Direct measurement of endosomal pH in living cells of the rat yolk sac epithelium by laser confocal microscopy.
Endocytosis of dual-fluorescent dextran by epithelial cells was observed in the perfused rat yolk sac using a confocal laser scanning microscope. Endosomal pH was quantified from confocal images using a dual-fluorescence ratiometry technique. Changes in endosomal pH were followed over time up to 32 min with and without Brefeldin A treatment. After 4 min of endocytosis without Brefeldin A, endosomes had a pH of 6.1 +/- 0.3. After 16 min and 32 min, their pH levels varied widely from 4.0 to 6.6. When Brefeldin A (10 microM) was added to the perfusion medium, endosomal pH remained fairly stable in the range of 6.0 to 6.2 from 4 min to 32 min. The corresponding endosomal structures were examined by electron microscopy after endocytic labeling with horseradish peroxidase. After 4 min, small endocytic vesicles and large endosomal vacuoles with tubular extensions were labeled. After 16 min and 32 min, multivesicular bodies and dense lysosomal structures were progressively labeled, but their labeling was almost undetectable after Brefeldin A treatment. These results suggested that the pH within the sorting compartment of early endosomes is 6.1 +/- 0.3. This is the first quantitative measurement of pH within sorting endosomes in intact cells of the living yolk sac epithelium.
Ichimura
T
T
Department of Anatomy, Kagawa Medical University, Japan.
Hatae
T
T
Ishida
T
T
D000818:Animals; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry*; D004848:Epithelium / Q000737:chemistry; D019793:Fluorescein; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal / Q000379:methods*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines; D014617:Vacuoles / Q000737:chemistry; D015017:Yolk Sac / Q000737:chemistry*
D003517:Cyclopentanes; D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; D012235:Rhodamines; D020126:Brefeldin A; C005358:tetramethylrhodamine; D006735:Horseradish Peroxidase; D019793:Fluorescein