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REAL-TIME ANALYSIS OF TRAIL/CHX-INDUCED CASPASE ACTIVITIES DURING APOPTOSIS INITIATION

Christian T Hellwig1, Barbara F Kohler 1, Anna-Kaisa Lehtivarjo2, Heiko Dussmann1, Michael J Courtney2, Jochen HM Prehn1, Markus Rehm 1*

From Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland1; Department of Neurobiology, University of Kuopio, Kuopio,

Finland2 Running head: TRAIL-induced kinetics of apoptosis initiation

Address correspondence to: Dr. Markus Rehm, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, RCSI York House, York Street, Dublin 2, Ireland. Phone: +353-1-402-8563. Fax: +353-1-402-2447. E-mail: mrehm@rcsi.ie

Employing single-cell 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 signalling dynamics during apoptosis initiation via the 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 to 600 min after TRAIL/CHX addition. Caspase-8-like activities persisted for 15 to 60 min before eventually inducing mitochondrial outer membrane permeabilisation (MOMP). Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, MOMP 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 signalling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.

INTRODUCTION Tumour necrosis factor-related apoptosis inducing ligand (TRAIL)* is a potent cytotoxic ligand

* Abbreviations: Cyan fluorescent protein (CFP); cycloheximide (CHX); Cytochrome c (cyt-c);

inducing apoptosis preferentially in tumour cells (1). New TRAIL-based treatment regimes for adjuvant chemotherapies therefore are currently being studied in phase I and II clinical trials (2). TRAIL binding to its cognate death receptors TRAIL-R1 and -R2 induces receptor trimerization. At their cytoplasmic domains, TRAIL-R1 and -R2 recruit the adaptor protein Fas-associated death domain (FADD) into the so called death inducing signalling complex (DISC). Via interaction of their death effector domains, FADD recruits procaspases-8 and -10 to the DISC, resulting in activation and processing of these initiator proteases (3). While in some cell lines caspases-8/-10 can directly activate effector caspase-3 (type I signalling) (4), the majority of cells require caspases-8/-10 to initiate apoptosis by cleaving the BH-3 only protein Bid (type II signalling). Truncated Bid (tBid) then translocates to mitochondria and induces Bax/Bak-dependent mitochondrial outer membrane permeabilization

Death inducing signalling complex (DISC); Fas-associated death domain (FADD); Fluorescence resonance energy transfer (FRET); Green fluorescent protein (GFP); Mitochondrial membrane potential (∆ΨM); Mitochondrial outer membrane permeabilisation (MOMP); Staurosporine (STS); Tetramethyl-rhodamine-methylester (TMRM); truncated Bid (tBid); Tumour necrosis factor-related apoptosis inducing ligand (TRAIL); X-linked inhibitor of apoptosis protein (XIAP); Yellow fluorescent protein (YFP); z-Val-Ala-Asp(O-methyl)-fluoromethyl ketone (zVAD-fmk).

http://www.jbc.org/cgi/doi/10.1074/jbc.M802889200The latest version is at JBC Papers in Press. Published on June 3, 2008 as Manuscript M802889200

Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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(MOMP) (5-8). MOMP results in the release of mitochondrial intermembrane space proteins such as cytochrome-c and Smac from the mitochondria into the cytosol, mitochondrial depolarisation and subsequent apoptosis execution by effector caspases, such as caspase-3, -7, and -6 (9-11). Independent of the choice of stimulus or the dose applied, the induction of MOMP and the subsequent execution of apoptosis by effector caspases were shown to be kinetically invariant all-or-none signalling processes that guarantee cell death over a wide range of key protein concentrations. (9,12,13). In the case of effector caspase activation, this switch-like response was shown to emanate as a systems property from feedback signalling loops in the caspase activation network (13,14). In contrast, little is known about the intracellular signalling dynamics during apoptosis initiation leading to the kinetically invariant activation of MOMP. Especially, since it is conceivable that both at physiological as well as therapeutic conditions cells can be exposed to TRAIL receptor ligands over a wide range of different concentrations, it is unclear how this variability can be translated into a clear cell death decision. Here, we address this question by using a fluorescence resonance energy transfer (FRET)-based approach to monitor the real time kinetics of caspase-8/-10 activation and activity via the TRAIL receptor pathway using single cell time-lapse imaging. We found that TRAIL exposure induced dose-dependent kinetics of caspase-8/-10 activation and activity. We furthermore identified a threshold mechanism that permits to integrate such differential caspase activities into an all-or-none decision of apoptosis execution. Importantly, we performed these analyses excluding the potential contribution of high abundant downstream effector caspases with similar substrate specificities.

EXPERIMENTAL PROCEDURES Materials. Embryo-tested mineral oil and cycloheximide (CHX) were from Sigma-Aldrich (Tallaght, Dublin, Ireland). TRAIL was from Leinco Technologies (St. Louis, Missouri, USA). TMRM was from MobiTec (Göttingen). Staurosporine (STS) was from Alexis (San Diego,

CA, USA). The broad spectrum caspase inhibitor z-Val-Ala-Asp(O-methyl)-fluoromethyl ketone (z-VAD-fmk) was purchased from Bachem (St Helen’s, UK). G418 was from Invitrogen (Paisley, UK). Molecular Cloning of the IETD FRET probe. A DEVDase-responsive ECFP-DEVD-Venus FRET cassette was obtained from pSCAT3 (15) (a generous gift of Masayuki Miura, RIKEN Brain Research Institute, Wako, Saitama, Japan) by digestion with BamHI and HindIII. This was subcloned into pTK-RL (Promega, WI), digested at the same sites and treated with shrimp alkaline phosphatase (SAP, US Biologicals/Amersham Pharmacia Biotech). The resulting plasmid was digested with SacI and KpnI to remove the DEVDase substrate cassette and ligated with excess annealed oligos forming an IETDase substrate cassette. This IETD cassette was generated using the following complementary oligonucleotides 5’-G AGC GGA ATC GAG ACC GAT GGTAC-3’ and 5’-C ATC GGT CTC GAT TCC GCT CAGCT-3’. This generated a cassette consisting of a coding sequence for IETD flanked NH2-terminally by a flexible glycine-serine dipeptide and COOH-terminally by a KpnI site (encoding glycine-threonine), and a flexible serine-glycine-serine tripeptide. The oligo sequences immediately adjacent to the overhangs were designed to destroy the SacI site upon ligation with corresponding vector overhangs, and to generate a diagnostic PvuII site to exclude multiple concatenated oligos. The resulting pTK-reverse SCAT8 vector was digested with BamHI and HindIII to obtain the ECFP-IETD-Venus FRET cassette. This was ligated into pcDNA3.1 vector to generate pSCAT8. Cell Culture and Transfection. All cells were cultured in RPMI 1640 medium supplemented with penicillin (100 µg/ml), streptomycin (100 µg/ml) and 10 % fetal calf serum (Sigma-Aldrich). Cells were transfected with 500 ng of pSCAT8 plasmid DNA, and 4 µl Metafecten (Biontex Laboratories, Munich, Germany) per milliliter of serum-free medium at 37 °C for 4 h. For the generation of stable cell lines cells were selected in

