Assessing Autophagic Flux in 2D and 3D Cell Culture Models with a Novel Plate-Based Assay

1266-B 1323-D 1236-B www.promega.com • Decrease of trafficking to proteasome and increase in protein levels 0 2 4 6 8 0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 N L -B R D 4 H E K 2 9 3 S ta b le + H T -P S M D 3  M d B E T 1 B R E T R a tio (m B U ) 0 0 2 1 0 0 6 4 1 0 0 6 6 1 0 0 6 0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 N L -B R D 4 H E K 2 9 3 S ta b le + H T -P S M D 3  M d B E T 1 R L U 0 0 .0 0 .5 1 .0 1 .5 2 .0 0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 N L -B R D 4 H E K 2 9 3 S ta b le + H T -C R B N  M d B E T 1 B R E T R a tio (m B U ) 0 0 1 0 0 0 0 0 0 2 0 0 0 0 0 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 5 0 0 0 0 0 0 0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 N L -B R D 4 H E K 2 9 3 S ta b le + H T -C R B N  M d B E T 1 R L U 0 0 2 0 4 0 6 0 8 0 5 1 0 1 5 c M y c R e c ru itm e n t to P ro te a s o m e T im e (m in .) B R E T R a tio (m B U ) N L -P S M D /H T -c M y c + 1 0  M C D 5 3 2 N L -P S M D /H T -c M y c + D M S O Monitoring functional mechanisms of protein degradation in living cells Abstract and introduction Monitoring interactions with E3 Ligases HiBiT Technology for endogenous studies Protein:Protein interactions in living cells Monitoring proteosomal trafficking Degradation studies with β-catenin Summary Here we present mechanistic cellular studies on several proteins targeted for degradation via the ubiquitin proteasome pathway in mammalian cells. Using bioluminescence resonance energy transfer with NanoLuc® and HaloTag® fusions, termed NanoBRET, we can monitor changes in interactions of proteins targeted for degradation, including dynamic recruitment to E3 ligases and real-time trafficking to the 26S proteasome using inhibitors or PROTAC based compounds. We show also the ability to quantitate protein levels over a significant dynamic range by monitoring NanoLuc luminescent levels (RLUs) of several proteins, either expressed exogenously or as endogenous knock-in fusion proteins using CRISPR/Cas9. Live cell monitoring of protein:protein interactions and degradation is shown for BRD4, β-catenin, cMyc, and HIF1α. These combined approaches deconvolute the complicated processes involved in proteosomal recruitment and are a powerful strategy for understanding and following protein degradation. NanoBRET™ Protein:Protein Interactions Bioluminescence Resonance Energy Transfer A:B interaction Luminescent Donor Protein A Fluorescent Acceptor Protein B + NanoLuc (NL) HaloTag (HT) Energy Transfer  NanoBRET sensitivity allows for detection of decrease of HIFα at proteasome after phenanthroline treatment  Significant range of HIF1α stabilization measured  HiBiT, 11aa, allows for rapid tagging of endogenous proteins via CRISPR/Cas9 Kristin Riching, Steven Edenson, Sarah Mahan, Jacqui L. Méndez-Johnson, Nancy Murphy , Chris Eggers, Brock Binkowski, Marie Schwinn, Thomas Machleidt, Keith Wood, Danette L. Daniels, and Marjeta Urh Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, U.S.A. Dose response degradation of BRD4 NL-B-catenin Levels 0 5 10 15 20 25 30 No compound AZD2858 (10uM) 0 200,000 400,000 600,000 800,000 1,000,000 1,200,000 1,400,000 1,600,000 No compound AZD2858 (10uM) mBU RLU 0 2 4 6 8 10 12 14 16 18 20 No compound AZD2858 (10uM)  Measure decrease and correlative protein interaction increases after treatment with B-catenin stabilizer compound AZD2858 AZD2858 Selective GSK3 inhibitor and Wnt signaling activator NanoBRET for real-time kinetic interactions with 26S Proteasome NL-cMyc HT-PSMD cMyc:PSMD + CD532 +/- CD532 – CD532  Measure changes in proteosomal recruitment of cMyc after addition of AURKA inhibitor, CD532  CD532 known to increase ubiquitination of cMyc and promote degradation NanoBRET for real-time kinetic interactions with E3 