Distribution and Apoptotic Function of Outer Membrane Proteins Depend on Mitochondrial Fusion

Transcription

1 Molecular Cell, Volume 54 Supplemental Information Distribution and Apoptotic Function of Outer Membrane Proteins Depend on Mitochondrial Fusion David Weaver, Verónica Eisner, Xingguo Liu, Péter Várnai, László Hunyady, Atan Gross, and György Hajnóczky

2 A B Mean Fig. S1 GFP-Bak WB: α-bak α GFP 50 >3x mean: 3.6% of cells Bak 25 Cells C GFP Fluorescence (a.u.) D GFP-Bak TMRE E Pearson s Correlation Coefficient 5 µm Bak Mfn1 -/- Bak Mfn2 -/- Bak Bak

3 Figure S1, related to Figure 1: Fusion-deficient cells show heterogeneous distribution of Bak. (A) Western blots of membrane fraction of MEFs transfected with GFP-Bak. The membrane was probed first for Bak, then stripped and reprobed for GFP to identify the GFP-Bak band. The intensity of the GFP-Bak band in the Bak blot is ~2% of the endogenous Bak. (B) Histogram of the GFP-Bak expressing cells from the same transfection by average fluorescence determined by imaging (y-axis, log scale). Cells were plated and stained with Hoescht to determine by automated couting a transfection efficiency of 19% (5,427 of 24,979 cells). Thus we estimate the GFP-Bak expression in the average expressing cell is ~10% of endogenous Bak, while 3.6% of cells were greater than 3x the mean (estimated >30% endogenous). (C) Analysis of the expression levels of fluorescent proteins from the colocalization experiments with GFP- Bak and mcherry-omp25 in and (Fig1C). Scatter plots with linear regression fits of detected instensity (left), the illumination power employed (center), and estimated expression as the ratio intensity divided by illumination (right), are shown, where each point represents one cell. To combine the two-color images to a single value we used the Euclidean distance (sqrt(x^2+y^2)). For intensity, the Euclidean distance for each mcherry-positive pixel was determined and the median pixel value for each cell is used, while for illumination, the individual laser powers were normalized to their experimental means before calculating the Euclidean measure. Data from two experimental days is shown. (D) Image from a 3D reconstruction of an MEF expressing GFP-Bak (green) and loaded with TMRE (red). Arrows indicate organelles containing GFP but without TMRE. (E) Summary of the Pearson s correlation coefficients of GFP-Bak and mcherry-omp25 expressed in, Mfn1 -/-, Mfn2 -/- and MEFs.

4 Fig. S2 A α Bak α mthsp70 B C D 20 α Bax E tbid: tbid: 0nM 500s 25nM 500s α cyto c Membrane SN Membrane SN 0nM, 500s α VDAC2 25nM, 500s α Bak tbid: 25 0nM 500s Oligomerized Bak (% of max) tbid: 2.5nM 100s F 2.5nM 100s 2.5nM 500s 2.5nM 500s Bak/Bax -/- sirna: NT MFN1 25nM 500s 25nM 500s α MFN1 0 min. α Prohibitin 5nM tbid 6 min. mcherry-omp25 GFP-Bak α-cyto c (Alexa405) Pearson s Correlation Coefficient 5µm sirna: NT MFN1