the presence of 50 µg/ml G418 (Invitrogen) for 3-4 weeks, and fluorescent clones were enriched.

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Generation of stable Bid depleted cells. Three different shRNA sequences specific for human Bid mRNA were designed using the Dharmacon siRNA design tool (http://www.dharmacon.com/sidesign/). The following 19-nucleotide sequences were chosen and subjected to BLAST analysis to avoid significant homology to other human genes: Bid-1 sense (5’AAGCTGTTCTGACAAC AGC-3’) corresponding to nt 78-97 downstream of the Bid mRNA start codon, Bid-2 sense (5’- AAGGAGAAGACCATGCTGG-3’) homologous to nt 430-449, and Bid-3 sense (5’- AAGAATAGAGGCAGATTCT-3’spanning nt 210-229. The Bid specific shRNA duplexes along with a scrambled control sequence were ligated into the pSilencer 2.1-U6 hygro vector (Ambion, Cambridgeshire, UK) via their BamH I and Hind III sites. To generate stable knock down cell lines HeLa cells were transfected with the different shRNA constructs using Metafectene (Biontex, Munich, Germany) according to the manufacturer’s instructions. 24h post transfection the cells were serially diluted, transferred to 96-well plates and stable clones were selected using hygromycin B (160 µg/ml). Bid expression in HeLa cells stably expressing Bid siRNA was compared to the Bid expression in parental HeLa cells by western blotting and the clone with the strongest Bid depletion selected for this study. After background subtraction, chemiluminescence intensities from n = 3 independent whole cell protein extracts were densitometrically measured using AlphaEase FC software (Alpha Innotech, San Leandro, CA, USA). Adenoviral Infection. The generation of adenoviruses for XIAP expression and control viruses as well as the infection procedure were described previously (13,16). In brief, parental and Bid-depleted HeLa cells expressing the SCAT8 FRET probe were grown on WillCo dishes (Willco BV, Amsterdam, The Netherlands). Cells were washed twice with PBS and infected at m.o.i. (multiplicity of infection) of 1,000 in serum free medium for 2 hrs and subsequently cultured in full growth medium. Experiments were carried out >24 hrs post infection. Preparation of Whole Cell Extracts and Western Blotting. Cells were collected at 1000 rpm for 3

min and washed with phosphate-buffered saline (PBS). The cell pellet was resuspended in lysis buffer (62.5 mM Tris-HCl, pH 6.8, 10 % (v/v) glycerin, 2 % (w/v) SDS, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin

A, 1 µg/ml leupeptin, and 5 µg/ml aprotinin) and heated at 95 °C for 20 min. Protein content was determined with the Pierce Micro-BCA Protein Assay (Pierce, Northumberland, UK). An equal amount of protein (20 µg) was loaded onto SDS-polyacrylamide gels. Proteins were separated at 100 V for 2.5 h and then blotted to nitrocellulose

membranes (Protean BA 83; 2 µm; Schleicher & Schuell, Dassel, Germany) in transfer buffer (25 mM Tris, 192 mM glycine, 20 % methanol (v/v) and 0.01 % SDS) at 18 V for 60 min. The blots were blocked with 5 % non-fat dry milk in TBST (15 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 0.1 % Tween 20) at room temperature for 1 h. Membranes were incubated with the following antibodies: a rabbit polyclonal caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA); a mouse monoclonal caspase-8 antibody (Alexis Biochemicals, San Diego, CA, USA); a mouse monoclonal GFP antibody (Clontech Laboratories, Palo Alto, CA); a goat polyclonal Bid antibody (R&D Systems, Abingdon, UK); a mouse monoclonal α-tubulin antibody (Sigma-Aldrich); a mouse monoclonal β-actin antibody (Sigma-Aldrich). Membranes were washed with TBST three times for 5 min and incubated with anti-mouse or anti-rabbit peroxidase-conjugated

secondary antibodies (Jackson Laboratories, PA, USA) for 1 h. Blots were washed and developed using the enhanced chemiluminescence detection

reagent (Amersham Biosciences, Buckinghamshire, UK). Chemiluminescence was detected at 12 bit dynamic range using a Fuji LAS 3000 CCD system (Fujifilm UK Ltd., Bedfordshire, UK). Determination of caspase-3-like protease activity. After exposure to staurosporine, TRAIL/CHX or vehicle, culture medium was aspirated and cells were lysed in 200 µl lysis buffer (10 mM HEPES pH 7.4, 42 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5% CHAPS and 1% protease inhibitor cocktail (Sigma-Aldrich)). Fifty microliters of this extract were added to 150 µl reaction buffer (25 mM HEPES pH 7.5, 1 mM EDTA, 0.1% CHAPS, 10% sucrose,

