ENDO-LYSOSOMAL ACIDIFICATION IN DICTYOSTELIUM-DISCOIDEUM AMEBAS - EFFECTS OF 2 ENDOCYTOSIS INHIBITORS - CAFFEINE AND CYCLOHEXIMIDE

Fluid-phase endocytosis (pinocytosis) is highly active in amoebae of t he cellular slime mould Dictyostelium discoideum as it provides an eff icient entry of nutrients in axenic strains. Detailed kinetic analyses were conducted using fluorescein-labeled dextran (FITC-dextran) as fl uid-phase marker and pH probe. Cells were first pulsed with FITC-dextr an during a short period then chased by suspension in probe-free mediu m. Chase kinetics were characterized by a lag phase of about 40 min be fore pseudo-first order FITC-dextran efflux and thus reflected the pro gression of the probe cohort through the various endosomal compartment s along the endosomal pathway. Temporal evolution of endo-lysosomal pH showed a rapid acidification (T1/2 is similar to 10 min) to pH 5.0 fo llowed by an increase up to pH 6.2 to 6.3. The effects of cycloheximid e and caffeine, two inhibitors of endocytosis in Dictyostelium amoebae , on the evolution of endosomal pH during fluid-phase endocytosis, hav e 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 conse quence of an elevation of cytosolic [Ca2+]. Furthermore, it allowed th e functional identification of a caffeine-insensitive terminal segment of the endocytic pathway. It corresponded to a recycling, postlysosom al compartment at pH 6.2 to 6.3 with an apparent volume of 160 mum3/am oebae.