Utilization of iron by hemoglobin-producing cells is highly efficient. The acquisition of iron from plasma requires the binding of diferric transferrin (Tf) to its cognate receptor (Tf-R) on the erythroid cell membrane, followed by internalization of the Tf - Tf-R complexes via receptor-mediated endocytosis. Through a poorly understood mechanism, iron is targeted to mitochondria, the site of heme biosynthesis. We believe that a direct interaction between iron-containing endosomes and mitochondria is essential for iron transfer to mitochondria and its efficient incorporation into heme. / In order to illustrate the interaction between endosomes and mitochondria, we have employed flow cytometry. Flow cytometry analysis of reticulocytes (erythrocyte precursors which still synthesize hemoglobin) stained with fluorescent dyes specific to mitochondria and endosomes revealed three distinct populations: mitochondria, endosomes and a population labeled with both dyes. This double-labeled population suggests a population composed of endosomes associated with mitochondria. Using non-fluorescent diferric-Tf, we were able to remove the double population, leaving only the endosomal and the mitochondrial population. This finding has confirmed that the double population is the result of the interaction between the two organelles. / Additionally, we established a cell-free assay consisting of fluorescent mitochondria and endosomes isolated from erythroid cells. Using confocal microscopy, we demonstrated a colocalization between the two organelles. We repeated the assay using fluorescent mitochondria and endosomes isolated from HeLa spinner cells. Using the mitochondrial uncoupler CCCP, we were able to significantly reduce the colocalization between the two organelles, indicating that the interaction between the organelles is specific and that the mitochondrial potential is a requirement for organellar interaction. / Based on our results from flow cytometry and confocal microscopy, we conclude that a specific and direct interaction exists between the two organelles.

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