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3 mM DTT). The reaction buffer was supplemented with 10 µM Ac-DEVD-AMC, the preferred fluorigenic substrate for effector caspases-3 and -7. Production of fluorescent AMC was monitored over 120 min using a fluorescent plate reader (excitation 360 nm, emission 465 nm). Autofluorescence of blanks containing lysis buffer only were subtracted. Protein content was determined using the Pierce Coomassie (Bradford) Protein Assay Kit (Pierce Biotechnology, Illinois, USA). Caspase activity was expressed as change in fluorescent units per hour and per µg protein. Epifluorescence Microscopy and Digital Imaging. Cells were cultivated on 12 mm glass-bottom dishes (Willco BV, Amsterdam, The Netherlands) in 1 ml of medium for at least overnight to let them attach firmly. Cells were equilibrated with 30 nM TMRM in 200 µl RPMI 1640 medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% fetal calf serum, buffered with N-2-hydroxyl piperazine-N'-2-ethane sulfonic acid (HEPES, 10 mM; pH 7.4), covered with mineral oil, and placed in a heated (37°C) incubation chamber that was mounted on the microscope stage. Cells were treated on stage with 1 µM STS or 10 – 1000 ng/ml TRAIL/1 µg/ml CHX. Fluorescence was observed using an Axiovert 200 M inverted microscope equipped with a 40x NA 1.3 oil immersion objective (Carl Zeiss, Jena, Germany), polychroic mirror and filter wheels in the excitation and emission light path containing the appropriate filter sets (CFP, excitation 436 ± 10 nm, emission 480 ± 20 nm; YFP, excitation 500 ± 10 nm, emission 535 ±15 nm; FRET, excitation 436 ± 10 nm, emission 535 ± 30 nm; TMRM, excitation 530 ± 25 nm, emission 592.5 ± 22.5 nm; dichroic mirrors for CFP (FRET), YFP and TMRM; Semrock, Rochester, NY, USA). Images were recorded using a back illuminated, cooled EM CCD camera (Andor Ixon BV 887-DCS, Andor Technologies, Belfast, Northern Ireland). The imaging setup was controlled by MetaMorph 7.1r1 software (Molecular Devices Ltd., Wokingham, UK). Confocal Microscopy and acceptor bleaching. To confirm resonance energy transfer within the IETD FRET probe, the acceptor Venus was bleached and the donor dequenching was analyzed. HeLa cells expressing the FRET probe were placed on the

heated stage of a LSM 510 Meta confocal microscope equipped with a Plan-Apochromat 63x NA 1.4 oil immersion DIC objective (Carl Zeiss, Jena, Germany). The emission spectra were recorded at 10.3 nm step size using the 405 nm laser line (attenuated to 1.0 %), the 405/514 multichroic beamsplitter and the Meta detector in the range of 442-614 nm. Following each cycle of bleach scans with the non-attenuated 514 argon laser line (laser was run at 50 % of its maximal power), spectral images were recorded. Mitochondrial membrane potential (∆ΨM) and FRET disruption. After background subtraction, the cellular TMRM fluorescence intensity was calculated for each cell. Caspase cleavage kinetics were detected at the single-cell level by FRET analysis as described previously (12). Images were processed using MetaMorph 7.1r1 software (Molecular Devices Ltd., Wokingham, UK). CFP/YFP and FRET/YFP emission ratio traces were obtained by dividing the fluorescence intensity values in the CFP and FRET channels by the YFP emission of single cells after background subtraction. The ratiometric readout automatically corrects for unspecific changes in fluorescence intensities that affect all channels in parallel such as slight drifts in optical focus or cellular movements. Statistics. Data are given as means ± s.d. or s.e.m. For statistical comparison Student’s t test or ANOVA and subsequent Tukey test were used for normal distributed data. Otherwise Mann-Whitney U test or Kruskal-Wallis H test were used. P values smaller than 0.05 were considered to be statistically significant. Kruskal-Wallis H tests returning statistical significances were subsequently followed up with Bonferroni adjusted Mann-Whitney U tests.

RESULTS Acceptor bleaching confirms resonance energy transfer in a new IETDase FRET probe. Previously, recombinant FRET probes based on green fluoresecent protein (GFP) variants were successfully employed to analyze effector caspase activity in single living cells (12,13,15,17). Here, we developed a new FRET probe based on cyan fluorescent protein (CFP) and Venus, an enhanced

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yellow fluorescent protein, which are interconnected by a short linker containing a preferred caspase-8/-10 recognition site IETD (18,19). Upon CFP excitation, energy is transferred to the acceptor fluorophore Venus (Fig.1A). Cleavage of the linker disrupts the resonance energy transfer as the distance between donor and acceptor increases and results in enhanced CFP emission (Fig.1B). The probe was generated from a previously described FRET probe template for effector caspases which provided a very high signal to noise ratio upon proteolytic cleavage (15). This high signal to noise ratio facilitates the detection of low caspase activities as expected in the case of caspase-8/-10 during apoptosis initiation. Efficient resonance energy transfer was observed when expressing the IETD FRET probe in HeLa cervical cancer cells: Upon photobleaching the acceptor fluorophore Venus in individual cells, CFP emission significantly increased as observed in cellular fluorescence emission profiles (Fig. 1C-F). Bid depletion can prevent apoptosis execution but not IETD probe cleavage following TRAIL exposure. Biochemically it has been shown that besides caspases-8 and -10, downstream effector caspases-3 and -6 can cleave IETD recognition sites as well (19). Similar overlapping specificities were reported for synthetic caspase inhibitors such as IETD- and DEVD-fmk, effectively preventing the selective inhibition of individual caspases (18,19). Using the above described new IETD FRET probe, we therefore investigated whether we could establish a model system that enabled us to temporally separate the downstream execution phase from caspase-8/-10 dependent signalling during the initiation phase of TRAIL-induced apoptosis. HeLa cells are type II signalling cells and consequently depend on the mitochondrial pathway for the activation of effector caspases during death receptor induced apoptosis (20). We generated HeLa cells stably depleted of Bid expression by siRNA transfection to impair MOMP and subsequent effector caspase activation (Fig.2A). Bid expression was reduced to approx. 4.5 +/- 3.0 % of the wild type expression level as densitometrically quantified from Western blots.

