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Iron Uptake in Drosophila Sg4 Cells Does Not Require Endocytosis
Author(s) -
Najera Diana,
Coca Michelle,
Gorman Maureen
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.05370
Subject(s) - endocytosis , pinocytosis , dmt1 , endocytic cycle , endosome , microbiology and biotechnology , transferrin receptor , transferrin , receptor mediated endocytosis , clathrin , biology , chemistry , ferrous , biochemistry , transporter , cell , intracellular , organic chemistry , gene
Iron is essential for all organisms including insects; however, at high levels it is toxic. Therefore, the transport and uptake of iron must be well‐regulated, and in insects this process is poorly understood. In mammals, the most widely known mechanism of iron uptake is receptor‐mediated endocytic uptake of Fe 3+ ‐transferrin. In the acidified endosome, iron is released from transferrin, reduced by a ferric reductase to Fe 2+ , and transported across the endosome membrane through divalent metal transporter 1 (DMT1). The goal of this project was to determine whether iron uptake by cultured Drosophila Sg4 cells (an isolate of embryonically derived S2 cells) requires endocytosis. To test this hypothesis, we blocked endocytosis and then measured cellular iron content. We performed RNAi‐mediated knockdown of rab5, which is expected to block both clathrin‐mediated and caveolar endocytic pathways, and we also treated cells with one of three chemical inhibitors: 200 nM bafilomycin‐A1, which inhibits clathrin‐mediated endocytosis in S2 cells, 5 mM methyl‐beta‐cyclodextrin, which blocks caveolae‐mediated endocytosis, and 1 µM cytochalasin D, which blocks phagocytosis and macropinocytosis. The rab5 RNAi and chemical treatments caused a slight decrease in cell growth and viability, but they did not decrease iron content. The results of these experiments suggest that endocytosis is not required for iron uptake by Sg4 cells. To test our hypothesis using a different strategy, we are evaluating the role in iron uptake of an insect homolog of DMT1, Malvolio (Mvl). Based on previous studies, Mvl appears to be a divalent metal transporter that is present mainly in endosome membranes. We used RT‐PCR to verify that Sg4 cells express Mvl, and we are currently performing RNAi to assess the effect of Mvl knockdown on cellular iron content. Given that blocking endocytosis had no effect on cellular iron content, if Mvl participates in endocytic uptake of protein‐bound iron, we expect that knockdown of Mvl will also have no effect on cellular iron content.