Volume 35, Issue 6, Pages 830-840 (September 2009) Characterization of Cytoplasmic Caspase-2 Activation by Induced Proximity  Lisa Bouchier-Hayes, Andrew Oberst, Gavin P. McStay, Samuel Connell, Stephen W.G. Tait, Christopher P. Dillon, Jonathan M. Flanagan, Helen M. Beere, Douglas R. Green  Molecular Cell  Volume 35, Issue 6, Pages 830-840 (September 2009) DOI: 10.1016/j.molcel.2009.07.023 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 C2-CARD BiFC Is Induced by Components of the PIDDosome (A) HeLa cells were transiently transfected with C2-CARD VN (20 ng) and C2-CARD VC (20 ng) along with 500 ng of expression plasmids encoding PIDD, RAIDD, FADD, or Apaf-1. All wells also received pshooter.dsRed-mito (10 ng) as a reporter for transfection. Twenty-four hours after transfection, the percentage of dsRed-mito-positive (red) cells that were Venus positive (green) was determined from a minimum of 300 cells per well. Results represent triplicate counts, with error bars representing standard deviation. (B) Representative confocal images of cells from (A) are shown. Scale bars represent 10 μm. (C) HeLa cells were transiently transfected as in (A) with the indicated amounts of expression plasmid. Forty-eight hours after transfection, the percentage of Venus-positive cells was determined as in (A). (D) Representative images of cells from (C) are shown. Scale bars represent 10 μm. Molecular Cell 2009 35, 830-840DOI: (10.1016/j.molcel.2009.07.023) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Induced Proximity of Caspase-2 Is Induced by Various Stressors (A) HeLa cells were transiently transfected with the indicated amounts of each of C2-CARD VC, C2-CARD VN, and pshooter.dsRed-mito (10 ng). Twenty-four hours later, cells were either left untreated or heated for 1 hr at 45°C. All cells were treated with qVD-OPH (20 μM). Cells were assessed 24 hr after treatment for the percentage of cells that were Venus positive (green), determined from a minimum of 300 cells per well. Results represent triplicate counts, with error bars representing standard deviation. (B) Representative images of cells from (A) are shown. Scale bars represent 10 μm. (C) Schematic representation of the caspase-2 BiFC constructs. (∗) indicates the active cysteine that was mutated to alanine in C2-FL. (D) HeLa cells were transiently transfected with expression plasmids with C2-CARD, C2-Pro (20 ng of each), or C2-FL BiFC plasmid pair (100 ng of each) along with pshooter.dsRed-mito (10 ng). Cells were treated and assessed as in (A). (E) HeLa cells were transiently transfected with plasmids encoding C2-CARD VC (20 ng), C2-CARD VN (20 ng), and dsRed-mito (10 ng) for 24 hr followed by treatment with anti-Fas (250 ng/ml)/CHX (10 μg/ml), TNF (10 ng/ml)/CHX (10 μg/ml), etoposide (5 μM), taxol (10 μg/ml), or vincristine (50 μg/ml) in the presence of qVD-OPH (20 μM). Representative confocal images were taken 24 hr after treatment. Scale bars represent 10 μm. (F) Cells were transfected as in (E) for 24 hr followed by treatment with etoposide (Etop, 5 μM), TNF (10 ng/ml)/CHX (10 μg/ml), taxol (Tax, 1 μg/ml) or heat shock (45°C, 1 hr), cytochalasin B (CytoB, 20 μg/ml), or vincristine (50 μg/ml), with qVD-OPH (20 μM). Cells were assessed as in (A). Molecular Cell 2009 35, 830-840DOI: (10.1016/j.molcel.2009.07.023) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Kinetics of Heat Shock-Induced C2-CARD BiFC (A) HeLa cells were transiently transfected with plasmids encoding C2-CARD VN (20 ng), C2-CARD VC (20 ng), and dsRed-mito (10 ng). Twenty-four hours later, cells were incubated at 45°C for 1 hr plus qVD-OPH (20 μM). The cells were returned to 37°C on a confocal microscope, and images were taken every 6 min for 16 hr. Frames from the movie show BiFC (green) of a representative cell. (B) HeLa cells were transfected and treated as in (A). Confocal images from a time-lapse with a 3 × 1.5 μm z stack are shown. Scale bars represent 10 μm. (C) HeLa cells were transfected as in (A). Twenty-four hours later, cells were either left untreated or heated at 42°C or 45°C for 1 hr with qVD-OPH (20 μM). The cells were returned to 37°C on a confocal microscope, and images were taken every 15 min for 16 hr. The average intensity of Venus was measured at each time point. Each line of the graph is an average of 21 (untreated), 35 (42°C), or 41 (45°C) individual cells, and error bars represent SEM. Scale bars represent 10 μm. Molecular Cell 2009 35, 830-840DOI: (10.1016/j.molcel.2009.07.023) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Heat Shock-Induced Caspase-2 BiFC Is Cytoplasmic (A) HeLa cells were transiently transfected with expression plasmids encoding C2-FL VC (100 ng) or C2-FL VN (100 ng) or with the indicated BiFC plasmid pairs (20 ng of each) along with dsRed-mito (10 ng). Twenty-four hours after transfection, cells were left untreated or heated at 45°C for 1 hr with qVD-OPH (20 μM). Cells were assessed for BiFC (green) 24 hr after treatment. (B) HeLa cells were transfected as in (A), and 24 hr after transfection, cells were either left untreated or treated with vincristine (25 μg/ml) with qVD-OPH (20 μM). Cells were assessed 24 hr after treatment. Nuclei were stained with Draq5 (blue). Images are 3D isosurface rendering reconstructions composed from 0.1 μ (A) or 0.2 μm (B) serial confocal images through the z plane of the cell. Scale bars represent 10 μm. Molecular Cell 2009 35, 830-840DOI: (10.1016/j.molcel.2009.07.023) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Heat Shock-Induced C2-CARD BiFC Requires RAIDD (A) D83A/E87A C2-CARD mutant binds RAIDD with less efficiency than wild-type. 293T cells were transiently transfected with plasmids encoding FLAG-RAIDD and HA-C2-CARD VC (WT), HA-C2-CARD VC D83A/E87A (MT), or a control protein (HA-FRB-VC). FLAG-RAIDD was immunoprecipitated from cell lysates with anti-FLAG-agarose and blotted for the presence of C2-CARD with an anti-HA antibody. (∗) indicates immunoglobulin light chain. (B) HeLa cells were transiently transfected with plasmids encoding C2-CARD VN (20 ng) and C2-CARD VC (20 ng) or the D83A/E87A mutant C2-CARD BiFC pair (20 ng of each) along with pshooter.dsRed-mito (10 ng), and 24 hr later, cells were incubated at 45°C for 1 hr with qVD-OPH (20 μM). The cells were returned to 37°C on a confocal microscope, and images were taken every 10 min for 20 hr. Each line of the graph represents the average intensity of Venus of 15 (untreated), 29 (WT C2-CARD, 1 hr at 45°C), or 22 (MT C2-CARD, 1 hr at 45°C) individual cells. Error bars represent SEM. (C) Representative images from the time lapse are shown. Scale bars represent 10 μm. (D) RAIDD−/− MEF and RAIDD+/+ MEFs from littermate embryos were transiently transfected with the indicated amounts of expression plasmids encoding C2-CARD pair and dsRed-mito (10 ng). Twenty-four hours posttransfection, cells were left untreated or heat shocked for 1 hr at 44°C with qVD-OPH (20 μM). The percentage of Venus-positive cells was determined at 24 hr from a minimum of 300 cells per well. Results represent triplicate counts, with error bars representing standard deviation. (E) RAIDD−/− and RAIDD+/+ MEFs were transiently transfected with the plasmids encoding the C2-CARD Venus pair (250 ng of each) and dsRedmito (10 ng) with the indicated amounts of an expression plasmid encoding RAIDD. Twenty-four hour posttransfection cells were left untreated or heat shocked for 1 hr at 44°C with qVD-OPH (20 μM). The percentage of Venus-positive cells was determined as in (D). (F) Representative images of cells from (E) are shown. Scale bars represent 10 μm. Molecular Cell 2009 35, 830-840DOI: (10.1016/j.molcel.2009.07.023) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Caspase-2 Is Activated by Induced Proximity Upstream of Mitochondria to Induce Apoptosis (A) HeLa cells or HeLa cells stably expressing Bcl-xL were heated for 1 hr at 45°C. Apoptosis was assessed by flow cytometry for Annexin V binding at the indicated times. Error bars represent