To investigate whether Bid depletion was sufficient to impair effector caspase activation during death receptor induced apoptosis, we next biochemically characterized the response of parental and Bid depleted HeLa cells to TRAIL/CHX (hereafter referred to as TRAIL) and intrinsic apoptosis stimulus staurosporine (STS). TRAIL was administered in combination with 1 µg/ml CHX to suppress the activation of translation dependent survival signalling in response to TRAIL exposure (3). In both parental and Bid depleted cells procaspase-8 was processed into active subunits with apparently identical kinetics in response to TRAIL (Fig.2B). While parental cells can activate caspase-3, Bid depleted HeLa cells did not show any reduction in procaspase-3 and active subunits were only detected in minute amounts (Fig.2B,C). To analyze whether the observed caspase-3 processing results in caspase-3-like activity, we employed fluorigenic DEVD-AMC substrate assays. In these assays Bid depleted HeLa cells exhibited massively reduced cleavage rates following death receptor stimulation with TRAIL, suggesting that downstream apoptosis execution is significantly inhibited in these cells (Fig.2D). In contrast, Bid depletion did not influence the activation of caspase-3 or caspase-8 (Fig.2E), which is largely activated downstream of caspase-3 during STS induced apoptosis (20). Neither was caspase-3 like activity significantly affected in fluorigenic assays (Fig.2F). Next, we tested whether the IETD FRET probe can still be cleaved in Bid depleted cells during TRAIL induced apoptosis. Following TRAIL exposure, the full length probe was cleaved into fragments corresponding to the molecular sizes of CFP-linker and Venus, as shown by Western blotting (Fig.2G). Addition of caspase inhibitor zVAD-fmk blocked FRET substrate cleavage, showing this to be a caspase dependent process (Fig.2G). Taken together these results show that Bid depletion efficiently inhibited apoptosis execution in response to TRAIL addition. Bid depletion however did not impair the processing of procaspase-8 neither did it prevent IETD FRET probe cleavage. TRAIL-induced caspases-8-like activities can persist for hours without causing immediate apoptotic cell death in Bid depleted HeLa cells.

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To analyze profiles of caspase-8-like activities in single living cells by time-lapse imaging, we then expressed the IETD FRET probe in the Bid depleted HeLa cells. Following administration of TRAIL, we observed that probe cleavage set on individually in single cells rather than it being a synchronous response throughout the whole population in the field of view (Fig.3A). Probe cleavage was detected as an intensity increase in the CFP/YFP emission ratio images (Fig.3A) and a decrease in FRET/YFP emission ratio images (not shown). Previous studies in HeLa cells have shown a loss in mitochondrial membrane potential (∆ΨM) in response to MOMP-induced cyt-c release (9,11,21,22). Using tetramethyl-rhodamine-methylester (TMRM) as a ∆ΨM sensitive dye in parallel with the FRET probe, we found that in Bid depleted cells mitochondria either did not depolarize (Fig.3A) or only at very late times during or after FRET probe cleavage (not shown). Importantly, classical apoptotic morphology such as cellular shrinkage or membrane blebbing could not be observed as long as the cells did not depolarise. Corresponding to our biochemical analyses (Fig.2), this indicates that MOMP dependent mitochondrial depolarisation and apoptosis execution are efficiently inhibited by Bid depletion. We next plotted the CFP/YFP and FRET/YFP emission ratios of individual Bid depleted cells to graphically follow substrate cleavage over time. Probe cleavage set on following an initial lag time, as observed by an increase in the CFP/YFP and a concomitant decrease in the FRET/YFP ratios (Fig.3B). In response to 100 ng/ml TRAIL, probe cleavage on average set on 56 +/- 25 min after drug addition and lasted for 5.5 +/- 2.8 hours (data are mean +/- s.d. from n = 20 cells analysed). To exclude the possibility that type-I like direct activation of caspase-3 by caspases-8/-10 contributed to the activity measured (4), in additional control experiments we employed adenoviral overexpression of XIAP, the most efficient intracellular inhibitor of downstream caspases-3, -7, and -9. XIAP overexpression did not affect IETDase activity in Bid depleted cell in response to TRAIL (Supplementary Figure 1). These findings demonstrate that HeLa cells individually activate IETDases in response to

TRAIL exposure, and that this caspase-8-like activity can be a surprisingly stable response that can persist for hours without causing immediate apoptotic cell death. TRAIL-induced profiles of caspase-8-like activities upstream of MOMP in parental HeLa cells. We next analysed caspase-8-like activities in parental HeLa cells following TRAIL exposure. In contrast to Bid depleted cells, we observed that parental HeLa cells cleaving the FRET probe subsequently underwent cellular condensation and membrane blebbing characteristic for apoptotic cell death (Fig.4A). Furthermore, in parental cells substrate cleavage was accompanied by MOMP, as shown by pronounced mitochondrial depolarisation (Fig.4A). Plotting the temporal profiles for CFP/YFP and FRET/YFP ratios for individual cells indicated that after an initial lag time the IETD FRET probe is cleaved at a low rate until eventually the IETDase activity sharply increased. Comparing the highest slopes during times of low and high rates of substrate cleavage suggested that IETDase activity increased approx. 12-fold (n = 16 cells exposed to 100 ng/ml TRAIL analyzed) (Fig.4B). We then closer examined whether this increase in activity temporally was related to MOMP and mitochondrial depolarisation. Indeed, when analyzing the TMRM fluorescence and CFP/YFP ratios for individual cells we observed that probe cleavage increased very soon after onset of mitochondrial depolarisation (Fig.4C). On average, the increased rate in substrate cleavage was observed within 4.1 +/- 2.2 min after MOMP (mean +/- s.d. from n = 23 cells), thus closely resembling times between MOMP and effector caspase activation reported for HeLa cells before (13). All cells that activated IETDases proceeded through to MOMP and increased substrate cleavage, followed by cellular shrinkage and apoptotic membrane blebbing. These results, and the results shown in Fig. 3 and Supplementary Fig. 1 thus suggest that during TRAIL induced apoptosis, low IETDase activities attributable to initiator caspases caspases-8/-10 can be observed upstream of MOMP, and that IETDase activities downstream of MOMP dramatically increase within living cells. This increase in activity may be largely subject to

