rather than by mechanical physical compression has so far not been observed.…”

“…The planarization of flipper probes by physical compression from the surrounding has been explored extensively to image membrane order and tension in living cells [18,27,31,32] . Planarization of flipper probes by packing forces in single crystals has been reported as well [28] .…”

“…Flipper probes 1 – 3 with negatively and positively charged headgroups have been reported as tension probes targeting plasma membrane, [18] early endosomes and mitochondria ( Figure 1). [32] They were prepared following the reported procedures.…”