Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

Summary

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

Abstract

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

Introduction

The caspase protease family are known for their critical roles in apoptosis and innate immunity ¹. Because of their importance, determining when, where, and how efficiently specific caspases are activated can provide crucial insights into the mechanisms of caspase activation pathways. The imaging based protocol described here enables the visualization of the earliest steps in the caspase activation cascade. This technique takes advantage of the dynamic protein:protein interactions that drive caspase activation.

The caspases can be divided into two groups: the initiator caspases (caspase-1, -2, -4, -5, -8, -9, -10, and -12) and the executioner caspases (caspase-3, -6, and -7). The executioner caspases are present in the cell as preformed dimers and are activated by cleavage between the large and small subunit ². When activated, they cleave numerous structural and regulatory proteins resulting in apoptosis ³. The initiator caspases are the first caspases to be activated in a pathway and generally trigger the activation of executioner caspases. In contrast to executioner caspases, initiator caspases are activated by dimerization ^4,5. This dimerization is facilitated by recruitment of inactive monomers to specific large molecular weight complexes known as activation platforms. Assembly of activation platforms is governed by a series of specific protein:protein interactions. These are mediated by conserved protein interaction motifs present in the proform of the initiator caspase and include the death domain (DD), death effector domain (DED) and caspase recruitment domain (CARD) ⁶ (Figure 1A). Activation platforms generally include a receptor protein and an adaptor protein. The receptor is usually activated upon binding of a ligand, inducing a conformational change that allows for the oligomerization of numerous molecules. The receptor then either recruits the caspase directly or adaptor molecules that in turn bring the caspase to the complex. Thus, numerous caspase molecules come into close proximity permitting dimerization. This is known as the induced proximity model ⁷. Once dimerized, the caspase undergoes autoprocessing, which serves to stabilize the active enzyme ^4,8. For example, assembly of the Apaf1 apoptosome is triggered by cytochrome c following its release from the mitochondria in a process called mitochondrial outer membrane permeabilization (MOMP). Apaf1 in turn recruits caspase-9 through an interaction that is mediated by a CARD present in both proteins ⁹. Similar protein interactions result in the assembly of the CD95 death inducing signaling complex (DISC) that leads to caspase-8 activation; the PIDDosome, which can activate caspase-2; and various inflammasome complexes that initiate caspase-1 activation ^10,11,12. Thus, initiator caspases are recruited to specific activation platforms by a common mechanism resulting in induced proximity and dimerization, without which activation will not occur.

Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based assay that was developed to measure this first step in the activation of initiator caspases, allowing direct visualization of caspase induced proximity following activation platform assembly. This method takes advantage of the properties of the split fluorescent protein Venus. Venus is a brighter and more photostable version of yellow fluorescent protein (YFP) that can be separated into two non-fluorescent and slightly overlapping fragments: the N-terminus of Venus (Venus N or VN) and the C-terminus of Venus (Venus C or VC). These fragments retain the ability to refold and become fluorescent when in close proximity ¹³. Each Venus fragment is fused to the prodomain of the caspase, which is the minimal portion of the caspase that binds to the activation platform. This ensures that the caspases do not retain enzymatic activity and therefore concurrent analysis of downstream events associated with endogenous events is possible. The Venus fragments refold when the caspase prodomains are recruited to the activation platform and undergo induced proximity. (Figure 1B). The resulting Venus fluorescence can be used to accurately and specifically monitor the subcellular localization, the kinetics, and the efficiency of assembly of initiator caspase activation platforms in single cells. Imaging data can be acquired by confocal microscopy or by standard fluorescence microscopy and can be adapted to a number of different microscopy approaches including: time-lapse imaging to track caspase activation in real time; high resolution imaging for precise determinations of subcellular localization; and end point quantification of efficiency of activation.

This technique was first developed to investigate the activation of caspase-2¹⁴. While the activation platform for caspase-2 is thought to be the PIDDosome, consisting of the receptor PIDD (p53-induced protein with a death domain) and the adaptor RAIDD (RIP-associated ICH-1/CAD-3 homologous protein with a death domain), PIDDosome-independent caspase-2 activation has been reported. This suggests that additional activation platforms for caspase-2 exist ^15,16. Despite not knowing the full components of the caspase-2 activation platform, the caspase BiFC technique has allowed for successful interrogation of the caspase-2 signaling pathways at the molecular level ^14,17. We have also successfully adapted this protocol for the inflammatory caspases (caspase-1, -4, -5 and -12) ¹⁸ and, in principle, the same approach should be sufficient to similarly analyze each of the remaining initiator caspases. This protocol could similarly be adapted to investigate other pathways were dimerization is a major activating signal. For example, STAT proteins are activated by dimerization following phosphorylation by Janus kinase (JAK) ¹⁹. Thus, the BiFC system could be used to visualize for STAT activation as well as many other pathways regulated by dynamic protein interactions. The following protocol provides step-by-step instructions for the introduction of the reporters into cells as well as methodologies for image acquisition and analysis.

Protocol

1. Preparation of cells and culture dishes

NOTE: Perform Steps 1-3 in a tissue culture laminar flow hood. Wear gloves.