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activation of effector caspases during apoptosis execution. No detectable IETDase activities upstream of MOMP during activation of the intrinsic apoptosis pathway by STS. To further analyse whether the post-MOMP increase in IETDase activity indeed was attributable to engagement of the apoptosis execution phase, we performed additional analyses in parental and Bid depleted HeLa cells treated with intrinsic death stimulus staurosporine (STS) (Fig.5). It was previously reported that the activation of effector caspases following MOMP is largely independent of the concentration and type of stimulus used to induce apoptosis (11,12), so that we expected largely identical IETDase activities downstream of MOMP during TRAIL and STS induced apoptosis. In response to STS, mitochondrial depolarisation preceded the onset of IETDase activity in HeLa cells and the slopes of FRET substrate cleavage downstream of MOMP closely resembled those observed downstream of MOMP during TRAIL induced apoptosis (Fig.5A,B, Fig.4C). Similar results were observed in Bid depleted HeLa cells upon STS exposure (Fig.5C,D). No cells were observed showing IETDase activity upstream of MOMP. The presence or absence of Bid apparently did not affect the caspase activity during apoptosis execution, as the time required for complete substrate cleavage in response to STS was largely identical in both groups (Fig.5E). Similar results were obtained with a DEVD FRET probe optimized for effector caspase-3 and -7 activities (Supplementary Figure 2). IETDase activities upstream of MOMP show dose-dependent differences in onset of substrate cleavage and caspase activity. We next analyzed in more detail how extracellular variability in TRAIL stimulation manifests within individual cells. To this end we first investigated the apoptosis initiation phase in response to TRAIL concentrations ranging from 10 to 1000 ng/ml. For all cells measured we determined the following parameters from the temporal IETDase profiles (Fig.6A): (i) The time required from TRAIL addition to caspase-8 activation, which biologically comprises the processes from TRAIL binding to its respective receptors, receptor

trimerization and DISC formation. (ii) The time from caspase-8 activation until mitochondrial depolarisation, indicating how long caspase-8-like activity needs to persist to induce MOMP. We found that the lag time between stimulus addition and caspase-8/-10 activation was indeed a dose-dependent response which was kinetically saturated at high TRAIL concentrations (Fig.7A). At high doses, the caspase-8/-10 response was observed on average 45 min following drug addition. In contrast, at submaximal TRAIL concentrations the caspase-8/-10 response was delayed by approx. 10 hours and became increasingly asynchronous between individual cells (Fig.7B). Quantifying the duration of caspase-8/-10 activity until MOMP indicated that higher stimulus concentrations resulted in an accelerated MOMP response, indicating that higher doses of TRAIL result in higher caspase-8/-10 activities (Fig.6C). While at submaximal doses caspase-8/-10 activity persisted for approx. 50 min, in response to high TRAIL doses approx. 15 min of caspase-8/-10 activity were sufficient to induce MOMP. MOMP is induced at 10% IETD FRET substrate cleavage in response to high and low TRAIL concentrations, regardless of the large variability in onset of substrate cleavage and caspase activity. To provide an explanation how these differences in IETDase activities upstream of MOMP are translated into an all-or-none-response at the level of MOMP, we quantified additional parameters for all cells analysed: We determined the amount of cleaved IETD FRET substrate at onset of mitochondrial depolarisation as a measure for the integrated caspase-8/-10 activity over time (Fig 6A, iii). We also determined the time from mitochondrial depolarisation to the subsequent increase in IETDase activity, which reflects the duration from MOMP until activation of effector caspases (Fig.6A, iv). Evaluating the amount of FRET probe cleaved at the time of MOMP we found that independent of the TRAIL dose applied, mitochondrial depolarisation occurred when approx. 10 % of the FRET substrate was cleaved (Fig.8A). The subsequent time between MOMP and effector caspase activation was dose independent as well, confirming that following MOMP apoptosis

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signalling proceeded in an all-or-none fashion (Fig.8B).

DISCUSSION TRAIL was shown to induce apoptotic cell death in transformed cells with high selectivity, rendering this cytotoxic ligand a prime candidate as a drug for novel anti-cancer therapies (1,23). To provide insight into how the consequences of TRAIL exposure manifest within the complex biological environment inside living cells, we analysed the intracellular dynamics of caspase-dependent signalling during apoptosis initiation and execution by time-lapse imaging of IETDase activity profiles. Our study showed that while the upstream caspase-8-like activity during TRAIL induced apoptosis is clearly a dose dependent response, this dose dependency can be translated into a dose independent all-or-none response of cell death execution. Our data demonstrate that this is functionally achieved at a conserved threshold of substrate cleavage, reflecting the integration of caspase-8/-10 activity over time. Upon TRAIL stimulation, procaspases-8 and -10 are recruited to and activated at the DISC. Both caspases-8 and -10 were described as closely related IETDases with largely overlapping substrate specificities. However, it was shown that TRAIL-induced apoptosis initiation seems to be largely dominated by caspase-8, as caspase-10, even though recruited to the DISC, cannot compensate for a loss in caspase-8 expression (24-28). The majority of IETDase activity we observed during the apoptosis initiation phase therefore can probably be attributed to caspase-8. Measuring the IETDase activities of caspases-8/-10 in presence of the complexity of the full caspase signalling network is challenging since other caspases were shown to possess significant affinities towards IETD recognition sites as well. In vitro assays identified that effector caspases-3 and -6 exhibited activities towards the supposedly specific caspase-8/-10 recognition site IETD which were twice as high as the activities of caspases-8/-10 themselves (19,29). Similarly, commercially available synthetic caspase inhibitors were shown being too unspecific to selectively inhibit effector caspases as they affect caspases-8/-10 as well

