A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Chu, Jun; Zhang, Zhihong; Zheng, Ying; Yang, Jie; Qin, Lingfeng; Lu, Jinling; Huang, Zhen‐Li; Zeng, Shaoqun; Luo, Qingming

doi:10.1016/j.bios.2009.06.008

Cited by 93 publications

(72 citation statements)

References 26 publications

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“…To date, over 10 fluorescent proteins have been used for the BiFC assay . For example, cyan (Hu and Kerppola 2003;Shyu et al 2006), green (Ghosh et al 2000), red (Chu et al 2009;Fan et al 2008;Jach et al 2006;Kodama and Wada 2009) and photoswitchable (Lee et al 2010) fluorescent protein-based BiFC assays are available. Using these fluorescence complementation technologies, advanced BiFC-based techniques such as multicolor BiFC and BiFC-based fluorescence (Förster) resonance energy transfer (FRET) assays have also been developed.…”

mentioning

confidence: 99%

“…Using these fluorescence complementation technologies, advanced BiFC-based techniques such as multicolor BiFC and BiFC-based fluorescence (Förster) resonance energy transfer (FRET) assays have also been developed. The multicolor BiFC assay is a method for visualization of two alternative (Hu and Kerppola 2003;Waadt et al 2008) or distinct (Chu et al 2009;Kodama and Wada 2009) protein complexes in a single cell using two different colored BiFCs (e.g. green and red).…”

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confidence: 99%

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A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Kodama

2011

Plant Biotechnology

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confidence: 99%

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confidence: 99%

A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Kodama

2011

Plant Biotechnology

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“…CA-GFP from pCA-GFP was isolated and ligated into the NheI and EcoRI sites of pT-CD8-IRES-SCN2B (a kind gift from Dr. Alfred George, Vanderbilt University) to produce pT-CA-GFP-IRES-SCN2B. The resulting plasmid was cut with SalI and NotI and the coding sequence for mLumin (20) (mKate2-S158A) was inserted, generating pT-CA-GFP-IRES-mLumin. All constructs were confirmed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Time-lapse, confocal imaging was carried out on a Zeiss LSM 510 Meta Confocal Microscope (Zeiss, Germany) over a 6-h period. GFP and mLumin (20) were excited at 488 nm and 543 nm, respectively, and fluorescence detected at 520/30 nm (for GFP) and 585LP nm (for mLumin) using a 40ϫ (1.3 N.A.) oil immersion objective lens.…”

Section: Methodsmentioning

confidence: 99%

“…To afford an internal, optically distinct fluorescent control, we generated the expression construct CA-GFP-IRES-mLumin that constitutively expresses both CA-GFP and the red fluorescent protein mLumin, which is a brighter, more photostable derivative of mKate (20). After addition of staurosporine to induce apoptosis, GFP fluorescence increased markedly (Fig.…”

Section: Ca-gfp Is An Apoptosis Reporter In Mammalianmentioning

confidence: 99%

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Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Nicholls

Chu

Abbruzzese

et al. 2011

Journal of Biological Chemistry

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Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.

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Suppression of TDO-mediated tryptophan catabolism in glioblastoma cells by a steroid-responsive FKBP52-dependent pathway

Ott

Litzenburger

Rauschenbach

et al. 2014

Glia

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Tryptophan catabolism is increasingly recognized as a key and druggable molecular mechanism active in cancer, immune, and glioneural cells and involved in the modulation of antitumor immunity, autoimmunity and glioneural function. In addition to the pivotal rate limiting enzyme indoleamine-2,3-dioxygenase, expression of tryptophan-2,3-dioxygenase (TDO) has recently been described as an alternative pathway responsible for constitutive tryptophan degradation in malignant gliomas and other types of cancer. In addition, TDO has been implicated as a key regulator of neurotoxicity involved in neurodegenerative diseases and ageing. The pathways regulating TDO expression, however, are largely unknown. Here, a siRNA-based transcription factor profiling in human glioblastoma cells revealed that the expression of human TDO is suppressed by endogenous glucocorticoid signaling. Similarly, treatment of glioblastoma cells with the synthetic glucocorticoid dexamethasone led to a reduction of TDO expression and activity in vitro and in vivo. TDO inhibition was dependent on the immunophilin FKBP52, whose FK1 domain physically interacted with the glucocorticoid receptor as demonstrated by bimolecular fluorescence complementation and in situ proximity ligation assays. Accordingly, gene expression profile analyses revealed negative correlation of FKBP52 and TDO in glial and neural tumors and in normal brain. Knockdown of FKBP52 and treatment with the FK-binding immunosuppressant FK506 enhanced TDO expression and activity in glioblastoma cells. In summary, we identify a novel steroid-responsive FKBP52-dependent pathway suppressing the expression and activity of TDO, a central and rate-limiting enzyme in tryptophan metabolism, in human gliomas.

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A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Cited by 93 publications

References 26 publications

A bright green-colored bimolecular fluorescence complementation assay in living plant cells

A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Suppression of TDO-mediated tryptophan catabolism in glioblastoma cells by a steroid-responsive FKBP52-dependent pathway

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