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

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. Self-assembled 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.