Ligases 0 5 0 1 0 0 1 5 0 0 5 1 0 1 5 2 0 2 5 N L -B R D 4 / H T -C R B N T im e (m in .) B R E T R a tio (m B U ) 1  M dB E T 1 D M S O NL-BRD4 HT-CRBN BRD4:CRBN + dBET1 – dBET1  Detect the induced and increased interaction of BRD4 with Cereblon E3 ligase after treatment with dBET1  Able to use the technology to study small molecule or PROTAC induced interactions with E3 Ligases Protein Interaction BRD4:PSMD Simultaneous measurement of BRD4 proteasomal interaction with BRD4 protein levels +/- dBET1 Protein Degradation NL-BRD4 Levels mBU RLU  Determine EC50 for BRD4:Proteasome and IC50 for loss of BRD4  In the same experiment follow new interaction as well as protein level Following interactions of β-catenin in the Wnt signaling pathway βcatenin:PSMD βcatenin:Tcf3 βcatenin Levels HiBiT (11 a.a.) LgBiT (17.6 kDa) -20 -19 -18 -17 -16 -15 -14 -13 -12 -11 1 2 3 4 5 6 7 8 9 10 11 log HiBiT or NanoLuc (moles) log RLUs, background subtracted HaloTag-HiBiT NanoLuc  High affinity interaction between HiBiT and LgBiT (KD = 700 pM) drives complementation in lysates or cells  HiBiT can be an N- or C-terminus fusion tag expressed either exogenously or endogenously via CRISPR/Cas9  Shows a 7-log range for detection of protein levels Detection of HiBiT Fusion proteins in Lysates NanoLuc® Binary Complementation Endogenously tagged HiBiT-HIF1α  Improved signal: background, decreased overlap  Greater assay window as compared to other BRET systems  NanoLuc enables BRET at low expression levels  Ratiometric, highly reproducible assay with excellent z’ factor mBU HIF1a-NL RLUs D M S O 1 0 0  M P h e n a n th ro lin e 0 .0 0 .5 1 .0 1 .5 2 .0 H e L a H if1 a -H iB iT 1 :1 L g B iT + H T -P S M D 3 B R E T R a tio (m B U ) D M S O 1 0 0  M P h e n a n th ro lin e 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 H e L a H if1 a -H iB iT 1 :1 L g B iT + H T -P S M D 3 R L U Proteasome recruitment and cellular protein quantitation of CRISPR/Cas9 endogenously tagged clonal HiBiT-HIF1α Protein Interaction HIF1α:LgBiT:PSMD Endogenous Stabilization HiBiT-HIF1α Levels  NanoBRET technology is a highly sensitive method for detecting live cell protein:protein interactions  NanoBRET assays for protein degradation can monitor in real time:  Protein:E3 ligase recruitment  Protein:Proteasome trafficking  PROTAC induced interactions  NanoLuc and/or HiBiT can be used for quantitative monitoring in cells of proteins targeted for degradation  HiBiT technology is highly amenable for use in CRISPR/CAS9, allowing for the study of endogenously tagged proteins dBET1 HiBiT technology consists of an 11amino acid high affinity peptide which complements with a larger fragment, LgBiT, to generate NanoLuc luminescence Research use only, not for diagnostic use Corresponding author: kristen.riching@promega.com 1349-E www.promega.com Real-Time High Throughput Detection of Annexin V Binding and Caspase-3 Activity Using a Plate Reader Terry Riss, Kevin Kupcho, John Shultz, Jim Hartnett, Robin Hurst, Wenhui Zhou, Michael R. Slater, Brock Binkowski, Ryutaro Akiyoshi and Andrew Niles Promega Corporation, Madison, WI, 53711 and 2Olympus Corp., Tokyo, Japan Abstract # February 2018 1. Introduction 4. Real-Time Imaging of Luminescent Annexin V Binding and Fluorescent DNA Dye Staining 6. Comparison of Real-Time GloSensor™ 30F and Endpoint Caspase-Glo® 3/7 Assays 2. Real-Time Detection of Apoptosis using Annexin V Enzyme Complementation Assay 5. Real-Time Live Cell Detection of Caspase Activity 7. Performance of GloSensor™ Caspase Assay Delivered at Different MOI using BacMam 3. Real-Time Detection of Apoptosis and Necrosis 8. Conclusions The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay is a homogeneous method to detect the kinetics of phosphatidylserine (PS) exposure as a marker of apoptosis and DNA staining to detect necrosis. The GloSensor™ luciferase contains DEVD sequence