5 Figure S2, related to Figure 2: Dynamics of tbid-induced OMM permeabilization in fusion deficient cells. (A,B,C,D) Analysis of the effect of tbid in suspensions of permeabilized and cells. (A) Western blot of Bak and VDAC2 in the membrane fractions and mitochondrial HSP70 as a loading control. (B) Representative blot of Bak in bismaleimidohexane (BMH)-treated membrane fractions after different tbid treatments. Background was subtracted by the Rolling Ball technique in ImageJ (radius, 20 pixels) to aid the quantification shown in the bar graph below. Three upper bands (dimeric, trimeric and tetrameric Bak) indicated by the box on the blot were measured and the total intensity was normalized to the range of the time control (0nM tbid, 500s) and maximum response (25nM, 500s). Three pairs of samples, each, were quantified after 100s and 500s of 2.5nM tbid for the means shown in the bar chart. (C) Blot cyto c in the supernatant fraction of time control and high tbid conditions. (D) Blot of Bax in membrane and supernatant fractions in time control and high tbid concentrations. (E) Representative images MEFs used for the analysis in Fig2D. Cells express GFP-Bak (green) and mcherry- OMP25 (red) and immunostained for cyto c with AlexaFluor 405 secondary antibody (cyan), at 0 min. (top) and 6 min. (bottom) of treatment with 5nM tbid. (F) Results of acute silencing of MFN1 in Bax/Bak -/- cells. Western blots of MFN1 in cells treated with sirna against Mfn1 or not targeting sirna (NT) for 72 hrs (Prohibitin is included as loading control) and quantification of the correlation of GFP-Bak and mcherry-omp25 fluorescence in the same cells as shown in Fig1.

6 A Bak -/- +25nM tbid +FCCP B Bak -/- 9 Fig. S3 α-mthsp70 37 α VDAC2 C 34 Vdac2 -/- α MTCH2

7 Figure S3, related to Figure 3: Absence of Bak is the cause of tbid insensitivity in VDAC2 -/- MEFs. (A) Mean traces ± SEM of normalized TMRM fluorescence in permeabilized Bak -/- MEFs transiently expressing Bak and marker (GFP-OMP25) (red, n = 44 cells) or marker alone (black, n = 45). 25nM tbid is added at 0 sec in the presence of oligomycin and FCCP at 600 sec to evoke complete depolarization. (B) Western blot of VDAC2 in the membrane fractions of permeabilized Bak -/- and MEFs plus mthsp70 as loading control. (C) Blot of MTCH2 from membrane fractions of Vdac2-/- and MEFs.

8 A -4s H9c2 0s 4s 12s 16s 20s B HUSM -39s -1s 0s 4.5s 7s Fig. S4 mtdsred AKAP (34-63) PA-GFP 2µm AKAP(34-63)- PA-GFP 2µm mtdsred F AKAP(34-63)-PA-GFP (a.u.) F mtdsred (a.u.) Time (s)

9 Figure S4, related to Figure 4: Examples of OMM fusions and separability from IMM fusion. (A) Visualization of the separation of OMM and IMM fusion in a single H9c2 cell expressing AKAP(34-63)-PA-GFP and mtdsred. Photoactivation of PA-GFP in a subset of mitochondria also photobleaches DsRed in those same organelles allowing visualization of bidirectional transfer during fusion. AKAP(34-63)-PA-GFP is transferred at 0s while mtdsred is mixed between 8 and 12s as shown in time lapse images and graphs of the mean fluorescence of each organelle (solid line, GFP-acceptor; dotted line, GFP donor). (B) Example OMM fusion in HUSM. shusm cell expressing AKAP(34-63)-PA-GFP and mtdsred. One elongated mitochondrion displays photoactivated PA-GFP and photobleached DsRed (donor organelle); at time 0s AKAP(34-63)-PA-GFP is transferred to an acceptor mitochondrion and only 4.5 s later, mtdsred is transferred from the acceptor mitochondrion to the donor. Graphs at bottom show the mean fluorescence of each organelle (solid line, GFPacceptor; dotted line, GFP donor).