standard deviation. (B) HeLa or HeLa.Bcl-xL cells were transiently transfected with plasmids encoding C2-CARD pair (20 ng of each) along with dsRed-mito (10 ng) and 24 hr later were heated at 45°C for 1 hr with qVD-OPH (20 μM). Cells were returned to 37°C on a confocal microscope, and images were taken every 15 min for 16 hr. The average intensity of Venus was measured at each time point. Results are an average of 21 (HeLa) and 21 (HeLa-Bcl-xL) individual cells. Error bars represent SEM. (C) HeLa or HeLa.Bcl-xL cells that were stably expressing vector or caspase-2 fused to FKBP dimerization domain were left untreated, treated with ActD (1 μM) or the dimerization drug (AP20187, 200 nM) for 24 hr. Apoptosis was assessed as in (A). (D) HeLa cells stably expressing Omi-mCherry were transiently transfected with expression plasmids encoding C2-CARD VN (20 ng) and C2-CARD VC (20 ng). Twenty-four hours later, cells were incubated at 45°C for 1 hr. The cells were returned to 37°C on a fluorescent microscope, and images were taken every 6 min for 16 hr. Frames from the movie show representative cells undergoing BiFC (green) prior to release of Omi-mCherry from the mitochondria (red). Scale bars represent 10 μm. (E) Graphs of three representative cells from a movie are shown. Each point on Omi-mCherry graph (red squares) represents the punctate/diffuse index and is scaled and aligned to each point on the caspase-2 BiFC graph (green circles) that represents the average intensity of Venus in the cell at 10 min intervals. Arrows show the point of onset of MOMP. Molecular Cell 2009 35, 830-840DOI: (10.1016/j.molcel.2009.07.023) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Hsp90α Blocks Caspase-2 Activation (A) HeLa cells were transiently transfected with C2-CARD VC (20 ng), C2-CARD VN (20 ng), and dsRed-mito (10 ng). Twenty-four hours later, cells were left untreated or heated for 1 hr at 45°C for 24 hr in the presence or absence of cycloheximide (CHX, 10 μg/ml) with qVD-OPH (20 μM). Cells were assessed 24 hr later for the percentage of Venus-positive cells, determined from a minimum of 300 cells per well. Results represent triplicate counts with error bars representing standard deviation. (B) HSF-1+/+ or HSF-1−/− MEFs from littermate embryos were heat shocked at the indicated temperatures for 1 hr. Twenty-four hours later, apoptosis was assessed by flow cytometry for Annexin V binding. (C) HSF-1+/+ and HSF-1−/− MEFs were transiently transfected with the indicated amounts of expression plasmids encoding the C2-CARD Venus pair and dsRed-mito (10 ng). Twenty-four hours posttransfection, cells were left untreated or heat shocked for 1 hr at 43°C with qVD-OPH (20 μM). The percentage of Venus-positive cells was determined at 24 hr as in (A). (D) HSF-1+/+ and HSF-1−/− MEFs were transiently transfected with the plasmids encoding the C2-CARD Venus pair (250 ng of each) and dsRed-mito (10 ng). Twenty-four hours posttransfection, cells were left untreated or heat shocked for 1 hr at 43°C with qVD-OPH (20 μM), with or without 17-DMAG, as indicated. The percentage of Venus-positive cells was determined at 24 hr as in (A). (E) Representative images of cells from (D) are shown. Scale bars represent 10 μm. (F) HeLa cells were transfected with siRNA targeting human Hsp90α (100 nM) or control siRNA (100 nM). Twenty-four hour posttransfection cells were left untreated or heat shocked for 1 hr at 43°C. Lysates were made at 72 hr and assessed for Hsp90α and β expression. (G) HeLa cells were transiently transfected with the indicated amounts of expression plasmids encoding the C2-CARD Venus pair and dsRedmito (10 ng) along with the indicated siRNA (100 nM). Twenty-four hours posttransfection, cells were left untreated or heat shocked for 1 hr at 42°C or 1 hr at 43°C with qVD-OPH (20 μM). The percentage of Venus-positive cells was determined at 24 hr as in (A). Molecular Cell 2009 35, 830-840DOI: (10.1016/j.molcel.2009.07.023) Copyright © 2009 Elsevier Inc. Terms and Conditions