1. If using glass bottom dishes, coat the glass with fibronectin. Skip to Step 1.2 if using plastic dishes.
   1. Make a 0.1 mg/mL solution of fibronectin: dilute 1 mL of 1 mg/mL fibronectin solution in 9 mL of 1 X PBS.
   2. Cover the glass bottom of each well with 0.5-1 mL of fibronectin and incubate for 1-5 min at room temperature.
   3. Using a pipette, collect the fibronectin solution and store at 4 °C.
      NOTE: The fibronectin solution can be reused multiple times.
   4. Wash the glass once with 1-2 mL 1 X PBS, and aspirate off PBS.
2. Plate 1 x 10⁵ cells per well of a 6-well plate in 2 mL of the appropriate cell culture media (see Table 1 for cell numbers to use for different sized wells). If using a cell line that stably expresses the caspase BiFC components (see Discussion), plate 2 x 10⁵ cells and proceed to the treatment step (Step 3).
3. Allow cells to adhere to the dish overnight at 37 °C in a tissue culture incubator.

2. Transfection of cells to introduce the caspase BiFC components

1. Transfect cells with the appropriate transfection reagent.
   NOTE: This protocol outlines the instructions for Lipofectamine 2000, which has been optimized for minimal toxicity. This protocol has been successfully used in Hela cells, MCF7 cells and LN18 cells. If using additional transfection reagents follow the manufacturer's instructions and optimize as needed.
   1. Add 12 µL of the transfection reagent to 750 µL of reduced serum media in a sterile tube.
   2. Incubate at room temperature for 5 min.
   3. Add 10 ng of a reporter plasmid (a plasmid encoding a fluorescent protein, e.g. red fluorescent protein [RFP], that will label the transfected cells) to a sterile 1.5 mL tube for each well to be transfected.
   4. Add 20 ng of each caspase BiFC plasmid (e.g., 20 ng of caspase-2 prodomain [C2 Pro]-VC and 20 ng of C2 Pro-VN) to each tube (Table 2).
   5. Bring each tube up to a total volume of 100 µL by adding reduced serum media.
   6. Using a p200 pipette, add 100 µL of transfection reagent solution from Step 2.1.2 to the plasmid mixture in a dropwise manner.
   7. Incubate the plasmid-transfection reagent mix for 20 min at room temperature.
   8. Aspirate the media from the cells using a pipette or with suction and then, using a p1000 pipette, pipette 800 µL of serum free media gently down the side of the well.
   9. Using a p200 pipette, add 200 µL of the DNA-lipid complex dropwise to each well.
   10. Incubate the cells in a tissue culture incubator at 37 °C for 3 h.
   11. Taking care not to disrupt the monolayer, remove the serum free media containing the DNA-lipid complexes by aspiration using suction or with a pipette.
   12. Pipette 2 mL of pre-warmed (37 °C) complete growth media gently down the side of each well.
2. Incubate the cells at 37 °C in a tissue culture incubator for 24-48 h for optimal protein expression.

3. Induction of caspase activation

1. Treat with a drug or stimulus that induces caspase activation approximately 24 h post-transfection.
   1. Add the desired concentration of the drug to pre-warmed (37 °C) imaging media (complete growth media supplemented with Hepes [20 mM, pH 7.2-7.5] and 2-mercaptoethanol [55 µM]) and mix gently (Table 3).
   2. Aspirate the media from the cells using a pipette or with suction and then, using a pipette, pipette 2 mL of the solution from 3.1.1 gently down the side of the well.
   3. Add imaging media without the drug to one well as an untreated control.
2. Incubate the cells in a tissue culture incubator at 37 °C or proceed to Step 4.3 for a time-lapse experiment.

