1. Volume 54, Issue 5, Pages 870-878 (June 2014)
Distribution and Apoptotic Function of Outer Membrane Proteins Depend on Mitochondrial Fusion 
David Weaver, Verónica Eisner, Xingguo Liu, Péter Várnai, László Hunyady, Atan Gross, György Hajnóczky 
Molecular Cell 
Volume 54, Issue 5, Pages (June 2014)
DOI: /j.molcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2014 54, 870-878DOI: (10.1016/j.molcel.2014.03.048)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Fusion-Deficient Cells Show Heterogeneous Distributions of OMM Proteins
(A) Various modes of anchorage of proteins at the OMM, and specific proteins discussed in this work are noted (adapted from Walther and Rapaport [2009]).
(B) Representative reconstructions of 3D confocal micrographs of WT and Mfn1/2−/− cells expressing GFP-Bak and mCherry-OMP25 and the fluorescence frequency scatter plots generated from the corresponding image stacks. Automatic thresholding of the mCherry-OMP25 signal (x axis) was used to select the relevant pixels (see the Experimental Procedures).
(C) Means ± SEM of the calculated Pearson’s correlation coefficients in WT and Mfn1/2−/− cells of the GFP-tagged version of the noted constructs in comparison to mCherry-OMP25.
(D) Representative reconstructions of an MFN2-overexpressing Mfn1/2−/− cell and an Opa1−/− cell and a summation of mean correlation coefficients for noted conditions as in (C).
(E) Table showing the significance (p value) of each pair of the conditions shown above and the number of cells for each condition.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

4. Figure 2
tBid-Induced OMM Permeabilization Depends on the Even Distribution of Bak Maintained by Fusion
(A) Representative still images from a time course of cyto c-GFP release induced by 75 nM tBid in WT and Mfn1/2−/− cells and graphs for these cells. Solid lines (left axis) show normalized fluorescence traces. Dots show the calculated rates of cyto c release, and dashed lines show Gaussian fits to this data (right axis, R2 = and for WT and Mfn1/2−/−, respectively).
(B) Cell-wise, mean traces of cyto c-GFP release from permeabilized WT and Mfn1/2−/− MEFs induced by 75 nM tBid synchronized to the 50% release point.
(C) Cell-wise means ± SEM of the maximum rates of cyto c-GFP release calculated as shown in (A) for the noted cell-types and tBid concentrations. ∗∗∗p < in comparison to the same concentration of tBid in WT cells.
(D) Histograms of individual mitochondria from Mfn1/2−/− MEFs binned by the logarithm (base 2) of the GFP-Bak to mCherry-OMP25 fluorescence ratio, normalized by the mean for that cell and grouped by the presence or absence of cyto c, determined by immunofluorescence: left, 0 min (1,622 mitochondria from 11 cells), and center, 6 min treatment with 5 nM tBid (1,279 mitochondria from 10 cells). On the right, a scatter plot of the fractional change in cyto c-negative mitochondria by bin of the histograms covering two SD from the mean. A linear regression indicates an increasing probability of cyto c release with increasing GFP:mCherry ratio (slope, 14% ± 5%; p = 0.01; R2 = 0.382).
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 3
Rescue of tBid Sensitivity to Vdac2−/− Mitochondria by Bak Transfer during Fusion
(A) Representative example of a PEG-induced cell hybrid of Vdac2−/− and WT MEFs expressing mtYFP and mtCFP, respectively. The accompanying graph shows the time course of ΔΨm loss from each mitochondrial population as the normalized area of mitochondria containing TMRE signal.
(B) Summary of individual mitochondria identified in six WT Vdac2−/− cell hybrids.
(C) Image sequence showing the transfer of GFP-Bak during a fusion event in a WT Vdac2−/− cell hybrid expressing GFP-Bak and mtRFP, respectively. OMM fusion and Bak transfer, initiated by 0 s, precedes matrix fusion by at least 6 s.
(D) Time-lapse images of a fusion event showing the transfer of fluorescent proteins between mitochondria expressing BAD-GFP and AKAP(34–63)-RFP in a cell fusion experiment with H9c2 myoblasts; fusion can first be observed at 0 s.
(E) Transfer of a full-length AKAP construct during a fusion event in a hybrid of H9c2 cells expressing (full-length) AKAP-mCherry and AKAP(34–63)-GFP.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

6. Figure 4
Comparison of the Dynamics of Transfer of Different OMM Proteins
(A) Graph showing the mean kinetics of transfer ± SEM (open circles and dashed line) of AKAP(34–63)-PA-GFP (green) and PA-GFP-OMP25 (red) measured in separate photoactivation-bleaching experiments with mtDsRed (yellow) in HUSM cells. For each event, the interval between 50% transfer of the OMM construct and mtDsRed was determined, and the averages were used to merge the graphs. Sigmoidal fits for each probe are also shown (solid lines). Average intervals for tested combinations of OMM and matrix-targeted proteins are summarized in the table. ∗∗p = comparing AKAP(34–63)-PA-GFP and PA-GFP-OMP25.
(B) Time-lapse images of a single fusion event from a cell fusion experiment with mitochondria expressing AKAP(34–63)-GFP and mCherry-OMP25 at high temporal resolution with accompanying line scan of the fluorescence profile along the length of the AKAP donor mitochondrion (length, vertical axis; time, horizontal axis) as well as a graph of the mean fluorescence in the AKAP donor. The initiation points of OMP and AKAP(34–63) are indistinguishable.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions