Figure 8.

Depolymerization of Mitochondrial Actin by Lat B Induces Mitochondrial Membrane Potential Loss and Triggers the Release of Cytochrome C.

Purified mitochondria harvested from 1-d-old cotyledons treated with 1 M KCl. The mitochondrial membrane potential was assessed using the fluorescent probe DiOC₆, followed by flow cytometric analysis.

(A) and (B) Mitochondrial membrane potential loss caused by various concentrations of Lat B treatment in ADB buffer. The control treatment of DMSO (in ADB buffer) on mitochondria is shown in (B). CCCP (50 and 250 μM) treatment of mitochondria was used as a positive control and is also shown in (B).

(C) Mitochondria in F-actin stabilizing buffer FSB, with or without phalloidin pretreatment, showed no effect of Lat B on the mitochondrial membrane potential.

(D) Fluorescence images of isolated mitochondria treated with Lat B (60 μM) in ADB buffer. The Lat B treatment reducing the mitochondrial membrane potential is detected by staining of mitochondria by DiOC₆, and this effect can be rescued by pretreatment with phalloidin (5 μM) prior to Lat B (60 μM) treatment. CCCP, as a positive control, reduced the mitochondrial membrane potential completely. Three independent replicates were performed for (A) to (C), and eight independent repeats were conducted for (D). Similar results were obtained among the repeats. Differential interference contrast (DIC) images in the bottom row in (D) represent individual mitochondrion existing in each image of the top row correspondingly.

(E) and (F) Depolymerization of mitochondrial actin dynamics by Lat B triggers the release of cytochrome C in cotyledon mitochondria. Cotyledon mitochondrial samples were isolated and purified from mung bean seeds/seedlings that had been germinating for 12 h, 1 d, 2 d, and 3 d at 27°C. Purified mitochondrial samples were treated with 0, 20, or 60 μM Lat B in TM buffer for 30 min at 25°C. Immunoblot analysis of the mitochondrial pellet and supernatant was performed following the procedures described in Figure 1B. Detectable increases of cytochrome C released into the supernatant were observed in 12 h to day 2 cotyledon mitochondria caused by Lat B treatment in comparison to the control ([F], bottom row). This effect was not observed in mitochondria from 3-d-old seedlings. This study was repeated three times, and equivalent results were obtained each time. Samples of 12 h, day 1, and day 3 mitochondria are analyzed on the same PAGE gel, and the day 2 samples were analyzed on a separate gel.