title: Imaging of heme/hemeproteins in nucleus of the living cells expressing heme-binding nuclear receptors.
abstract: Several factors involved in the core circadian rhythm are PAS domain proteins, one of which, neuronal PAS2 (NPAS2), contains a heme-binding motif. It is thought that heme controls the transcriptional activity of core circadian factors BMAL1-NPAS2, and that the heme-binding nuclear receptor REV-erbα negatively regulates the expression of BMAL1. To examine the role of heme in the nucleus, we expressed nuclear hemeproteins including the nuclear localization signal-added cytoglobin, NPAS2 and REV-erbα. Then, the living cells expressing these proteins were treated with 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). The fluorescent signal derived from DCFH-DA was observed in the nucleus. When the cells were cultured with hemin, the signal of heme in the nucleus increased. Considering that DCFH-DA reacted with heme, we propose that the use of DCFH-DA could be useful in detection of the heme moiety of hemeprotein in vivo.
authors.affiliation: Department of Biotechnology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.
authors.forename: Ryuhei
authors.initials: R
authors.lastname: Itoh
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Fujita
authors.forename: Anfeng
authors.initials: A
authors.lastname: Mu
authors.forename: Dao Hoang Thien
authors.initials: DH
authors.lastname: Kim
authors.forename: Tran Tien
authors.initials: TT
authors.lastname: Tai
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagami
authors.forename: Shigeru
authors.initials: S
authors.lastname: Taketani
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism*; D000078842:Cytoglobin; D003593:Cytoplasm / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005914:Globins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1 / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods; D013194:Staining and Labeling
chemical_list: C000627265:CYGB protein, human; D000078842:Cytoglobin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006420:Hemeproteins; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1; C029569:diacetyldichlorofluorescein; D006418:Heme; D006427:Hemin; D005914:Globins