title: A reactivity-based probe of the intracellular labile ferrous iron pool.
abstract: Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe(II) pools are larger in cancer cells than in nontumorigenic cells.
authors.affiliation: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Benjamin
authors.identifier: 0000-0001-9409-1529
authors.initials: B
authors.lastname: Spangler
authors.affiliation: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Charles W
authors.initials: CW
authors.lastname: Morgan
authors.affiliation: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Shaun D
authors.identifier: 0000-0002-5800-0391
authors.initials: SD
authors.lastname: Fontaine
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Mark N
authors.initials: MN
authors.lastname: Vander Wal
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.|Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Chang
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.|Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Wells
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Renslo
mesh_terms: D005296:Ferrous Compounds / Q000032:analysis* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D015394:Molecular Structure; D011691:Puromycin / Q000737:chemistry / Q000494:pharmacology; D013141:Spiro Compounds / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D005296:Ferrous Compounds; D005456:Fluorescent Dyes; D013141:Spiro Compounds; D011691:Puromycin