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Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization - PubMed

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization

Ann Cassidy-Stone et al. Dev Cell. 2008 Feb.

Abstract

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.

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Figures

Figure 1
Figure 1. Chemical screen for mitochondrial division inhibitors

A. Growth phenotypes of mitochondrial fusion (fzo1-1) and division (Δdnm1) mutants. B. Chemical structure of the quinazolinone, mdivi-1. Important structural features are highlighted in red and were determined by comparing the efficacies of mdivi-1 like compounds shown in Figure 2. C. mdivi-1 suppresses the growth defect of mitochondrial fusion mutants, fzo1-1 and mgm1-5 at the restrictive temperature . D and E. mdivi-1 causes the formation of mitochondrial net-like structures (E, right panel, mdivi-1; left panel, DMSO) in Δprd1 Δprd3 yeast cells in a dose-dependent manner (D, representative experiment shown, n ≥ 100). In E, left panel is the DMSO control, right panel is mdivi-1. F. mdivi-1 has no effect on the F-actin cytoskeleton. Mitochondria are in red, and Phalloidin is in green. Left panel: DMSO control cells. Center panel: mdivi-1-treated cells. Right panel: Latrunculin-A and mdivi-1-treated cells. N=mitochondrial nets. Scale bar = 2μ.

Figure 2
Figure 2. Compounds related to mdivi-1 have different efficacies

Compounds (A-H) are grouped by their relative efficacy to form mitochondrial net-like structures in yeast. The structural differences between each molecule and mdivi-1 (A) are highlighted in red.

Figure 3
Figure 3. The target of mdivi-1 is the mitochondrial division dynamin, Dnm1

A. The dose-dependent effect of mdivi-1 on the GTPase activity of Dnm1, Dnm1 1-388 (Dnm1 GTPase domain) and dynamin-1. 100% activity: Dnm1, 50 min-1; Dnm1 1-388 0.9 min-1; dynamin-1 3.3 min-1 B. The effects of mdivi-1 analogs (see Fig. 2) on Dnm1 GTPase activity. C. Kinetic analysis of the effects of mdivi-1 on Dnm1 GTPase activity. Representative experiments shown in parts A-C. D. Analysis of Dnm1 self-assembly by negative stain electron microscopy. Representative images of Dnm1 incubated in the presence of GMPPCP (left panel) and Dnm1 pre-incubated with mdivi-1 prior to the addition of GMPPCP (right panel). Scale bar = 100 nm.

Figure 4
Figure 4. mdivi-1 inhibits mitochondrial division in mammalian cells by attenuating Drp1 self-assembly

A. Mitochondrial morphology in COS cells in the absence (left panel, DMSO control) and presence (right panels, 50 μM) of mdivi-1. B. The concentration of mdivi-1 required to produce mitochondrial net-like and perinuclear structures in COS cells increases in cells overexpressing Drp1 (light grey bars) as compared to cells transfected with a control empty vector (dark grey bars). C. The reticular morphology of mitochondria (left panel) in COS cells becomes fragmented upon addition of staurosporine (STS, center panel). mdivi-1 attenuates STS-induced mitochondrial fragmentation (50 μM, right panel). D. mdivi-1 (50 μM, compound A) and the active derivative, but not the inactive derivatives G and H (each at 50 μM), inhibit self-assembly of GFP-Drp1 stimulated by STS treatment (in green) in COS cells. The bottom panels are a representative region of each cell shown in the top panels and are magnified seven fold. Mitochondria are labeled with MitoTracker Red CMXRos and shown in red. All images were obtained using identical exposure and gain settings. Size bars are 10 μm.

Figure 5
Figure 5. mdivi-1 and its active analogs attenuate apoptosis

A. FACS analysis of staurosporine-treated Hela cells in the presence and absence of the active compound B (left panel) and inactive compound G (left panel). B. Analysis of the effects of mdivi-1 derivatives (B and F) on the release of cytochrome c stimulated by C8-Bid injection of HeLa cells. The injection marker is Texas Red (in red) and cytochrome c release is monitored with cytochrome c-GFP (in green). Size bar is 10 μm.

Figure 6
Figure 6. mdivi-1 and its active analogs inhibit activated Bax/Bak-dependent MOMP in vitro

A. Analysis of the effects of mdivi-1 and its analogs on MOMP by monitoring cytochrome c release in vitro from murine liver mitochondria. B. Analysis of the effects of mdivi-1 and its analogs on MOMP in vitro as in A. with murine liver mitochondria stimulated by C8-Bid. C. The effects of mdivi-1 and its analogs on C8-Bid stimulated MOMP depend on the presence of Bax. The assays were as described in A and B using mitochondria isolated from the livers of polydIdC-treated MxCre bak-/- baxf/- mice. D. mdivi-1 and its analogs do not directly inhibit Bid-activated Bax permeabilization of large unilamellar vesicles. Permeabilization is monitored by the release of encapsulated fluorescein-dextran by filtration analysis as described in methods.

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