ENDOCYTIC PATHWAY OF EXOGENOUS IRON-LOADED FERRITIN IN INTESTINAL 1

26 Ferritin, a food constituent of animal and vegetal origin, is a source of dietary iron. Its hollow 27 central cavity has the capacity to store up to 4,500 atoms of iron, so its potential as an iron donor 28 is advantageous to heme iron, present in animal meats, and inorganic iron of mineral or vegetal 29 origin. In intestinal cells, ferritin internalization by endocytosis results in the release of its iron into 30 the cytosolic labile iron pool. The aim of this study was to characterize the endocytic pathway of 31 exogenous ferritin absorbed from the apical membrane of intestinal epithelium Caco-2 cells, 32 using both transmission electron microscopy and fluorescence confocal microscopy. Confocal 33 microscopy revealed that endocytosis of exogenous Alexa-Fluor 488-labeled ferritin was initiated 34 by its engulfment by clathrin-coated pits and internalization into early endosomes, as determined 35 by co-distribution with clathrin and early endosome antigen 1 (EEA1). Alexa-Fluor 488-labeled 36 ferritin also co-distributed with the autophagosome marker microtubule-associated protein 1 light 37 chain 3 (LC3), and the lysosome marker lysosomal-associated membrane protein 2 (LAMP2). 38 Transmission electron microscopy revealed that exogenously added ferritin was captured in 39 plasmalemmal pits, double-membrane compartments and multivesicular bodies considered as 40 autophagosomes and lysosomes, respectively. Biochemical experiments revealed that the 41 lysosome inhibitor chloroquine and the autophagosome inhibitor 3-methyladenine (3-MA) 42 inhibited degradation of exogenously added I-labeled ferritin. This evidence is consistent with 43 a model in which exogenous ferritin is internalized from the apical membrane through clathrin44 coated pits, and then follows a degradation pathway consisting of the passage through early 45 endosomes, autophagosomes and autolysosomes. 46 47