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Imaging of hypoxia-inducible factor 1? 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-1?. SEPT9_i1 is a member of the conserved family of GTP-binding cytoskeleton septins. SEPT9_i1 stabilizes HIF-1? and facilitates its cytoplasmic-nuclear translocation. We utilized split yellow fluorescent protein (YFP) bimolecular fluorescence complementation (BiFC) methodology to monitor the interaction between HIF-1? and SEPT9_i1 in live cells. N-terminal (YN) and C-terminal (YC) split YFP chimeras with HIF-1? and SEPT9_i1 on both their amino and carboxyl termini were generated. HIF-1? and SEPT9_i1 chimeras were expressed in cancer cells and screened for functional complementation. SEPT9_i1-YN and YC-HIF-1? formed a long-lived highly stable complex upon interaction. The BiFC signal was increased in the presence of hypoxia-mimicking agents. In contrast, YC-?HLH-HIF-1? 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-1?, 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-1?. It was increased with fourchlorfenuron, a small molecule that increases the interaction between HIF-1? and SEPT9_i1. These results reconfirmed the interaction between HIF-1? 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.

SUBMITTER: Golan M

PROVIDER: S-EPMC5458251 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells.

Golan Maya M Mabjeesh Nicola J NJ

Oncotarget 20170501 19

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 ...[more]

PMID: 28380438

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Project description:Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1-158 amino acid residues, called Venus-N, and its C-terminal 159-239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is proportional to the strength of protein-protein interaction (Morell et al., 2008); and fourth, the BiFC signals are derived from intrinsic protein-protein interaction, rather than from extrinsic fluorophores that may not reflect true protein-protein interaction due to their nonspecific association with cellular macromolecules or subcellular compartments. However, some limitations of BiFC include slow maturation (T1/2 ~ 1 h) of an eventually stable BiFC complex (Hu et al., 2002), making it unsuitable for real-time observation of transient interaction that disappears prior to BiFC detection, and enhanced BiFC background at high expression levels due to fusion-independent association between two non-fluorescent fragments association. BiFC signals generated by in vivo protein-protein interaction can be validated by amino acid mutation introduced at the protein-protein contact surfaces. This imaging technique has been widely used in different cell types and organisms (Kerppola, 2006).

Project description:Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.

Dataset Information

Imaging of hypoxia-inducible factor 1? and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells.

Publications

Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells.

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