Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy.
The evolution of endo-lysosomal pH in Dictyostelium discoideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fluorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an extended lag phase lasting for 45-60 min and corresponding to intracellular transit of FITC-dextran cohort. During the chase period, endosomal pH decreased during approximately 20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20-40 min to reach pH 6.0-6.2. It was only at this stage that FITC-dextran was released back into the medium with pseudo first-order kinetics. A vacuolar H(+)-ATPase is involved in endosomal acidification as the acidification process was markedly reduced in mutant strain HGR8, partially defective in vacuolar H(+)-ATPase and in parent type strain AX2 by bafilomycin A1, a selective inhibitor of this enzyme. Our data suggest that endocytic cargo is channeled from endosomes to secondary lysosomes that are actively linked to the plasma membrane via recycling vesicles.
Aubry
L
L
Laboratoire de Biologie Cellulaire (URA 1130 CNRS), Département de Biologie Moléculaire et Structurale, Grenoble, France.
Klein
G
G
Martiel
J L
JL
Satre
M
M
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003911:Dextrans; D004023:Dictyostelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018942:Macrolides; D009550:Nigericin / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000900:Anti-Bacterial Agents; D003911:Dextrans; D018942:Macrolides; C015219:fluorescein isothiocyanate dextran; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D016650:Fluorescein-5-isothiocyanate; D009550:Nigericin