Volume 41, Issue 2, Pages 150-160 (January 2011) The Soluble Form of Bax Regulates Mitochondrial Fusion via MFN2 Homotypic Complexes  Suzanne Hoppins, Frank Edlich, Megan M. Cleland, Soojay Banerjee, J. Michael McCaffery, Richard J. Youle, Jodi Nunnari  Molecular Cell  Volume 41, Issue 2, Pages 150-160 (January 2011) DOI: 10.1016/j.molcel.2010.11.030 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Mammalian Mitochondrial Outer and Inner Fusion In Vitro (A) Schematic representation of content-mixing in vitro fusion assay (top) and fluorescent images of a fusion reaction with wild-type m-EGFP and m-dsRed mitochondria in the absence of exogenous energy (bottom, left) and with the addition of an energy regeneration system and nucleotides (bottom, right). Fusion events are indicated with arrows. (B) Energetic requirements of mitochondrial fusion in vitro. Fusion efficiency is described as a percentage of the standard reaction which contains an energy regeneration system, ATP, and GTP, and was performed in parallel. Error bars show mean + standard error of at least three experiments, and statistical significance was determined by paired t test analysis. ∗p < 0.05, ∗∗p = 0.002, ∗∗∗p = 0.0002, ∗∗∗∗p < 0.0001. (C) Western blot analysis of mitochondrial proteins during in vitro fusion reactions. Mitochondrial fusion reactions without (standard) or with valinomycin 1 mM (val), 1 mM nigericin (nig), or 100 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were stopped at the indicated times, and proteins were subject to SDS-PAGE and western analysis with the indicated antibodies. (D) Electron micrograph images of a fusion reaction with wild-type mitochondria in the absence of exogenous energy. Representative images of outer membrane fusion events are shown. Scale bar represents 5 μm. (E) Energetic requirements of mitochondrial outer membrane fusion in vitro. Outer membrane fusion efficiency for each condition is described as a percentage of the standard reaction performed in parallel. Error bars show mean + standard error of at least two independent experiments, and statistical significance (∗p = 0.0012) was determined by one-way ANOVA analysis. Molecular Cell 2011 41, 150-160DOI: (10.1016/j.molcel.2010.11.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Analysis of the Role of MFN1, MFN2, and OPA1 in Mitochondrial Fusion (A) Mitochondria were isolated from wild-type Mfn1−/−Mfn2+/+, Mfn1+/+Mfn2−/−, Mfn1−/−Mfn2−/−, or OPA1−/− cells and subjected to either S2 fusion conditions (black bars) or S1 fusion conditions (gray bars) to assess mitochondrial inner and outer versus mitochondrial outer membrane fusion efficiency, respectively. Data are expressed as a percent of the wild-type control reactions performed in parallel. Error bars show mean + standard error of at least three independent experiments, and paired t-test analysis was used to determine statistical significance. ∗p = 0.0002, ∗∗p = 0.0017, ∗∗∗p = 0.0095. (B) Mitochondria isolated from the indicated cell lines were subjected to S2 fusion conditions in heteroallelic reactions (gray bars) to assess the requirement for each protein on mitochondria fusion partners. For comparison, results are shown with data from homotypic fusion experiments performed in parallel from (A) (light gray bars) and wild-type control (black bar). For both, error bars show mean + standard error of at least three independent experiments and paired t test analysis was performed to determine statistical significance. ∗p = 0.0049. Molecular Cell 2011 41, 150-160DOI: (10.1016/j.molcel.2010.11.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Bax Regulates Mammalian Mitochondrial Fusion In Vitro (A) Mitochondria were isolated from wild-type cells and subjected to S2 fusion conditions in the presence of the indicated cytosol-enriched fraction (black bars) or the indicated concentration of recombinant, purified Bax or Bcl-xL (gray bars). Data are expressed as fold stimulation of a standard reaction that was performed in parallel. Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p ≤ 0.05, ∗∗p ≤ 0.004, ∗∗∗p = 0.008. (B) The indicated amounts of purified, recombinant Bax (top) and Bcl-xL (bottom) and the equivalent amount of either cytosol-enriched extract (wild-type or Bak−/−Bax−/−) or recombinant, purified Bax or Bcl-xL added to one fusion reaction were subject to SDS-PAGE and western analysis with the indicated antibody. Some lanes of the same exposure from the same blots were rearranged to generate the figure. (C) Mitochondria isolated from either wild-type or Bak−/−Bax−/− cells were subjected to S2 fusion conditions under standard conditions or with the addition of either wild-type MEF cytosol-enriched fraction or 150 nM purified, recombinant Bax. After 60 min, the fusion reactions were stopped; fusion was quantified and is expressed as a percent of wild-type control reactions performed in parallel. Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p ≤ 0.05. Molecular Cell 2011 41, 150-160DOI: (10.1016/j.molcel.2010.11.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Soluble Bax Stimulates Mitochondrial Fusion In Vitro (A) Cytosol-enriched fractions prepared from wild-type, Bak−/−Bax−/− cells, or Bak−/−Bax−/− cells expressing the indicated GFP-Bax variant were separated by SDS-PAGE and subject to western analysis with Bax-specific antibody (top panel). Mitochondria were isolated from Bak−/−Bax−/− cells and subjected to standard S2 fusion conditions in the absence (black bar) or presence of wild-type MEF cytosol-enriched fraction or cytosol- enriched fractions from Bak−/−Bax−/− cells expressing the indicated GFP-Bax variant (gray bars). Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p = 0.04, ∗∗p ≤ 0.025. (B) Mitochondria isolated from wild-type MEFs were subject to S2 fusion conditions in the presence of the indicated amount of recombinant, purified Bax1-2/L-6 or wild-type Bax. After 60 min, the fusion reactions were stopped; fusion was quantified and is expressed as a fold induction of each standard reaction, all performed in parallel. Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p = 0.05, ∗∗p = 0.003. Molecular Cell 2011 41, 150-160DOI: (10.1016/j.molcel.2010.11.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Apoptotic Active Bax Cannot Stimulate Mitochondrial Fusion (A) Mitochondria were isolated from wild-type cells and subjected to S2 fusion conditions, in the presence of cytosol-enriched fractions collected from wild-type MEFs treated with DMSO or 2 μM STS for 4 hr (left), or of wild-type MEF cytosol-enriched fraction containing either DMSO or 2 μM STS (right). Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p = 0.017, ∗∗p = 0.008, ∗∗∗p = 0.005. (B) Mitochondria were isolated from wild-type cells and subjected to S2 fusion conditions (standard), in the presence of either 150 nM recombinant, purified Bax or Bcl-xL, with (gray bars) or without (black bars) 20 nM recombinant, purified tBid. After 60 min, fusion was quantified and expressed as a fold induction of each standard reaction performed in parallel. Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p = 0.01, ∗∗p = 0.03, ∗∗∗p = 0.007. (C) Samples from (B) were split and analyzed for MOMP by monitoring cytochrome c release from the mitochondrial fraction (P) to the supernatant fraction (S), by western analysis. (D) Mitochondria were isolated from wild-type cells and subjected to S2 fusion conditions in the absence (standard) and presence of 20 nM tBid. Samples were split and analyzed for either MOMP (top panel, as in C) or fusion (bottom panel). Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p = 0.0086. Molecular Cell 2011 41, 150-160DOI: (10.1016/j.molcel.2010.11.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Bax Positively Regulates MFN2 Homotypic Fusion Activity In Vitro and In Cells (A) Mitochondria were isolated from the indicated cell lines and subjected to S2 in vitro fusion conditions in the absence (black bars) or presence (gray bars) of 150 nM recombinant, purified Bax. After 60 min, the fusion reactions were stopped; fusion was quantified and is expressed as a fold induction of each standard reaction, performed in parallel (i.e., with the absence of Bax). Error bars show mean + standard error of at least three independent experiments, and paired t test analysis was performed to determine statistical significance. ∗p = 0.03, ∗∗p = 0.02. (B) Upper panel: Representative images of the indicated cell lines, expressing empty vector (vector) or GFP-Bax92-94Ala. Scale bar is 10 μm. Lower panel: Quantification of mitochondrial morphology of GFP-positive cells expressing the GFP-Bax variants shown. Error bars show mean + standard error of at least three experiments, and paired t test analysis was performed to determine statistical significance. ∗p = 0.04, ∗∗p = 0.01. Molecular Cell 2011 41, 150-160DOI: (10.1016/j.molcel.2010.11.030) Copyright © 2011 Elsevier Inc. Terms and Conditions