4. Caspase BiFC data acquisition

1. Caspase BiFC for single time point acquisition
   1. Turn on the microscope and fluorescent light source following the manufacturer's instructions.
   2. Find the cells under the RFP filter and focus on the reporter gene fluorescence.
   3. Using a hand-tally counter, count all of the RFP-positive cells in the visual field. Record the number.
   4. While maintaining the same visual field, switch to the GFP filter (or YFP filter if available) and, using a hand-tally counter, count the number of red cells that are also green (Venus-positive). Switch back and forth between the two filters to ensure that only the red cells are evaluated for Venus-positivity. Record the number (Figure 3).
   5. Count at least 100 RFP-positive cells from each of three individual areas of the plate.
   6. In each area, calculate the percentage of Venus-positive transfected cells (i.e. red cells that are also green). Average the resulting percentages to get the standard deviation.
2. Three-dimensional imaging of caspase BiFC
   1. Turn on the microscope following the manufacturer's instructions and launch the acquisition software.
   2. Select the 60x or 63x oil objective and place a drop of oil on the objective
   3. Place the culture dish on the microscope stage using the correct plate holder.
   4. Using either an epifluorescence light source and the microscope eye piece or the confocal lasers and the computer screen, navigate to a field of interest.
   5. Focus on the reporter fluorescence using the RFP laser or filter.
   6. If epifluorescence has been used up to this point, switch the light source to the confocal lasers and visualize the live image of the cells as acquired by the camera and displayed by the acquisition software on the computer screen.
   7. Using the joystick control and the focus wheel, fine tune the focus and position of the cells so that the cells are in the center of the view finder. Choose cells that are separated from each other and are not overlapping.
   8. Input the percentage laser power to 50% and the exposure time at 100 ms for both Venus and RFP as a starting point to determine the optimal settings to use for the experiment.
   9. Turn on the live capture and inspect the resulting image and the accompanying display histograms for both channels.
   10. If the signal is low and the image is hard to make out, increase the percentage laser power and/or exposure time in increments until the signal in the image looks good.
   11. Ensure that the signal does not reach saturation. Inspect the display histogram to make sure a distinct peak is visible for each fluor. If the peak encompasses the entire histogram display, input lower laser power and exposure settings (Figure 2).
   12. Visualize control (untreated or non-transfected) cells under the same conditions to ensure that the signal is specific.
       NOTE: Single color transfectants can also be used to control for crossover, but if the signals are spatially distinct (e.g. mitochondrial versus cytosolic) this is not necessary.
   13. Select or open the Z stack module in the microscope software.
   14. Using the focus knob, approximate the focal position corresponding to the center of the cell.
   15. Adjust the focus in one direction until the cell is no longer visible, select this as the top position.
   16. Adjust the focus in the opposite direction until the cell is again no longer visible and select this as the bottom position.
   17. Choose the optimal step size (usually approximately 20 µm) and start the acquisition to instruct the camera to automatically take a single image in each channel at each of the 20 µm steps between the top and bottom positions.
   18. Use 3D rendering software to reconstruct the 3D image (Figure 4, Movie 1).
3. Time-lapse imaging of caspase BiFC
   1. At least 1 h prior to the experiment, turn on the microscope and set the temperature to 37 °C.
   2. Select the 40x, 60x, or 63x oil objective and place a drop of oil on the objective.
   3. Place the culture dish on the microscope stage using the correct plate holder.
   4. If a CO₂ source is available, place the CO₂ delivery device (usually a plexiglass lid attached to the tube that delivers the CO₂) on top of the plate holder. Set the CO₂ level to 5% and turn on the CO₂ controller from within the software. If a CO₂ source is not available, include 20 mM Hepes to buffer the media.
   5. Navigate to a field of transfected cells and visualize the live image of the cells as acquired by the camera and displayed by the acquisition software on the computer screen using the RFP laser.
   6. Following Steps 4.2.8-4.2.12, input the settings for percentage laser power and exposure time for the RFP laser. Keep these values as low as possible while still being able to detect the fluorescent signal.
   7. Using a positive control sample (see Table 3 for examples), follow Steps 4.2.8-4.2.12 to input the settings for percentage laser power and exposure time for the YFP laser light. Keep these values as low as possible while still being able to detect Venus signal.
   8. For each well of the plate, choose a number of different positions that contain one or more cells expressing the reporter.
   9. Input the time interval between each frame of the time-lapse and total number of frames to be taken. Ensure there is time to acquire all the selected positions before the next frame is to be taken.
   10. Re-visit each position and correct and update the focus as needed.
   11. To correct for focal drift that can result from changes in temperature or external vibrations, turn on the focus drift correction system (if available).
   12. Begin the time-lapse and save the data.
   13. Analyze the data using available imaging software (Figure 5, Movie 2).

Results

An example of caspase-2 BiFC induced by DNA damage is shown in Figure 3. Camptothecin, a topoisomerase I inhibitor, was used to induce DNA damage and caspase-2 activation. The red fluorescent protein mCherry was used as a reporter to show that the cells express the BiFC probe and to help visualize the total number of cells. Venus fluorescence is shown in green and the large puncta represent caspase-2 induced proximity. These cells can be counted to determine ...

Discussion

This protocol discusses the use of split fluorescent proteins to measure caspase induced proximity. Split Venus was chosen for this technique because it is very bright, highly photostable and the refolding is fast ¹³. Thus, the analysis of Venus refolding upon caspase induced proximity can provide close to real-time estimations of caspase protein interaction dynamics. Venus is split into two slightly overlapping fragments, the N terminus of Venus (Venus-N or VN) comprising amino acids 1-173 and th...

Materials

Name	Company	Catalog Number	Comments
6-well Uncoated No. 1.5 20 mm glass bottom dishes	Mattek	P06G-1.5-20-F
Human Plasma Fibronectin Purified Protein	Millipore	FC010-10MG
DPBS	Sigma	D8537-6x500ML
Lipofectamine 2000 reagent	Invitrogen	11668019
OPTI MEM I	Invitrogen	31985088
C2-Pro VC plasmid	Addgene	49261
C2-Pro VN plasmid	Addgene	49262
Inflammatory caspase BiFC plasmids			available by request from LBH
HeLa cells stably expressing the C2-Pro BiFC components			available by request from LBH
DsRed mito plasmid	Clontech	632421	similar plasmids that can be used as fluorescent reporters can be found on Addgene
HEPES	Invitrogen	15630106
2 Mercaptoethanol 1000X	Invitrogen	21985023
q-VD-OPH	Apex Bio	A1901