Endocytic pathway of exogenous iron-loaded ferritin in intestinal epithelial (Caco-2) cells.
Ferritin, a food constituent of animal and vegetal origin, is a source of dietary iron. Its hollow central cavity has the capacity to store up to 4,500 atoms of iron, so its potential as an iron donor is advantageous to heme iron, present in animal meats and inorganic iron of mineral or vegetal origin. In intestinal cells, ferritin internalization by endocytosis results in the release of its iron into the cytosolic labile iron pool. The aim of this study was to characterize the endocytic pathway of exogenous ferritin absorbed from the apical membrane of intestinal epithelium Caco-2 cells, using both transmission electron microscopy and fluorescence confocal microscopy. Confocal microscopy revealed that endocytosis of exogenous AlexaFluor 488-labeled ferritin was initiated by its engulfment by clathrin-coated pits and internalization into early endosomes, as determined by codistribution with clathrin and early endosome antigen 1 (EEA1). AlexaFluor 488-labeled ferritin also codistributed with the autophagosome marker microtubule-associated protein 1 light chain 3 (LC3) and the lysosome marker lysosomal-associated membrane protein 2 (LAMP2). Transmission electron microscopy revealed that exogenously added ferritin was captured in plasmalemmal pits, double-membrane compartments, and multivesicular bodies considered as autophagosomes and lysosomes. Biochemical experiments revealed that the lysosome inhibitor chloroquine and the autophagosome inhibitor 3-methyladenine (3-MA) inhibited degradation of exogenously added (131)I-labeled ferritin. This evidence is consistent with a model in which exogenous ferritin is internalized from the apical membrane through clathrin-coated pits, and then follows a degradation pathway consisting of the passage through early endosomes, autophagosomes, and autolysosomes.
Facultad de Ciencias, Departamento de Biología, Universidad de Chile, Santiago, Chile.
Elmer
E
Antileo
Carolina
C
Garri
Victoria
V
Tapia
Juan Pablo
JP
Muñoz
Mario
M
Chiong
Francisco
F
Nualart
Sergio
S
Lavandero
Juan
J
Fernández
Marco T
MT
Núñez
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001343:Autophagy; D018938:Caco-2 Cells; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis; D005293:Ferritins / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D033921:Vesicular Transport Proteins / Q000378:metabolism
D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C025946:3-methyladenine; D005293:Ferritins; D007501:Iron; D000225:Adenine