(18,19). We observed an approx. 12-fold increase in IETDase activity following MOMP, which seems to support the hypothesis that the majority of intracellular IETDase activity reflects effector caspase activity. We cannot exclude the possibility that activation of additional caspase-8/-10 via caspase-3/-6 dependent feed back loops downstream of MOMP might contribute to this increased IETDase activity as well (30). However, this contribution may be relatively small as our biochemical characterizations suggested that the efficiency of procaspase-8 processing was independent of whether or not apoptotic signalling could proceed beyond MOMP (Fig.2). Our experiments demonstrate that the contribution of downstream effector caspases needs to be excluded when determining the activities of initiator caspases-8/-10 based on substrate cleavage. We accomplished this in living cells by separating initiation and execution phases by parallel detection of MOMP. Similar to the cell-to-cell asynchrony in apoptosis execution that can be observed for individual cells within a population (12,31), we found that also the upstream activation of initiator caspases is a response that occurs individually at the single cell level. These asynchronies were most prominent at submaximal TRAIL concentrations. Such asynchronies may arise from cell-to-cell differences in cell cycle phases or gene and protein expression patterns (32) and highlight the requirement to analyze signalling at the single cell level. A limited number of “activated” receptors at low TRAIL doses could reduce the probability of receptor trimerization. This stochastic factor could contribute to the observed cell-to-cell variability in caspase-8/-10 activation time. Several mechanisms may further reduce the level of activated receptors and increase the variability of the onset time of the response. TRAIL decoy receptors (TRAIL-R3 and TRAIL-R4) might negatively regulate the cellular responsiveness to TRAIL by scavenging TRAIL before it can bind to TRAIL-R1 or -R2. In addition, TRAIL-R4 was shown to oligomerize with TRAIL-R2, resulting in a non-functional DISC as a potential second level of regulation mediated by decoy receptors (33,34). Probably more important, the binding of c-FLIP, an inactive caspase-8 homologue, might significantly delay or even fully inhibit formation of mature DISC

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complexes if it is expressed sufficiently high in relation to procaspase-8 (35,36). Indeed, it was recently shown in type I cells that c-FLIP controls the apoptotic response in a threshold dependent manner which is most prominent following low dose stimulation of CD95 death receptors (37,38). While type I cells can activate apoptosis execution by direct positive feedback between caspases-8 and -3, type II cells such as HeLa cells require activation of MOMP for efficient apoptosis execution (20). The question therefore remains whether in type II cells the activation of caspase-8 at the DISC already represents a cell death decision. The caspase-8/-10 activities we observed were dependent on the TRAIL dose and therefore differed from the dose-independent all-or-none signalling during effector caspase activation downstream of MOMP. However, caspases-8/-10 activation was not a transient response but instead activities were detected to persist for hours in Bid depleted cells. This suggests that a cell death decision can be made at the level of DISC formation during TRAIL induced apoptosis, as long as the subsequent signalling steps leading to MOMP and apoptosis execution are not blocked. In our experiments we deliberately uncoupled caspase signalling from parallel transcription/translation dependent survival signalling pathways that can be activated upon TRAIL receptor stimulation by the addition of CHX. We therefore may have suppressed NF-κB and/or survival kinase signalling cascades which otherwise might have allowed for additional modification of the caspase-8/-10 response (39). Regardless of the TRAIL concentration used, MOMP coincided with a FRET probe cleavage of approx. 10 % under our experimental conditions. We found that at this threshold, dose-dependent signalling kinetics were translated into a dose-independent apoptosis execution. This finding suggests that the MOMP decision threshold could correspond to the accumulation of a conserved critical amount of tBid. The requirement of the signalling network for such a threshold arises from the biological necessity to be insensitive to low amounts of tBid. Such low levels of intrinsic proapoptotic noise could arise e.g. from zymogenic activities of procaspases or accidental caspase auto-activation (40) and could sufficiently be filtered out by a threshold mechanism. It is

noteworthy that the observed threshold for the MOMP decision was found at the lower end of the substrate cleavage range. This effectively means that HeLa cells have the possibility to cleave much more Bid than actually required for MOMP induction. This additional capacity can be crucial to override potential changes in the balance of pro- and anti-apoptotic Bcl-2 family members towards compositions that otherwise would not allow MOMP to proceed. Corresponding to our findings, previous studies have shown that indeed submaximal amounts of tBid seem sufficient to effectively induce mitochondrial permeabilisation and that a large proportion of Bid cleavage during extrinsically induced apoptosis seems to occur downstream of MOMP (6,41). However, post-translational modifications of Bid may additionally delay or inhibit MOMP in some scenarios. It has been reported that Bid phosphorylation as well as impaired degradation of the NH2-terminal fragment following Bid cleavage can further modify cellular susceptibility to active caspase-8/-10. (42-45). Indeed, non-lethal activation of caspases-8/-10 was shown to be a prerequisite for NF-κB activation during lymphocyte activation (46) as well as for monocyte and trophoblast differentiation (47-49). Correspondingly, our analyses in Bid depleted cells have shown that caspase-8/-10 activation in absence of MOMP in itself does not cause an immediate cell death response. In these scenarios caspase-8/-10 activation is clearly not a cell death decision. Based on analyses within living cells, our study thus provides mechanistic insight into how the initiation network of extrinsic apoptotic signalling is balanced to avoid unwanted induction of apoptosis execution and at the same time is sensitive enough to guarantee robust cell death responses following TRAIL administration.

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FOOTNOTES Acknowledgements: This research was supported by grants from the Academy of Finland (Grant 203520) to MJC, and from Science Foundation Ireland to JHMP (03/RP1/B344) and MR (05/RFP/BIM056; 07/RFP/BICF601). The authors wish to thank Dr. JB Schulz (University of Tübingen, Germany) and Dr. Masayuki Miura (RIKEN Brain Research Institute, Japan) for supplying plasmids and adenoviral vectors, and Aidan Spring for excellent technical assistance.

