Endo-lysosomal acidification in Dictyostelium discoideum amoebae. Effects of two endocytosis inhibitors: caffeine and cycloheximide.

Fluid-phase endocytosis (pinocytosis) is highly active in amoebae of the cellular slime mould Dictyostelium discoideum as it provides an efficient entry of nutrients in axenic strains. Detailed kinetic analyses were conducted using fluorescein-labeled dextran (FITC-dextran) as fluid-phase marker and pH probe. Cells were first pulsed with FITC-dextran during a short period then chased by suspension in probe-free medium. Chase kinetics were characterized by a lag phase of about 40 min before pseudo-first order FITC-dextran efflux and thus reflected the progression of the probe cohort through the various endosomal compartments along the endosomal pathway. Temporal evolution of endo-lysosomal pH showed a rapid acidification (T1/2 approximately 10 min) to pH 5.0 followed by an increase up to pH 6.2 to 6.3. The effects of cycloheximide and caffeine, two inhibitors of endocytosis in Dictyostelium amoebae, on the evolution of endosomal pH during fluid-phase endocytosis, have been investigated. Cycloheximide fully blocked the cellular transit of FITC-dextran but acidification of endo-lysosomal compartments still took place. Caffeine increased endo-lysosomal pH, probably as a consequence of an elevation of cytosolic [Ca2+]. Furthermore, it allowed the functional identification of a caffeine-insensitive terminal segment of the endocytic pathway. It corresponded to a recycling, postlysosomal compartment at pH 6.2 to 6.3 with an apparent volume of 160 microns 3/amoebae.