Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Wu, Xiaoli; Simone, James; Hewgill, Derek; Siegel, Richard M.; Lipsky, Peter E.; He, Liusheng

doi:10.1002/cyto.a.20300

Cited by 56 publications

(46 citation statements)

References 53 publications

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“…We showed that not only sialic acids, but also fucose, could be successfully used for FRET detection. These findings open up the possibility of further applications of this FRET technique, such as simultaneous detection of different glycoforms on the same protein using, for example, a double FRET technique to detect two FRET pairs sharing one fluorophore [37][38][39] .…”

Section: Discussionmentioning

confidence: 95%

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

et al. 2012

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Analyses of mice lacking glycosyltransferase have suggested that their pathological phenotypes are not attributable to the overall change of the sugar modification, but instead the result of changes of the glycan structures on a specific 'target' glycoprotein. Therefore, detecting or monitoring the glycosylation status of a specific protein in living cells is important, but no such methods are currently available. Here we demonstrate the detection of glycoforms of a specific glycoprotein using the fluorescence resonance energy transfer technique. using model proteins, we detect characteristic fluorescence resonance energy transfer signals from the specific glycoform-bearing target glycoprotein. We also show that, upon insulin removal, sialylated glycoforms of green fluorescent protein-tagged GLuT4 seem to be internalized more slowly than non-sialylated GLuT4. This novel analytical imaging tool allows studying the roles of specific glycan modifications of a protein of interest.

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Section: Discussionmentioning

confidence: 95%

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

et al. 2012

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“…Although caspase-3 and -7 are structurally similar and can be inhibited by Z-VAD-FMK, caspase-6 shows very different characteristics and cannot be inhibited by Z-VAD-FMK (33). In addition, caspase-6 is activated by both caspase-3-dependent and -independent cascades (36). Moreover, FAM-DEVD-FMK used for measuring caspase-3 activity can also bind to caspase-7 and -6.…”

Section: Discussionmentioning

confidence: 99%

Activation of caspases and apoptosis in response to low-voltage electric pulses

Matsuki

Takeda²,

Ishikawa³

et al. 2010

Oncol Rep

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Abstract. Few studies have examined apoptosis induced by low-voltage electric pulses (LVEPs). LVEP-induce changes in membrane potential that are below the membrane breakdown threshold and increase membrane permeability without electroporation (pore formation) through the transport of extracellular substances via phagocytosis. We demonstrated that propidium iodide uptake and apoptosis increased in accordance with the duration and number of LVEPs in B16 cells, which showed relatively good viability under mild electric field conditions. We showed that LVEP-induced apoptosis was achieved through caspase-8 and -9 activation and subsequent caspase-3 activation. Long-duration LVEPs caused only mild cell damage, such that the apoptosis ratio (apoptosis/total cell death) in electric pulse-treated cells was similar to that in non-treated control cells. To assess the relative degree of caspase dependency in LVEP-induced apoptosis, the apoptosis rate and caspase-3 activity were analyzed using a pan-caspase inhibitor (Z-VAD-FMK). Z-VAD-FMK treatment inhibited, but did not abolish, LVEP-induced apoptosis, indicating that caspases other than caspase-3 participate in this pathway. Moreover, LVEP treatment inhibited cell growth, suggesting that LVEP treatment may be a valuable anticancer therapy. Although the mechanism of LVEP-induced apoptosis remains unclear, it may be related to dysfunctional membrane transport of Ca 2+ and other extracellular substances involved in caspase activation.

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“…These reporters monitor a change in the FRET intensity upon caspase cleavage (13)(14)(15). The multimodality reporter uses copies of the caspase-recognition sequence to link a fluorescent protein, a bioluminescent protein, and a positron emission tomography reporter, which can each be more readily detected after caspase cleavage (16).…”

mentioning

confidence: 99%

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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Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Cited by 56 publications

References 53 publications

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

Activation of caspases and apoptosis in response to low-voltage electric pulses

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

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