that restricts the conformation of the molecule in an inactive confirmation. Caspase cleavage at DEVD activates luciferase to generate a real-time luminescent signal. U20S were forward transduced by the addition of BacMam GloSensor™ 30F Caspase particles at ratios of 30, 60, 120 and 240 particles per cell (multiplicity of infection, MOI) for 16 h. Serial dilutions of recombinant human TRAIL were added to replicate wells to induce an apoptotic response during an 18 h exposure. Bright-Glo™ lytic reagent was added to reveal the magnitude of GloSensor™ 30F activation for each MOI. • The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay detects the kinetics of apoptosis and secondary necrosis in real time using a plate reader. • The assay requires one homogenous reagent addition step and no other washes or processing steps. • The non-lytic assay reagents enable multiplexing with other assay chemistries such as endpoint methods to measure caspase-3/7 activity as an orthogonal marker of apoptosis. • The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay enables luminescent time lapse imaging of cells undergoing apoptosis to illustrate heterogeneity among the population of cells and the “real-time” kinetics of apoptosis. • The GloSensor™ Caspase-3/7 Biosensor containing the DEVD sequence is expressed in cells in an inactive form. • During apoptosis, caspase-3/7 cleavage at the DEVD sequence enables folding of GloSensor™ luciferase to form an active enzyme to generate light and detection of apoptosis in real-time. • Stable expression of GloSensor™ luciferase or BacMam delivery of plasmid can be used to generate cells for this real-time caspase assay. A plate containing U2OS cells was forward transduced for 18 hr with BacMam GloSensor™ 30F Caspase particles at a MOI of 60, then exposed to serial dilutions of rhTRAIL prepared in medium containing luciferin. Luminescence was recorded from the GloSensor™ plate at indicated times. Six parallel plates were prepared with U20S cells treated with the same rhTRAIL concentrations. Caspase-Glo ® 3/7 Reagent (endpoint lytic assay) was added to one of the six parallel plates at the indicated times (3.5-72hr) and luminescence recorded. Corresponding author: terry.riss@promega.com Progression from normal to apoptosis to secondary necrosis Multiplexing real-time apoptosis (annexin V binding to PS) and secondary necrosis (DNA staining dead cells) assays from the same sample of cells. The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay reagent was added once at time zero to DLD-1 cells treated with rhTRAIL. Luminescence from annexin V binding indicating apoptosis (blue) and fluorescence from DNA staining indicating necrosis (green) were recorded repeatedly from the same samples. 1 hr after staurosporine exposure. No staining. 3:45 hr. Note slight cell shape change. 6 hr. Cell membrane is decorated with Annexin V fusion proteins enabling reconstitution of luciferase to generate light indicating apoptosis. 9 hr. Fluorogenic DNA dye has penetrated cell membrane to stain nucleus green indicating 2° necrosis. 12 hr. More intense staining of nucleus with DNA dye. Luminescent signal remains on outer membrane. U2OS cells were cultured in presence of Apoptosis and Necrosis Assay reagent, exposed to 1 µM staurosporine and photographed at indicated times using fluorescent and luminescent modes with an Olympus LV200 microscope. Data provided by Olympus. We developed two homogeneous real-time assays for detecting apoptosis that can be recorded using a plate-reading luminometer. The first assay is based on binding of annexin V to phosphatidyl serine (PS) which becomes exposed on the outer leaflet of the cell membrane during apoptosis. We engineered two fusion proteins composed of annexin V linked to a small or large subunit of NanoBiT® luciferase. When the fusion proteins bind in close proximity