Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells

Activation of caspase-3 is a central event in apoptosis. We have developed a GFP-based FRET (fluorescence resonance energy transfer) probe that is highly sensitive to the activation of caspase-3 in intact living cells. This probe was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD. The linker design was optimized to produce a large FRET effect. Using purified protein, we observed a fivefold change in the fluorescence emission ratio when the probe was cleaved by caspase-3. To demonstrate the usefulness of this method, we introduced this FRET probe into HeLa cells by both transient and stable transfection. We observed that during UV-induced apoptosis, the activation of caspase-3 varied significantly between different cells; but once the caspase was activated, the enzyme within the cell became fully active within a few minutes. This technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process.     

Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation

Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.     

High throughput ratio imaging to profile caspase activity: potential application in multiparameter high content apoptosis analysis and drug screening

Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based.     

Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase-3

Activation of effector caspases is considered to be the final step in many apoptosis pathways. We transfected HeLa cells with a recombinant caspase substrate composed of cyan and yellow fluorescent protein and a linker peptide containing the caspase cleavage sequence DEVD, and we examined the cleavage kinetics at the single-cell level by fluorescence resonance energy transfer (FRET) analysis. Caspase activation in response to tumor necrosis factor-alpha, staurosporine, or etoposide resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The time to caspase activation varied among individual cells, depending on the type of treatment and concentration used. However, once initiated, disruption of the FRET signal was always rapid (相似文献   

Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects

Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton (H+) and chloride ion (Cl-) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an H+- and Cl--insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.     

Comparison of caspase-3 activation in tumor cells upon treatment of chemotherapeutic drugs using capillary electrophoresis

Caspases play important roles in cell apoptosis. Measurement of the dynamics of caspase activation in tumor cells not only facilitates understanding of the molecular mechanisms of apoptosis but also contributes to the development, screening, and evaluation of anticancer drugs that target apoptotic pathways. The fluorescence resonance energy transfer (FRET) technique provides a valuable approach for defining the dynamics of apoptosis with high spatio-temporal resolution. However, FRET generally functions in the single-cell level and becomes ineffective when applied in the high throughput detection of caspase activation. In the current study, a FRET sensor was combined with capillary electrophoresis (CE) to achieve a high throughput method for cellular caspase detection. The FRET-based CE system is composed of a homemade CE system and a laser source for detecting the dynamics of caspase-3 in various cells expressing sensors of caspase-3 that have been treated with anticancer drugs, such as cell cycle-independent drug cisplatin and specific cell cycle drugs camptothecin and etoposide, as well as their combination with tumor necrosis factor (TNF). A positive correlation between the caspase-3 activation velocity and drug concentration was observed when the cells were treated with cisplatin, but cells induced by camptothecin and etoposide did not show any apparent correlation with their concentrations. Moreover, different types of cells presented distinct sensitivities under the same drug treatment, and the combination treatment of TNF and anticancer drugs significantly accelerated the caspase-3 activation process. Its high throughput capability and detection sensitivity make the FRET-based CE system a useful tool for investigating the mechanisms of anticancer drugs and anticancer drug screening.     

The Use of a Stably Expressed FRET Biosensor for Determining the Potency of Cancer Drugs

Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.     

Imaging FRET between spectrally similar GFP molecules in single cells

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.     

利用荧光共振能量转移技术监测高通量低能量激光诱导细胞凋亡过程中caspase-3活性的动态变化

荧光共振能量转移可用于对生物大分子之间的距离进行定性、定量检测。应用荧光共振能量转移技术对高通量低能量激光诱导肺腺癌细胞凋亡过程中caspase-3的激活过程进行实时动态监测。实验结果表明:高通量低能量激光可以诱导肺腺癌细胞(human lung adenocarcinoma cell,ASTC-a-1)凋亡。同时荧光共振能量转移技术是一个有效的监测caspase-3在凋亡过程中活性动态变化的方法。     

Characterization of Puma-dependent and Puma-independent neuronal cell death pathways following prolonged proteasomal inhibition

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model.     

Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFalpha-induced apoptosis

In this study, we reported the first measurement of the dynamics of activation of caspase-8 in a single living cell. This measurement was conducted using a specially developed molecular sensor based on the FRET (fluorescence resonance energy transfer) technique. This sensor was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a linker containing a tandem caspase-8-specific cleavage site. The change of the FRET ratio upon cleavage was larger than 4-fold. Using this sensor, we found that during TNFalpha-induced apoptosis, the activation of caspase-8 was a slower process than that of caspase-3, and it was initiated much earlier than the caspase-3 activation. Inhibition of caspase-9 delayed the full activation of caspase-3 but did not affect the dynamics of caspase-8. Results of these single-cell measurements suggested that caspase-3 was activated by caspase-8 through two parallel pathways during TNFalpha-induced apoptosis in HeLa cells.     

FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death

Two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) and emission spectral imaging (ESI) are powerful tools for fluorescence resonance energy transfer (FRET) measurement. In this study, we use these two techniques to analyze caspase-3 activation inside single living cells during anticancer drug-induced human lung adenocarcinoma (ASTC-a-1) cell death. TPE-ESI of SCAT3, a caspase-3 indicator based on FRET, was performed inside single living cell stably expressing SCAT3. The TPE-ESI measurement was performed using 780 nm excitation which was considered to selectively excite the donor ECFP of SCAT3 by measuring the emission ratio of 526 to 476 nm. The emission peak at 526 nm disappeared and that of 476 nm increased after STS or bufalin treatment, but taxol treatment did not induce a significant change for the SCAT3 emission spectra, indicating that caspase-3 was activated during STS- or bufalin-induced cell apoptosis, but was not involved in taxol-induced PCD. Fluorescence lifetime of ECFP inside living cells was acquired using FLIM. The lifetime of ECFP was the same as that of the control group after taxol treatment, but increased from 1.83 ± 0.02 to 2.05 ± 0.03 and 1.90 ± 0.03 ns, respectively after STS and bufalin treatment, which agree with the results obtained using TPE-ESI. Taken together, TPE-FLIM and ESI analysis were proved to be valuable approaches for monitoring caspase-3 activation inside single living cells. W. Pan and J. Qu contributed equally to this study.     

Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix-targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis.     

Integrity of the methylation cycle is essential for mammalian neural tube closure

BACKGROUND: Closure of the cranial neural tube during embryogenesis is a crucial process in development of the brain. Failure of this event results in the severe neural tube defect (NTD) exencephaly, the developmental forerunner of anencephaly. METHODS: The requirement for methylation cycle function in cranial neural tube closure was tested by treatment of cultured mouse embryos with cycloleucine or ethionine, inhibitors of methionine adenosyl transferase. Embryonic phenotypes were investigated by histological analysis, and immunostaining was performed for markers of proliferation and apoptosis. Methylation cycle intermediates s-adenosylmethionine and s-adenosylhomocysteine were also quantitated by tandem mass spectrometry. RESULTS: Ethionine and cycloleucine treatments significantly reduced the ratio of abundance of s-adenosylmethionine to s-adenosylhomocysteine and are, therefore, predicted to suppress the methylation cycle. Exposure to these inhibitors during the period of cranial neurulation caused a high incidence of exencephaly, in the absence of generalized toxicity, growth retardation, or developmental delay. Reduced neuroepithelial thickness and reduced density of cranial mesenchyme were detected in ethionine-treated but not cycloleucine-treated embryos that developed exencephaly. Reduced mesenchymal density is a potential cause of ethionine-induced exencephaly, although we could not detect a causative alteration in proliferation or apoptosis prior to failure of neural tube closure. CONCLUSIONS: Adequate functioning of the methylation cycle is essential for cranial neural tube closure in the mouse, suggesting that suppression of the methylation cycle could also increase the risk of human NTDs. We hypothesize that inhibition of the methylation cycle causes NTDs due to disruption of crucial reactions involving methylation of DNA, proteins or other biomolecules.     

Auxin-induced Rapid Degradation of Inhibitor of Caspase-activated DNase (ICAD) Induces Apoptotic DNA Fragmentation,Caspase Activation,and Cell Death: A CELL SUICIDE MODULE*

Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms.     

Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein

Activation of effector caspases is a final step during apoptosis. Single-cell imaging studies have demonstrated that this process may occur as a rapid, all-or-none response, triggering a complete substrate cleavage within 15 min. Based on biochemical data from HeLa cells, we have developed a computational model of apoptosome-dependent caspase activation that was sufficient to remodel the rapid kinetics of effector caspase activation observed in vivo. Sensitivity analyses predicted a critical role for caspase-3-dependent feedback signalling and the X-linked-inhibitor-of-apoptosis-protein (XIAP), but a less prominent role for the XIAP antagonist Smac. Single-cell experiments employing a caspase fluorescence resonance energy transfer substrate verified these model predictions qualitatively and quantitatively. XIAP was predicted to control this all-or-none response, with concentrations as high as 0.15 microM enabling, but concentrations >0.30 microM significantly blocking substrate cleavage. Overexpression of XIAP within these threshold concentrations produced cells showing slow effector caspase activation and submaximal substrate cleavage. Our study supports the hypothesis that high levels of XIAP control caspase activation and substrate cleavage, and may promote apoptosis resistance and sublethal caspase activation in vivo.     