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FIGURE LEGENDS

Figure 1: Acceptor bleaching confirms resonance energy transfer in the IETDase FRET probe. (A) The intact probe. Energy is transferred from the excited CFP to the acceptor fluorophore Venus by resonance energy transfer. (B) Cleavage of the IETD linker region results in disruption of energy transfer and an increased CFP emission. (C-F) Acceptor bleaching confirms FRET in individual living cells. (C,D) HeLa cells expressing the IETD-FRET probe were monitored by spectral confocal microscopy and are displayed colour-coded according to their emission spectrum upon CFP excitation. In (D) the acceptor fluorophore Venus was selectively bleached from three individual cells, which resulted in increased emission in the blue wavelength range. The region for subsequent spectral analysis (see E,F) is shown in red. Scale bars = 10 µm. (E,F) Spectral analysis. Emission spectra from the region shown in (C) and (D) demonstrate an increase of CFP emission upon acceptor photobleaching. Figure 2: Caspase-8 dependent cleavage of the IETD FRET probe in living cells. (A) Stable knockdown of Bid expression in HeLa cells. HeLa cells stably expressing a Bid-specific siRNA or unspecific scrambled siRNA (ctrl) were probed for Bid expression by Western Blot analysis. Tubulin was used as a loading control. (B) Analysis of TRAIL responsiveness of parental and Bid-depleted HeLa cells. Time course analysis of cells treated with 100 ng/ml TRAIL/1 µg/ml CHX. As controls, cells were treated with TRAIL/CHX plus caspase-inhibitor z-VAD-fmk (50 µM). Whole cell extracts were analysed by immunoblotting for caspase-8 processing into p45 and p18 active subunits and for caspase-3 processing into p20 and p17 active subunits. β-actin was used as a loading control. (C) Comparison of caspase-3 processing into p20 and p17 active subunits in parental and Bid-depleted HeLa cells. Cells were treated with 100 ng/ml TRAIL/1 µg/ml CHX. β-actin served as a loading control. (D) Bid depletion impairs effector caspase activation upon TRAIL treatment. Following TRAIL treatment (100 ng/ml + 1 µg/ml CHX), caspase-3-like activity was measured in extracts of parental and Bid-depleted HeLa cells by cleavage of fluorigenic Ac-DEVD-AMC substrate. Data are means + s.d. from triplicates. * significant (p<0.05, Student’s t-test). Experiment was repeated with similar results. (E) Analysis of STS responsiveness of parental HeLa and Bid-depleted HeLa cells. Time course analysis of cells treated with 1 µM STS. As controls, cells were treated with STS plus caspase-inhibitor z-VAD-fmk (50 µM). Whole cell extracts were analysed by immunoblotting for caspase-8 processing into p45 and p18 active subunits and for caspase-3 processing into p20 and p17 active subunits. β-actin was used as a loading control. (F) Bid depletion does not impair effector caspase activity upon STS treatment. Following STS treatment for 4 hours, caspase-3-like activity was measured in extracts of parental and Bid-depleted HeLa cells by cleavage of a fluorigenic Ac-DEVD-AMC substrate. Data are means + s.d. from triplicates (n.s. = not significant; Student’s t-test). Experiment was repeated with similar results. (G) Cleavage of the IETDase FRET probe. Bid-depleted HeLa cells stably expressing the IETD-FRET probe were analyzed by immunoblotting. Probe expression and cleavage was analysed in control and TRAIL/CHX (100 ng/ml + 1 µg/ml) treated cells using an anti-GFP antibody. Caspase specificity of probe cleavage was analyzed by additional treatment with the broad spectrum caspase-inhibitor z-VAD-fmk (50 µM). β-actin was used as a loading control. Figure 3: TRAIL-induced profiles of intracellular IETDase activity in absence of MOMP. (A) Pseudo coloured CFP/YFP emission ratio images of a group of Bid-depleted HeLa cells expressing the IETDase FRET probe treated with TRAIL/CHX (100 ng/ml/1 µg/ml). IETDase activity resulted in FRET probe cleavage and displayed as an increase in the CFP/YFP emission ratio. TMRM fluorescence was used as an indicator for the mitochondrial membrane potential (∆ΨM). Time stamps indicate time after stimulus addition. Scale bar = 10 µm. (B) Quantitative analysis of time-lapse microscopy. The profiles of IETD FRET probe cleavage for three

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representative Bid-depleted HeLa cells were plotted against time. Probe cleavage caused an increase in the CFP/YFP emission ratio and a concomitant decrease in FRET/YFP emission ratio. Arrows indicate onset of IETDase activity. Similar traces were recorded for n = 20 cells. Figure 4: TRAIL-induced profiles of IETDase activities in parental HeLa cells. (A) Pseudo coloured CFP/YFP emission ratio images of a group of parental HeLa cells expressing the IETDase FRET probe. Cells were treated with TRAIL/CHX (100 ng/ml/1 µg/ml). IETDase activity resulted in FRET probe cleavage and displayed as an increase in the CFP/YFP emission ratio. Mitochondrial depolarisation indicates MOMP and displayed as a loss in TMRM fluorescence. Cellular membrane blebbing of apoptotic cells can be seen at late time points in CFP/YFP emission ratio images. Arrows in the TMRM images point out individual depolarised cells. Time stamps indicate time after stimulus addition. Scale bar = 10 µm. (B) Quantitative analysis of time-lapse microscopy. Profiles of IETD FRET probe cleavage for two representative HeLa cells following TRAIL treatment are shown plotted against time. Probe cleavage results in an increase in the CFP/YFP emission ratio and a concomitant decrease in FRET/YFP emission ratio. Arrows indicate onset of probe cleavage. After an initial period of low IETDase activity, a rapid increase in IETDase activity is observed. Similar traces were recorded for n = 23 cells. (C) For the cells shown in (B), the ∆ΨM profile (TMRM fluorescence) is shown in relation to the increase in IETDase activity (CFP/YFP emission ratio). Onset of mitochondrial depolarisation is indicated by grey arrows. Arrowheads indicate the time when IETDase activity increases. Similar traces were recorded for n = 23 cells. Figure 5: IETDase activities during STS-induced apoptosis execution. (A,B) Quantitative analysis of MOMP and IETDase activity in individual parental (A) and Bid-depleted (B) HeLa cells treated with 1 µM STS. The temporal ∆ΨM profiles (TMRM fluorescence) for representative cells are shown in relation to the increase in IETDase activity (CFP/YFP emission ratio). Onset of mitochondrial depolarisation and IETDase activation are indicated by grey and black arrows, respectively. Similar traces were recorded for n = 16 and 59 cells, respectively. (C,D) For the cells shown in (A) and (B), FRET disruption is shown by the increase in CFP/YFP emission and the concomitant decrease in FRET/YFP emission ratio. (E) IETDase activity following MOMP is independent of Bid expression. The time required for completion of FRET substrate cleavage was quantified for all parental and Bid depleted HeLa cells measured. Data are from n = 16 and 59 cells, respectively. n.s. = not significant, Mann-Whitney U test. Figure 6: Schematic presentation of all parameters quantified for individual parental HeLa cells during TRAIL/CHX-induced apoptosis. (A) For a typical cell, the graph shows IETD FRET probe cleavage as the CFP/YFP emission ratio in black and ∆ΨM as TMRM fluorescence intensity in grey. The following parameters were determined for each cell analysed: (i) The time from stimulus addition to caspase-8/-10 activation. (ii) The time from caspase-8/-10 activation to MOMP. (iii) The percentage of cleaved substrate at the time of MOMP. (iv) The time from MOMP to effector caspase activation. Figure 7: Dose-dependent differences in onset of IETD substrate cleavage and IETDase activity upstream of MOMP. HeLa cells were treated with doses of 10, 100, or 1000 ng/ml TRAIL plus 1 µg/ml CHX. (A) The time from stimulus addition to caspase-8/-10 activation was quantified for all parental or Bid depleted HeLa cells measured (n = 16-32 cells per group; data are shown means +/- s.d.). * significant delay in comparison to higher doses (p < 0.001); One way ANOVA. (B) The time from caspase-8/-10 activation to induction of MOMP was analyzed for all parental HeLa cells measured (n = 20-27 cells per group; data are means +/- s.e.m.). * significant (p < 0.01), Kruskal-Wallis H test followed by Bonferroni adjusted Mann-Whitney U tests.