on the surface of apoptotic cells, the reconstituted NanoBiT generates light to report apoptosis in real-time. Multiplexing with a DNA binding dye reports 2° necrosis in real-time from the same sample. The second approach relies on expression of a modified firefly luciferase (GloSensor™ Caspase-3/7 Biosensor) containing the DEVD amino acid sequence that is cleaved by caspase-3. GloSensor is inactive in viable cells. Upon induction of apoptosis, caspase-3 cleavage of the DEVD sequence enables GloSensor to fold into an active conformation and generate a luminescence in real-time as the population of cells undergoes apoptosis. These real-time homogeneous apoptosis assay methods represent an improvement over endpoint assay methods by providing kinetic data from the same sample of live cells in real time using a standard plate reading luminometer. For Research Use Only. Not for use in diagnostic procedures. Caspase-Glo® 3/7 Endpoint Assay GloSensor TM Real-Time Assay www.promega.com Real-Time Image Analysis of Apoptotic to Necrotic Process in the Same Cells by Microscopy Ryutaro Akiyoshi1 , Mitsunori Ota2 , Tsutomu Kudoh2 , Kevin Kupcho2 , Thomas Machleidt 2 , Andrew Niles2 and Hirobumi Suzuki1 1Olympus Corporation, 2-3 Kuboyama-cho, Hachioji-shi, Tokyo, Japan; 2Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711 Abstract # 437650 February 2018 1. Introduction 4. Real-Time Imaging of Apoptotic to Necrotic Process in the Same Cells by Microscopy 5. Time-Course Analysis of Apoptotic to Necrotic Process 3. Real-Time Detection of Apoptosis using Annexin V Enzyme Complementation Assay 6. Conclusions To detect externalization of PS and secondary necrosis (late apoptosis), we applied RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay Kit (Promega corp.) to the U2OS stable cell line in usage as prescribed in the kit protocol. This kit contains equal ratios of two annexin V molecules expressed as fusions with large or small subunits of NanoBiT luciferase (Promega corp.) and time-released substrate. When PS is externalized on the cell membrane, annexin V binds to PS as a dimmer on the cell surface and NanoBiT Luciferase is reconstructed, and bioluminescence light (λmax=463 nm) is generated. This kit also contains a cell-impermeant, profluorescent DNA dye, which detects necrosis by fluorescent light (λmax=525nm) with the dye binding to nuclear DNA. • Our imaging method makes possible real-time analysis of the apoptotic to necrotic process by visualizing the same cell samples using VivoGlo™ Caspase-3/7 Assay and RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay. • This method revealed heterogeneous responses of apoptotic to necrotic process in individual cells, and that suggests importance of single cell analysis. • However, this result does not conflict with total cell analysis, and that would also strengthen plate-reader based screening results. Namely, before and after screening, reliability of the assay conditions and efficacy of the candidates can be confirmed by image analysis. Corresponding author: ryutaro_akiyoshi@ot.olympus.co.jp Apoptosis is an indispensable process for normal tissue development and homeostasis, which allows cells to undergo timely programmed cell death. When apoptosis is induced, in most cases, caspase-3/7 is activated and the cell membrane is compromised by rearrangement of phospholipid. As activation of caspase-3/7 always leads to apoptotic death of the cells, the caspase activity is considered to be a reliable apoptosis marker. In addition, externalization of phosphatidylserine (PS) in the cell membrane is also a typical apoptosis marker. Therefore, the two markers have been assessed by enzymatic and flow cytometric endpoint assays. However, major disadvantages of these assays are (1) it obtains only a single result for each set of endpoints, and therefore (2) it is impossible for real-time measurement of live cells. In order to overcome these disadvantages, we combined bioluminescence and fluorescence microscopy and tried to detect the apoptotic to necrotic process on the same live cell samples. 