title: Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells.
abstract: Hypoxia-inducible factor 1 (HIF-1) is a major mediator of the hypoxic response involved in tumor progression. We had earlier described the interaction between septin 9 isoform 1 (SEPT9_i1) protein and the oxygen-regulated subunit, HIF-1α. SEPT9_i1 is a member of the conserved family of GTP-binding cytoskeleton septins. SEPT9_i1 stabilizes HIF-1α and facilitates its cytoplasmic-nuclear translocation. We utilized split yellow fluorescent protein (YFP) bimolecular fluorescence complementation (BiFC) methodology to monitor the interaction between HIF-1α and SEPT9_i1 in live cells. N-terminal (YN) and C-terminal (YC) split YFP chimeras with HIF-1α and SEPT9_i1 on both their amino and carboxyl termini were generated. HIF-1α and SEPT9_i1 chimeras were expressed in cancer cells and screened for functional complementation. SEPT9_i1-YN and YC-HIF-1α formed a long-lived highly stable complex upon interaction. The BiFC signal was increased in the presence of hypoxia-mimicking agents. In contrast, YC-ΔHLH-HIF-1α chimera, which lacked the helix-loop-helix domain that is essential for the interaction with SEPT9_i1 as well as the expression of SEPT9_i1 252-379 amino acids fragment required for the interaction with HIF-1α, significantly reduced the BiFC signal. The signal was also reduced when cells were treated with 17-N-allylamino-17-demethoxygeldanamycin, an HSP90 inhibitor that inhibits HIF-1α. It was increased with fourchlorfenuron, a small molecule that increases the interaction between HIF-1α and SEPT9_i1. These results reconfirmed the interaction between HIF-1α and SEPT9_i1 that was imaged in live cells. This BiFC system represents a novel approach for studying the real-time interaction between these two proteins and will allow high-throughput drug screening to identity compounds that disrupt this interaction.
authors.affiliation: Prostate Cancer Research Laboratory, Department of Urology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Maya
authors.initials: M
authors.lastname: Golan
authors.affiliation: Prostate Cancer Research Laboratory, Department of Urology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Mabjeesh
mesh_terms: D045744:Cell Line, Tumor; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D057054:Molecular Imaging / Q000379:methods; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D058112:Septins / Q000235:genetics / Q000378:metabolism*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011993:Recombinant Fusion Proteins; D058112:Septins
keywords: bimolecular fluorescence complementation; cancer; hypoxia-inducible factor 1α; imaging; septin 9