We would like to acknowledge all the previous members of the Bouchier-Hayes lab who contributed to the development of this technique. This work was funded in part by a Texas Children&#39;s Hospital Pediatric Pilot award to LBH. We thank Joya Chandra (MD Anderson, Houston, Texas) for permission to include data published in collaboration with her team. The development of the reagents described was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the NIH (NIAID P30AI036211, NCI P30CA125123, and NCRR S10RR024574) and the assistance of Joel M. Sederstrom

1. Preparation of cells and culture dishes
NOTE: Perform Steps 1-3 in a tissue culture laminar flow hood. Wear gloves.
<ol>
	<li>If using glass bottom dishes, coat the glass with fibronectin. Skip to Step 1.2 if using plastic dishes.
	<ol>
		<li>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.</li>
		<li>Cover the glass bottom of each well with 0.5-1 mL of fibronectin and incubate for 1-5 min at room temperature.</li>
		<li>Using a pip...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+mCherry+as+a+new+red+bimolecular+fluorescence+complementation+system+for+visualizing+protein-protein+interactions+in+living+cells.">Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells.</a> Biochem Biophys Res Commun. 367 (1), 47-53 (2008).</li><li>Chu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+far-red+bimolecular+fluorescence+complementation+system+that+allows+for+efficient+visualization+of+protein+interactions+under+physiological+conditions.">A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions.</a> Biosens Bioelectron. 25 (1), 234-239 (2009).</li><li>Karbowski, M., Youle, R. 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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...

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.

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

lighting up the pathways to caspase activation using bimolecular fluorescence complementation

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.

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 ...

Watch this Scientific Journal Video about Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation at JoVE.com

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

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.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

The overall goal of this methodology is to visualize one of the first steps in the activation of an initiator caspases. This step is called induced proximity and occurs when caspase molecules come together to form active dimers during apoptotic cell death. This method can help answer key questions in the caspase field such as when, where, and how efficiently specific initiator caspases can get be activated.The main advantage of this technique is that one can visualize the assembly of caspase activation complexes in living cells in real time. This biomolecular fluorescence complementation method uses fragments of the thyroxine protein, Venus, that have infused to a caspase. The fragments are not fluorescent on their own but when the caspase undergoes induced proximity, this brings the two Venus halves together and they light up.To transfect the cells, first add 12 microliters of the transfection reagent to 750 microliters of reduced serum media in a steril tube. Incubate the mixture at room temperature for five minutes. Then add 10 nanograms of a reporter plasmid to a steryl 1.5 milliliter tube for each well to be transfected.Add 20 nanograms of each caspase BiFC plasma to each tube. Bring each tube up to a total volume of 100 microliters by adding reduced serum media. Using a P200 pipette, add 100 microliters of transfection reagent solution to the plasmid mixture in a dropwise manner.After incubating the plasmid transfection reagent mix for 20 minutes at room temperature, aspirate the medium from the cells using a pipette or with suction. Then using a P1000 pipette, pipette 800 microliters of serum-free media gently down the side of the well. Using a P200 pipette, add 200 microliters of the DNA lipid complex dropwise to each well.Incubate the cells in a tissue culture incubator at 37 degrees Celsius for three hours. Following incubation, remove the serum-free media containing the DNA lipid complexes by aspiration using suction or with the pipette taking care not to disrupt the monolayer. Pipette two milliliters of pre-warmed complete growth media gently down the side of each well before incubating the cells as described in the text protocol.Prior to data acquisition, perform induction of caspase activation as described in the text protocol. Turn on the microscope following the manufactures instructions and launch the acquisition software. Select the 60x or 63x oil objective and place a drop of oil onto the objective.Then place the culture dish on the microscope stage using the correct plate holder. Turn on the heater and set the temperature to 37 degrees Celsius. Focus on the reporter fluorescence using the RFP laser or filter.Input the percent laser power at 50%and the exposure time at 100 milliseconds for both Venus and RFP as a starting point to determine the optimal settings to use for the experiment. Turn on the live capture and inspect the resulting image and the displayed histograms for both channels. 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 and the image looks good.Next, select or open the Z stack module in the microscope software. Adjust the focus in one direction until the cell is not longer visible. Select this as the top position.Adjust the focus in the opposite direction until the cell is again no longer visible and select this as the bottom position. Choose the optimal step size and start the acquisition. If needed adjust the display histogram to improve the visual appearance of the fluorescent signal.Finally, use three dimensional rendering software to reconstruct the 3D image. If a carbon dioxide source is available, place the carbon dioxide delivery device on top of the plate holder. Set the carbon dioxide level to 5%and turn on the carbon dioxide controller from within the software.Navigate to a field of transfected cells. 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. Next, input the settings for percentage laser power and exposure time for the RFP laser.Then, input the setting for percentage laser power and exposure time for the YFP laser light. For each well of the plate, choose a number of different positions that contain one or more cells expressing the reporter. Input the time interval between each frame of the time laps and total number frames to be taken.Revisit each position and correct and update the focus as needed. Begin the time lapse and save the data. Finally, analyze the data using available imaging software as described in the text protocol.Shown here is an example of caspase-2 BiFC following DNA damage. Cells were treated with a topoisomerase one inhibitor:Camptothecin. The red fluorescent protein, mCherry, was used as a reporter to show that the cells express the BiFC probe and help visualize the total number of cells.Venus fluorescence is shown in green and the large puncta represent caspase-2 induced proximity. To provide high resolution visualization of the subcellular localization of the caspase activation complex, single cells can be imaged in three dimensions. caspase-2 activation is shown here in green and the nucleus is shown in red.The orthoganal slices view of the cell is shown where the xy plane, the yz plane and the xz plane can be visualized side-by-side. The three dimensional image can also be displayed in a movie where the cell is rotated around the y axis. To provide kinetic data of caspase BiFC time lapse imaging can be used.This movie shows caspase-2 activation in green following treatment with the proteasome inhibitor:Bortezomib. The mitochondria are shown in red. After watching this video you should have a good understanding of how to use caspase BiFC to track the assembly of the caspase activation complexes in real time and to distinctly determine the size, shape and localization the complexes produced.This protocol outlines a few microscope based approaches to collecting the results to the caspase BiFC experiment;however, it should be noted that a number of variations and combinations of these approaches can be used to creatively interrogate caspase activation pathways. While attempting this procedure, it is important to remember to include controls from microscopy induced artifacts such as photo toxicity and photo bleaching. The accompanying text protocol includes tips to accomplish this.Deciding which acquisition and analysis method to use will be determined by the perimeters to be explored and the availability of instrumentation and imaging software. Following this procedure, imaging analysis approaches can be used to interpret the data. You can measure changes in fluorescence intensity over time or the percentage of fluorescent cells.Number of foci or object size can also be calculated.

lighting-up-pathways-to-caspase-activation-using-bimolecular

Research

JoVE Journal

Biochemistry

8.9K Views.  Baylor College of Medicine. The overall goal of this methodology is to visualize one of the first steps in the activation of an initiator caspases. This step is called induced proximity and occurs when caspase molecules come together to form active dimers during apoptotic cell death. This method can help answer key questions in the caspase field such as when, where, and how efficiently specific initiator caspases can get be activated.The main advantage of this technique is that one can visualize the assembly of c...