LKT detected by flow cytometry of purified mitochondria from LKT-treated BL-3 cells. Mitochondria were stained with Mitotracker Red and FITC-conjugated anti-LKT MAb. (A) Mitochondria with positive staining for both LKT and Mitotracker Red. (B and C) Mitochondria from BL-3 cells not treated with LKT (B) and mitochondria from LKT-treated cells stained with Mitotracker Red and an isotype-matched primary Ab as controls (C). The data represent the results of three similar experiments that were performed.

Dynamin-2 and β-actin are required for targeting of LKT to mitochondria. (A) Representative immunoblot of cell lysates from BL-3 cells transfected with dynamin-2 siRNA, untreated cells (control [Con cells]), or cells transfected with scrambled siRNA (Con siRNA). (B) BL-3 cells were transfected with dynamin-2 siRNA or scrambled siRNA or were not treated (Con Cells) before incubation with LKT (0.5 U) at 37°C for 1 h. Mitochondria were purified and lysed, and the lysates were immunoblotted for LKT and porin-2 (mitochondrial marker). (C) BL-3 cells were pretreated with cytochalasin D (CytD) (2 μg/ml for 1 h at 37°C) before they were treated with LKT. Mitochondria were then isolated and immunoblotted for LKT and porin-2. The controls included cells treated with cytochalasin D alone and untreated BL-3 cells.

LKT on mitochondria isolated from BL-3 cells detected by scanning electron microscopy and transmission electron microscopy. Mitochondria isolated from BL-3 cells were incubated with 0.5 U LKT (A and B) or RPMI medium (control) (C), stained with anti-LKT colloidal gold, and visualized by scanning electron microscopy. The micrographs show areas of LKT binding (clusters of gold beads in panels A and B) and sloughing of the MOM, leaving punched-out areas (arrows in panels A and B). (D) Mitochondrion with a disrupted outer membrane and cristae (black arrows) and normal mitochondrion with an intact double membrane (white arrow). Bars = 200 nm.

LKT binds to mitochondria from BL-3 cells and RAW 264.7 cells. (A) Immunopurified mitochondria from 10⁶ BL-3 cells were incubated for 30 to 60 min with LKT (0.5 U). Lysates were prepared and immunoblotted for LKT and the mitochondrion-specific porin-2 protein. Con, control. (B) BL-3 cells were protein transfected with anti-LKT MAbs (5 μg of MM601 or MM605) or with anti-β-actin MAb as a negative control using the Pro-ject protein transfection reagent. The cells were then incubated with LKT (0.5 U) for 1 h. Mitochondria were immunopurified and lysed, and the lysates were immunoblotted for LKT and porin-2. (C) Single BL-3 cell stained for LKT (green), mitochondria (red), and colocalization of LKT with mitochondria (yellow). (D) RAW 264.7 cells were protein transfected with LKT (0.5 U) using the Pro-ject protein transfection reagent and stained for LKT (green) and mitochondria (red) as described above. Colocalization of LKT with mitochondria is indicated by yellow. Bars = 10 μm.

Movement of dynamin-2 to the cell periphery in LKT-treated BL-3 cells. (A) BL-3 cells (10⁶ cells) were incubated with LKT (0.5 U) for 30 or 60 min and then fixed, permeabilized, stained for cytoplasmic dynamin-2 (arrows), and visualized by confocal microscopy. (B) BL-3 cells were incubated with CSA (5 μM for 30 or 60 min at 37°C). Mitochondria were isolated and lysed, and the lysates were immunoblotted for dynamin-2 and porin-2. (C) BL-3 cells preincubated with CSA were treated with LKT for 30 or 60 min. Their mitochondria were then isolated and lysed, and the lysates were immunoblotted for LKT, dynamin-2, and porin-2. (D) Mitochondrial (Mito) dynamin-2 and LKT levels in immunoblots as determined by densitometry. The bars indicate the means of three separate experiments, and the error bars indicate the standard errors of the means (*, P < 0.05). OD, optical density.

LKT binds to the mitochondrial matrix protein cyclophilin D. (A) BL-3 cells were protein transfected with anti-cyclophilin D Ab (5 and 10 μg) and then incubated with LKT (0.5 U for 1 h at 37°C), and cytotoxicity was measured as described previously. BL-3 cells transfected with anti-cyclophilin D Ab (10 μg) alone or LKT-treated untransfected cells served as controls. (B) BL-3 cells were protein transfected with anti-cyclophilin D Ab (5 or 10 μg) or anti-β-actin MAb (unrelated Ab control) and then incubated with LKT (0.5 U for 1 h at 37°C). BL-3 cells incubated with LKT alone (Con LKT) and untreated BL-3 cells served as controls. Mitochondria were then purified, lysed, and immunoblotted for LKT and porin-2. (C) BL-3 cells were protein transfected with anti-cyclophilin D Ab or anti-β-actin MAb (unrelated control Ab) and incubated with LKT (0.5 U for 1 h at 37°C). Mitochondria were isolated and lysed, and the lysates were immunoblotted for cytochrome c (Cyt C) and porin-2. (D) LKT was bound to agarose protein A/G beads coated with anti-LKT Ab and incubated overnight at 4°C with a preabsorbed mitochondrial lysate (Mito lysate) from untreated BL-3 cells. The beads were then washed five times in 0.05% Triton X-100-PBS wash buffer, and proteins were eluted with Laemmli buffer. The eluates were immunoblotted for cyclophilin D and LKT. LKT bound to agarose protein A/G beads alone and mitochondrial lysate from untreated BL-3 cells served as controls. (E) Immunoblot demonstrating that preincubation with CSA (5 μM for 30 min at 37°C) reduced binding of LKT (0.5 U for 30 min at 37°C) to purified mitochondria from untreated BL-3 cells.

CSA protects BL-3 cells against LKT-mediated cytotoxicity, mitochondrial membrane damage, and collapse of ψ_m. (A) BL-3 cells (10⁶ cells) were preincubated with CSA (2 or 5 μM) for 30 min before incubation with LKT (0.5 U) for 60 min. Cytotoxicity was measured using the Cell Titre 96 AQ one-assay system. (B) Mitochondria were immunopurified from untreated BL-3 cells, preincubated with CSA (5 μM) or with propranolol (Prop) (200 μM), and then incubated with LKT (0.2 U) for 30 min at 37°C. Immunoblot analysis of the resulting mitochondrial lysates demonstrated that CSA, but not propranolol, protected against LKT-mediated mitochondrial cytochrome c (Cyt c) release. Con, control. (C and D) BL-3 cells were preincubated with CSA or medium (control) and then incubated with LKT (0.5 U) for 1 h. Cells were then incubated with JC-1, a ψ_m-sensitive dye, at 37°C for 15 min and washed three times in PBS. Aggregated intramitochondrial JC-1 is red, and the monomeric cytoplasmic form is green. BL-3 cells were then analyzed by flow cytometry in the FL2 channel (absorption and emission maxima at 580 and 595 nm, respectively). The results show that CSA protected against collapse of ψ_m. The bars in panel D indicate the means of three separate experiments, and the error bars indicate the standard errors of the means. *, P < 0.05.