AJP - Cell Physiology, Vol 258, Issue 2 C309-C317, Copyright © 1990 by American Physiological Society
FITC-dextran as a probe for endosome function and localization in kidney
W. I. Lencer, P. Weyer, A. S. Verkman, D. A. Ausiello and D. Brown
Renal Unit, Massachusetts General Hospital, Boston.
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.