2. Real-Time Live Cell Detection of Caspase Activity We created a stable U2OS cell line that expresses Luc2 luciferase (Promega). To detect caspase-3/7 activity, the aminoluciferin incorporating the DEVD (Asp-Glu-Val-Asp) motif recognized by the caspase-3/7 (VivoGlo™ Caspase-3/7 substrate, Promega) was added into the culture medium. When caspase-3/7 is activated, liberated luciferin reacts with Luc2 and generates bioluminescent light (609nm). CMV luc2 CMV::luc2 construct U2OS cells Establishment of stable cell line Transfection Add into the medium Final conc. 1mM Induction of apoptosis luc2 VivoGlo substrate is cleaved by caspase-3/7 to produce aminoluciferin Light VivoGlo substrate (463nm) DNA dye (525nm) We observed apoptosis and necrosis of the U2OS cells by sequential bioluminescence and fluorescence imaging using a microscope, LV200 (OLYMPUS) after induction of apoptosis by 100nM staurosporine (STS). Fluorescence and bioluminescence signals from single cells were measured as an average value in a region of interest (ROI) enclosed for each cells of Fig.1 by Time-lapse Imaging Analysis software (TiLIA, Olympus). Similarly, signals in the whole visual field were measured as a plate reader-like analysis. (a) 0.5h 12h 18h 0h (e) (b) (f) (c) (g) (d) (h) 0h 0.5h 12h 18h Fig. 1 (a)-(d) Bioluminescence images of caspase-3/7 activity (red) and annexin V dimmerization (blue) indicating apoptotic process from 0 to 18h. (e)-(h) Fluorescence images of nucleus (green) indicating necrosis merged with phase contrast image from 0 to 18h. 0h: (a) No bioluminescence signals. (e) No morphological changes of the cells. 0.5h: (b) Caspase-3/7 activity increased as apoptotic initiation. (f) Cells were shrunk slightly. 12h: (c) Annexin V-NanoBiT dimmerization occurred. (g) Nucleus of penetrated cells were stained as 2nd necrosis. 18h: (d) Annexin V dimmerization signal decreased as cell membrane compromise. (h) Nucleus of all cells were stained as 2nd necrosis. Measurement conditions: The U2OS stable cells were seeded on a 35mm glass bottom dish. Luminescence images were acquired by the luminescence imaging system LUMINOVIEW (LV200, Olympus) attached with an electron multiplier charge-coupled device camera (ImagEM, Hamamatsu Photonics) binning 1×1 and EM-gain 1200. Each images were taken by 40x phase contrast objective lens [numerical aperture (NA) 0.75] at 100msec exposure (Phase contrast), 100msec exposure (fluorescence, Ex: BP490-500, Em: BP515-560), 3min exposure (Annexin V-NanoBiT, BP460- 510 filter), 5min exposure (luc2-caspase-3/7, 610ALP filter), 10min interval. Duration time of observation was 24 hours. The dish was kept at 37ºC under 5% CO2 in the humidified chamber during observation. Fig. 2 (a) Time-course analysis of bioluminescence and fluorescence signals in the whole area of the image (from total cells ). Necrosis Dye and NanoBiT signals were plotted in the primary axis, VivoGlo signals were shown in the secondary axis. Caspase-3/7 activity reached the first peak within 1 h after STS addition. Annexin V dimmerization signal rose in 1 h and reached the maximum in 12 h, and then decreased. Secondary necrosis signal rose in 7 h and increased for 24 h. (b)-(k) Time-course analysis for each cell in Fig. 1 (ROI 1 to 10). Caspase-3/7 activity reached the first peak within 1 h, and annexin V dimmerization signal rose in 1 h as same as total cells analysis (a). However, signal profile of annexin V dimmerization varied among cells and the maximum peak time from 9 to 14 h. Secondary necrosis signal rose after the maximum peak time of annexin V dimmerization. (a) 100μm (d) (b) (c) (e) (i) (j) (k) (f) (g) (h) 1212-C 1257-C 1358-D