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Figure 8: MOMP is induced at a dose-independent threshold of IETD FRET substrate cleavage. HeLa cells were treated with doses of 10, 100, or 1000 ng/ml TRAIL plus 1 µg/ml CHX. (A) The amount of cleaved FRET substrate at the time of MOMP was analyzed for all cells measured (n = 20-27 cells; data are shown as median +/- quartiles). n.s. = not significant; Kruskal-Wallis H-test. Circles indicate statistical outliers. (B) The time from MOMP induction to the increase in the rate of FRET probe cleavage, reflecting effector caspase activation, was analyzed for all parental HeLa cells measured (n = 20-27 cells; data are means +/- s.d.). n.s. = not significant, ANOVA and subsequent Tukey’s test.

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Supplementary Material: Figure Legend Supplementary Figure 1: XIAP over-expression inhibits IETDase activity downstream but not upstream of MOMP. (A) Profiles for ∆ΨM and IETD FRET probe cleavage following STS treatment of a Bid depleted HeLa cell infected with an adenovirus for XIAP overexpression. Following mitochondrial depolarisation as an indicator for MOMP, no IETDase activity was observed. Similarly, IETDase activity was either completely inhibited or significantly delayed and reduced following MOMP in n = 8 additional cells analyzed. Similar responses were reported before for parental HeLa cells overexpressing XIAP following adenoviral infection (13). Control infected cells instead responded just as non-infected HeLa Bid k/d cells (not shown). (B) Profiles for ∆ΨM and IETD FRET probe cleavage following 100 ng/ml TRAIL/ 1 µg/ml CHX treatment of a Bid depleted HeLa cell infected with an adenovirus for XIAP overexpression. No inhibition in IETDase activity was observed when compared to traces obtained from non-infected cells (Fig.3) or control infected cells (not shown). Mitochondrial depolarisation as an indicator for MOMP was not observed. Similar IETDase activities were recorded during apoptosis initiation for n = 5 additional cells. Figure Legend Supplementary Figure 2: Bid depletion does not impair DEVDase activity during STS induced apoptosis execution. (A) Duration of DEVDase substrate cleavage in individual HeLa cells. Parental or Bid depleted HeLa cells were transfected with a FRET probe containing a caspase-3 optimised DEVD cleavage site as described previously (12). Cells were exposed to 1 µM STS. Data from n = 18 and 20 cells are shown as average + s.e.m. Bid depletion did not impair substrate cleavage when compared to control cells (p = 0.39; Mann-Whitney U-test).

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[%]

CFP/YFP

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[TM

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ores

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FP/Y

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issi

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tio 6 min 60 min 180 min 300 min 360 minA

0

100

FRET-probe cleaved [%]

80

C

FP/Y

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FR

ET/

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emis

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[%]

0204060

10080

0

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Figure 5

Hela Bid k/d, 1 µM STS

parental Hela, 1 µM STSA B

C D

020406080

100

100 150 200 250 300 350

C

FP/Y

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FR

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FRET/YFP

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time after stimulus addition [min]

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C

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time after stimulus addition [min]

CFP/YFP

FRET/YFP0

20406080

100

120 150 180 210 240time after stimulus addition [min]

TMRM

CFP/YFP

CFP

/YFP

emis

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[%]

100175250325400475

∆

ΨM

[TM

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.]

020406080

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120 150 180 210 240time after stimulus addition [min]

TMRM

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CFP

/YFP

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Dur

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Figure 6

0

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0 100 200 300time after stimulus addition [min]

FRE

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i iiiii

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Figure 7

A

parentalBid k/d

Tim

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0100200300400500600700800900

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Figure 8

B

02468

101214161820

Tim

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[min

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TRAIL 10 100 1000 [ng/ml]

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A

0

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FRE

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J Courtney, Jochen HM Prehn and Markus RehmChristian T Hellwig, Barbara F Kohler, Anna-Kaisa Lehtivarjo, Heiko Dussmann, Michael

initiationReal-time analysis of TRAIL/CHX-induced caspase activities during apoptosis

published online June 3, 2008J. Biol. Chem. 

  10.1074/jbc.M802889200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2008/06/04/M802889200.DC1

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