10 Supplemental Experimental Procedures Reagents Unless otherwise specified, chemicals are from Sigma. Transient Expression and Silencing MEFs and H9c2 were transfected by electroporation with 2-5 million cells and 4-20µg of plasmid DNA in µl or chemically by Lipofectamine 2000 (Invitrogen) or XtremeGene (Roche) according to the manufacturers protocols. One million HUSM cells were transfected using Nucleofector Kit for Human Dermal Fibroblasts (Lonza) with 4-15 µg of plasmid DNA. All mtfp vectors use the targeting sequence of cyto c oxidase subunit VIII to achieve mitochondrial matrix localization. AKAP(34-63) is described previously (Csordas et al., 2010) and full-length AKAP1 was purchased from Invitrogen. OMP25 targeting sequence was provided by Manuel Rojo (Malka et al., 2005; Nemoto and De Camilli, 1999). BAD was described previously (Roy et al., 2009), Bak and BakC were provided by Katsuyoshi Mihara (Setoguchi et al., 2006) and untagged Bak by Donna George (Leu et al., 2004). MFN2-CFP was provided by Heidi McBride (Neuspiel et al., 2005). Cyto c-gfp was provided by Anna-Liisa Nieminen (Heiskanen et al., 1999). Silencing of MFN1 was accomplished by a pool of 4 sirna against mouse Mfn1 (Dharmacon SMARTpool) transfected by electroporation by means of a BTX-830 square-pulse generator in a 4-mm gap cuvette using a single 250-V 13-ms pulse. Immunoblot Analysis Western blots of cytosolic fractions or lysed membrane fractions were performed as described previously (Garcia-Perez et al., 2012). Antibodies employed were: rabbit polyclonal Anti-Bak (Millipore #06-536), monoclonal anti-gfp (Clontech #632381), monoclonal anti-mthsp70 (Thermo Scientific #MA3-028), rabbit polyclonal anti-bax (Santa Cruz, sc-493), MFN1 antibody (from Richard Youle), rabbit polyclonal anti-prohibitin (Abcam #ab28172), goat polyclonal anti-vdac2 (Abcam #ab37985), rabbit polyclonal anti-mtch2 (from Atan Gross), monoclonal anti-cytochrome c (BD Pharmingen #556433). Detection of bands was performed on a LI-COR Odyssey scanner using the manufacturer s fluorescent secondary antibodies. Buffers Extracellular medium (ECM): 120 mm NaCl, 5 mm NaHCO 3, 10 mm Na-Hepes, 4.7 mm KCl, 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 2 mm CaCl 2, and 10 mm glucose, ph 7.4. Intracellular medium (ICM): 120 mm KCl, 10 mm NaCl, 1 mm KH 2 PO 4, 20 mm Hepes/Tris, ph 7.2, supplemented with 2 mm MgATP, 10 μm EGTA/Tris, and 1 μg/ml each of antipain, leupeptin, pepstatin. Sodium-Hepes-EGTA (NaHE): 120 mm NaCl, 5mM KCl, 1 mm KH 2 PO 4, 0.2 mm MgCl 2, 20 mm NaOH/Hepes, 0.1 mm EGTA, ph 7.4.

