FITC-dextran as a probe for endosome function and localization in kidney.
Fluorescein isothiocyanate (FITC)-labeled endosomes were localized in kidney epithelial cells after tissue fixation and sectioning, and specific membrane transport properties of isolated endocytic vesicles were measured using the same probe. Rats were infused intravenously with 10 kDa FITC-dextran, and kidneys were fixed with paraformaldehyde lysine periodate. FITC-labeled vesicles were visualized in semithin (1 micron) frozen sections of excised tissue by epifluorescent microscopy and by electron microscopy after a photoconversion reaction. Most FITC-labeled endosomes were apically located in epithelial cells lining the urinary tubules. By immunocytochemistry the anti-lysosomal glycoprotein LGP 120 was absent from most of the FITC-labeled vesicles, although some colocalization was noted. The limiting membrane of FITC-labeled endosomes contained a vacuolar proton pump (pHmin = 6.23 +/- 0.033) and a water channel (osmotic water permeability coefficient, Pf = 0.052 +/- 0.005 cm/s) and was highly permeable to ethylene glycol and urea but relatively impermeable to glucose. Methods allowing the attribution of specific membrane functions to vesicles that can be visualized in the apical endocytic pathway of epithelial cells should be of general use for the study of endocytic pathways in a variety of systems.
Renal Unit, Massachusetts General Hospital, Boston.
W I
WI
Lencer
P
P
Weyer
A S
AS
Verkman
D A
DA
Ausiello
D
D
Brown
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000941:Antigens; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D003911:Dextrans; D004705:Endocytosis; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D007668:Kidney / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure*; D007700:Kinetics; D008854:Microscopy, Electron; D015388:Organelles / Q000502:physiology / Q000648:ultrastructure*; D011506:Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D051381:Rats; D011910:Rats, Brattleboro; D011919:Rats, Inbred Strains
D000941:Antigens; D003911:Dextrans; D005452:Fluoresceins; D011506:Proteins; C015219:fluorescein isothiocyanate dextran; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D016650:Fluorescein-5-isothiocyanate