Iron Transport in Intestinal Epithelial Cells Occurs by Transcytosis. A Fluorescent Metal-Sensor Study.
Authors: Ma Yuxiang , Yeh Mary , Yeh Kwo-yih , Glass Jonathan 
Published: April 2020

Abstract
The molecular mechanisms ensuring directionality of iron transport across the intestinal epithelium are still poorly understood. Iron is transported across the brush-border membrane (BBM) by the divalent metal transporter 1 (DMT1) and then must be targeted to specific organelles and other transporters for transport across the basolateral membrane (BLM). We have previously shown that in Caco2 cells grown in bicameral chambers with the addition of iron to the apical surface, DMT1 on the BBM undergoes endocytosis and fuses with endocytic vesicles derived from the basal surface. However, which cytosolic proteins mediate the endocytosis of DMT1 and the interaction of apical derived vesicles with basal derived vesicles are still unknown. The present study focuses on trying to confirm our hypothesis that iron absorption in the intestinal epithelium is through endocytic processes and involves a pathway in which apical derived vesicles fuse with basolateral-derived vesicles. Calcein is a fluorescent metal sensor whose fluorescence is quenched with chelation of iron. Calcein, which is hydrophilic and membrane-impermeable, is taken up by cells by endocytosis (Glickstein H et al. Blood First Edition Paper, prepublished online July 14, 2005). To monitor the flux of iron via transcytosis we took advantage of the properties of calcein and used Caco2 cells grown as a polarized cell layer in bicameral chambers as a model system to study iron flux. In the Caco2 model the apical chamber baths the brush border surface of the cells and the basal chamber baths the basolateral surface. When calcein was offered in the basal chamber along with apo-transferrerin (apo-Tf), calcein was found to undergo endocytosis and co-localize with apo-Tf in the sub-apical cytoplasm with a coefficient of co-localization of 36.8 ± 3.6 %. Under these conditions offering ferrous iron in the apical chamber caused the calcein to be quenched and the coefficient of co-localization decreased to 22.5 ± 5.7 %. The addition of a permeable iron chelator restored calcein fluorescence and the co-localization increased to 58.6 ± 20.5 %. Iron chelated to calcein, which markedly quenches calcein fluorescence, was then offered in the apical chamber as a source of iron. Apo-Tf was offered simultaneously in the basal chamber. After internalization calcein fluorescence was subsequently restored and calcein was observed to co-localize in vesicles with apo-Tf with a co-localization coefficient was 26.4 ± 5.8 %. These studies strongly suggest that iron is transported across the intestinal epithelium by transcytosis with apical derived vesicles fusing with basolateral-derived vesicles. Early endosome antigen 1 (EEA1) and the small GTPase, Rab5, are required for endosomal transport and fusion in mammalian cells and might provide directionality to vesicular transport from the membrane to the early endosomes. We examined if both EEA1 and Rab5 are involved in the transcytosis of DMT1 and iron and found that after iron feeding that there was increased co-localization of DMT1 with both EEA1 and Rab5 in the sub-apical compartment. Taken together these studies support our hypothesis that iron absorption in the intestinal epithelium is through endocytic processes and involves a transcytosis pathway.