11 Bak oligomerization Suspensions of cells (1.2 mg protein) were washed in NaHE (see Buffers ) on ice, then permeabilized with 40µg/ml saponin in 1.5ml ICM at 37 C; tbid was added 250s after the start of permeabilization. At the desired time point, cell suspension was diluted five-fold in ice cold NaHE and pelleted by centrifugation. The pellet was resuspended in 60µl of 1mM bismaleimidohexane (Thermo), 8mM EDTA in phosphate buffered saline (PBS) and incubated at room temperature for 30 minutes with constant agitation. The crosslinking reaction was stopped by lysis in RIPA buffer containing 50mM dithiothreitol and protease inhibitors. Live-cell imaging Intact cell imaging was performed in ECM containing 0.25% BSA. Permeabilized cell imaging was done in ICM(Garcia-Perez et al., 2012); after permeabilization with 40µg/ml digitonin or saponin, fresh ICM was added for imaging. Membrane potential measurements were performed in ICM containing 5nM TMRE and 5µg/ml oligomycin after pre-loading with 25nM TMRE in ECM with 2% BSA. All live-cell imaging was performed at 36 C except for colocalization experiments, which were done at room temperature to minimize movement of the organelles. The bulk of the imaging was performed on two laser scanning confocal microscopes, a Zeiss LSM780 and a BioRad Radiance The Radiance was coupled to an Olympus IX70 microscope and we used a 40x objective (Uapo340, 1.35NA); excitation light at 488nm (GFP) and 568nm (DsRed/RFP) was provided by a multi-line Kr/Ar ion laser while photoactivation/bleaching was accomplished with either 442nm He-Cd laser (Melles Griot) or by 2-photon excitation from a Ti-Sapphire pulsed laser (Spectraphysics Tsunami) with excitation maximum near 740nm. The LSM780 is equipped with an Ar laser for 458/488/514nm excitation used for CFP, GFP and YFP, respectively as well as a DPSS laser at 561nm (RFP, TMRE) and a HeNe at 594nm (mcherry) and 405nm diode laser (AlexaFluor 405, Hoescht 33342). Photoactivation/bleaching was performed by 2-photon excitation from a Chameleon (Coherent) pulsed laser at nm. Imaging was performed with a 63x oil-immersion objective (PlanApo, 1.4NA) for high resolution and 10x, 0.45NA for quantification of GFP-Bak expression. For colocalization and cyto c immunostaining experiments, Z-series stacks were acquired at the software-determined optimum spacing on the LSM780. Laser power was varied for GFP constructs and mcherry-omp25 to adjust for cell-to-cell expression levels, but not for the AlexaFluor 405 conjugated cyto c, while gain settings were left fixed to attempt to minimize sampling error variations (see FigS1B). Some PEG cell fusion experiments (Fig3D&E) were performed on a Nipkow, spinning-disk confocal (Solamere) described previously (Liu and Hajnoczky, 2011; Liu et al., 2009). High temporal resolution imaging (Fig5B) and TMRM imaging of Bak -/- cells (FigS3A) were performed on widefield fluorescence scope built on an Olympus IX80 inverted scope with a 60x objective (UPlanSApo, 1.35NA) and 40x (Uapo340, 1.35NA), respectively and using a DG-4 (Sutter) Xenon light source with 485/15nm and 577/25nm excitation filters (Chroma) for GFP and mcherry/tmrm, respectively, and a Hamamatsu ImageEM EM-CCD camera. Fast

12 imaging was accomplished with a dual-band dichroic/emission filter combination (Chroma 59022) and streaming acquisition using Metamorph software (Molecular Devices). PEG cell fusion Vdac2 -/- and cells transfected with mitochondrial matrix-targeted YFP and CFP, respectively were co-plated and fused by PEG 1500 (Fluka) as described previously (Liu et al., 2009). 30 to 90 minutes after PEG, the cells were loaded with TMRE (30nM) for imaging mitochondrial membrane potential. Immunocytochemistry Cells on coverslips were permeabilized by saponin in ICM (40µg/ml, 5 min. at 37 C) and buffer was changed to fresh ICM containing 5nM tbid and fixed by 3.5% paraformaldehyde immediately or after 6 min exposure to tbid (10 min. fixation at room temperature). Cells were further exposed to 100µg/ml digitonin (10 min., RT) to permeabilize the OMM. Samples were blocked with 3% BSA, 0.1% Tween-20 in PBS for 1 hr, then incubated 1 hr. with mouse monoclonal anti-cyto c antibody (BD Pharmingen #556432) in 1% BSA, 0.1% Tween-20 and 45 min with AlexaFluor 405 anti-mouse secondary antibody (Molecular Probes). Image Analysis To calculate the rate of cyto c release in and MEFs we smoothed each trace by a five-point (15s) running average, calculated the discrete differential of the smoothed trace, and fit a 3-parameter Gaussian curve to that differential, the amplitude of the fit representing the maximum rate of release. In general, the fits were quite good, with a standard error of less than 10% of the amplitude estimate. The few cases where a wave pattern was observed in cyto c release (Garcia-Perez et al., 2012) were excluded from the analysis of kinetics. Evaluation of the cyto c release from GFP-Bak and mcherry transfected cells (Figs2D&S2E) was accomplished by masking individual organelles from maximum intensity projections generated from five slices of the acquired z-stacks, where each slice is separated by 0.32µm. Cyto c immunostaining was thresholded automatically by the default method in ImageJ. The presence of any cyto c-positive pixels within a mask was scored as a cyto c containing mitochondrion. Overall distributions of the normalized log 2 (GFP:mCherry) ratios were very similar at 0 and 6 minutes of tbid: standard deviations of 0.53 and 0.55, respectively. To analyze tbid-induced mitochondrial depolarization in cell hybrids (Fig3A) we exploited the loss of motility and dynamics in permeabilized cells. Masks of the mitochondria deriving from each cell in the hybrid were generated by thresholding a single CFP/YFP still image acquired immediately before tbid addition. A mask of the hybrid mitochondria was generated by taking the intersection of the two raw masks and then the hybrid mask was subtracted from each raw resulting in masks of the three distinct classes of mitochondria. During the time-course following tbid addition, only YFP was acquired simultaneously with TMRE, and the YFP signal was used to correct for small drifts in the image position during the run for the purpose of masking using the Descriptor-based Registration plugin (translation only) in ImageJ (Preibisch

13 et al., 2010). Translation values determined by registering the YFP image used to generate the mask with YFP at the given time-point were applied to the three masks for calculating the TMRE-containing area to monitor depolarization. To calculate the mixing kinetics of fluorescent proteins in HUSM cells (Fig 4A), each mitochondrion in a fusion event was masked and the increase of fluorescence in each was measured, as well as the lag time between the half-maximal transfers of OMM PA-GFP and mtdsred. The average kinetics were calculated by synchronizing individual traces to the halfmaximum, which are represented on the graph according to the average lag time calculated from the individual events. Determination of the GFP-Bak transfection efficiency and expression (FigS1B) was determined by automatic masking and object counting in ImageJ. To determine the total number of cells, Hoescht images were thresholded and converted to masks, then segmented according to the watershed algorithm. The Analyze Particles routine was used to find count objects of area >20µm 2. Similarly the GFP image was thresholded, dilated to fill holes and Analyze Particles was used to generate masks which were applied to the original image to calculate the distribution of intensities. Statistics Data are shown as mean ± S.E.M. Significance was determined by student s t-test for comparisons of distributions compiled from multiple experiments and the z-test when comparing proportions among different populations. Precise p-values are shown for significant differences except where <.001.

14 Supplementary References Csordas, G., Varnai, P., Golenar, T., Roy, S., Purkins, G., Schneider, T.G., Balla, T., and Hajnoczky, G. (2010). Imaging interorganelle contacts and local calcium dynamics at the ERmitochondrial interface. Molecular cell 39, Garcia-Perez, C., Roy, S.S., Naghdi, S., Lin, X., Davies, E., and Hajnoczky, G. (2012). Bidinduced mitochondrial membrane permeabilization waves propagated by local reactive oxygen species (ROS) signaling. Proc Natl Acad Sci U S A 109, Heiskanen, K.M., Bhat, M.B., Wang, H.W., Ma, J., and Nieminen, A.L. (1999). Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells. The Journal of biological chemistry 274, Leu, J.I., Dumont, P., Hafey, M., Murphy, M.E., and George, D.L. (2004). Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nature cell biology 6, Liu, X., and Hajnoczky, G. (2011). Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress. Cell Death Differ 18, Nemoto, Y., and De Camilli, P. (1999). Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein. The EMBO journal 18, Neuspiel, M., Zunino, R., Gangaraju, S., Rippstein, P., and McBride, H. (2005). Activated mitofusin 2 signals mitochondrial fusion, interferes with Bax activation, and reduces susceptibility to radical induced depolarization. The Journal of biological chemistry 280, Preibisch, S., Saalfeld, S., Schindelin, J., and Tomancak, P. (2010). Software for bead-based registration of selective plane illumination microscopy data. Nat Methods 7,