Intracellular signaling dynamics during apoptosis execution in the presence or absence of X-linked-inhibitor-of-apoptosis-protein

X-linked-inhibitor-of-apoptosis-protein (XIAP) is the most potent intracellular inhibitor of caspases-9, -3 and -7. While highly elevated XIAP levels reduce the apoptotic response to various stimuli, the potency of physiological XIAP expression to control caspase activation and the consequences of XIAP deficiency on apoptosis execution remain controversial. We therefore analyzed parental and XIAP-deficient DLD-1 and HCT-116 colon cancer cells by employing fluorescence-based single-cell imaging of mitochondrial permeabilisation and effector caspase activation. Our results demonstrate that physiological XIAP expression maintains a transient "off"-state for effector caspase activation following mitochondrial permeabilisation. Loss of XIAP expression instead accelerated the caspase activation response, but did not enhance the measured caspase activity. Apoptosis execution kinetics were independent of activating the intrinsic or extrinsic pathway by either staurosporine or TRAIL, and corresponded to computational systems analyses of caspase activation dynamics. We confirmed a protective role of XIAP upstream of mitochondrial permeabilisation during TRAIL-induced apoptosis, however, once mitochondria permeabilised ultimately no cell could escape effector caspase activation, regardless of potential cell-to-cell variability within the populations or the presence of XIAP. Our study provides comprehensive kinetic and mechanistic insight into the rapid molecular dynamics during apoptosis execution in the presence or absence of physiological XIAP expression.     

Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis

Caspase activation and proteolytic cleavage of specific target proteins represents an integral step in the pathway leading to the apoptotic death of cells. Analysis of caspase activity in intact cells, however, has been generally limited to the measurement of end-point biochemical and morphological markers of apoptosis. In an effort to develop a strategy with which to monitor caspase activity, early in the cell death cascade and in real-time, we have generated cell lines that overexpress recombinant GFP-based caspase substrates that display a quantifiable change in their spectral properties when cleaved by group II caspases. Specifically, tandem GFP substrates linked by a caspase-sensitive cleavage site show diminished fluorescence resonance energy transfer (FRET), as a consequence of cleavage, due to physical separation of the GFP moieties in apoptotic cells. We have evaluated the influence of different caspase-sensitive linkers on both FRET efficiency and cleavage by caspase-3. We also demonstrate that caspase activity as well as inhibition by pharmacological agents can be monitored, with minimal manipulation, in intact adherent cells seeded in a 96-well cell culture dish. Finally, we have adapted this technology to a high throughput screening platform to identify novel small molecule and cell permeable inhibitors of apoptosis. Based on a biochemical analysis of the compounds identified it is clear that this assay can be used to detect drugs which inhibit caspases directly as well as those which target upstream components of the caspase cascade.     

Imaging calpain protease activity by multiphoton FRET in living mice

Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions.     

Single cell analysis of PKC activation during proliferation and apoptosis induced by laser irradiation

Laser irradiation has been shown to trigger cellular proliferation and apoptosis in various cell types. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. In present study, to monitor the protein kinase Cs (PKCs) activity in living cells in real time, we transfected and screened human lung adenocarcinoma cells (ASTC-a-1) stably expressing C kinase activity reporter (CKAR) constructed based on fluorescence resonance energy transfer (FRET) technique. The CKAR is a specific, reversible reporter of phosphorylation by PKCs and it can monitor the ongoing balance between PKCs and phosphatases. The increasing dynamics of PKCs activity is monitored during cell proliferation induced by low-power laser irradiation (LPLI) (0.8 J/cm2) in serum-starved ASTC-a-1 cells stably expressing CKAR reporter using FRET imaging on laser scanning confocal microscope and using spectrofluorometric analysis on a luminescence spectrometer, respectively. However, the decreasing dynamics of PKCs activity has been monitored in real time using FRET imaging for the cells treated with high fluence LPLI (60 J/cm2), which was previously found to induce cell apoptosis. Taken together, LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs. However, PKCs activity decreases during cell apoptosis induced by high fluence LPLI. Our results indicate that PKCs play an important role in the laser irradiation-induced biological effects.