DRP1 interacts directly with BAX to induce its activation and apoptosis
2022; Springer Nature; Volume: 41; Issue: 8 Linguagem: Inglês
10.15252/embj.2021108587
ISSN1460-2075
AutoresAndreas Jenner, Aida Peña‐Blanco, Raquel Salvador‐Gallego, Begoña Ugarte‐Uribe, Cristiana Zollo, Tariq Ganief, Jan Bierlmeier, Markus Mund, Jason E. Lee, Jonas Ries, Dirk Schwarzer, Boris Maček, Ana J. García‐Sáez,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle13 January 2022Open Access Source DataTransparent process DRP1 interacts directly with BAX to induce its activation and apoptosis Andreas Jenner Andreas Jenner orcid.org/0000-0002-6388-3053 Institute for Genetics, CECAD, University of Cologne, Cologne, Germany Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Formal analysis (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Aida Peña-Blanco Aida Peña-Blanco Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Raquel Salvador-Gallego Raquel Salvador-Gallego orcid.org/0000-0002-1447-2303 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Begoña Ugarte-Uribe Begoña Ugarte-Uribe Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Formal analysis (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Cristiana Zollo Cristiana Zollo orcid.org/0000-0002-8747-1344 Institute for Genetics, CECAD, University of Cologne, Cologne, Germany Contribution: Formal analysis (equal), Investigation (equal), Visualization (equal), Methodology (equal) Search for more papers by this author Tariq Ganief Tariq Ganief Interfaculty Institute of Cell Biology, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Investigation (equal), Visualization (equal), Methodology (equal) Search for more papers by this author Jan Bierlmeier Jan Bierlmeier orcid.org/0000-0002-7261-4744 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Methodology (equal) Search for more papers by this author Markus Mund Markus Mund orcid.org/0000-0001-6449-743X Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Contribution: Resources (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Jason E Lee Jason E Lee orcid.org/0000-0001-5757-5954 University of Colorado, Boulder, CO, USA Contribution: Resources (equal), Methodology (equal) Search for more papers by this author Jonas Ries Jonas Ries orcid.org/0000-0002-6640-9250 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Contribution: Resources (equal), Supervision (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Dirk Schwarzer Dirk Schwarzer orcid.org/0000-0002-7477-3319 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Supervision (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Boris Macek Boris Macek orcid.org/0000-0002-1206-2458 Interfaculty Institute of Cell Biology, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Supervision (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Ana J Garcia-Saez Corresponding Author Ana J Garcia-Saez [email protected] orcid.org/0000-0002-3894-5945 Institute for Genetics, CECAD, University of Cologne, Cologne, Germany Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Conceptualization (equal), Supervision (equal), Funding acquisition (equal), Writing - review & editing (equal), Project administration (equal), Writing - review & editing (equal) Search for more papers by this author Andreas Jenner Andreas Jenner orcid.org/0000-0002-6388-3053 Institute for Genetics, CECAD, University of Cologne, Cologne, Germany Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Formal analysis (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Aida Peña-Blanco Aida Peña-Blanco Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Raquel Salvador-Gallego Raquel Salvador-Gallego orcid.org/0000-0002-1447-2303 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Begoña Ugarte-Uribe Begoña Ugarte-Uribe Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Formal analysis (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Cristiana Zollo Cristiana Zollo orcid.org/0000-0002-8747-1344 Institute for Genetics, CECAD, University of Cologne, Cologne, Germany Contribution: Formal analysis (equal), Investigation (equal), Visualization (equal), Methodology (equal) Search for more papers by this author Tariq Ganief Tariq Ganief Interfaculty Institute of Cell Biology, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Investigation (equal), Visualization (equal), Methodology (equal) Search for more papers by this author Jan Bierlmeier Jan Bierlmeier orcid.org/0000-0002-7261-4744 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Methodology (equal) Search for more papers by this author Markus Mund Markus Mund orcid.org/0000-0001-6449-743X Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Contribution: Resources (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Jason E Lee Jason E Lee orcid.org/0000-0001-5757-5954 University of Colorado, Boulder, CO, USA Contribution: Resources (equal), Methodology (equal) Search for more papers by this author Jonas Ries Jonas Ries orcid.org/0000-0002-6640-9250 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Contribution: Resources (equal), Supervision (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Dirk Schwarzer Dirk Schwarzer orcid.org/0000-0002-7477-3319 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Supervision (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Boris Macek Boris Macek orcid.org/0000-0002-1206-2458 Interfaculty Institute of Cell Biology, University of Tübingen, Tübingen, Germany Contribution: Resources (equal), Supervision (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Ana J Garcia-Saez Corresponding Author Ana J Garcia-Saez [email protected] orcid.org/0000-0002-3894-5945 Institute for Genetics, CECAD, University of Cologne, Cologne, Germany Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Contribution: Conceptualization (equal), Supervision (equal), Funding acquisition (equal), Writing - review & editing (equal), Project administration (equal), Writing - review & editing (equal) Search for more papers by this author Author Information Andreas Jenner1,2, Aida Peña-Blanco2,†, Raquel Salvador-Gallego2,†, Begoña Ugarte-Uribe2,†, Cristiana Zollo1,†, Tariq Ganief3, Jan Bierlmeier2, Markus Mund4, Jason E Lee5, Jonas Ries4, Dirk Schwarzer2, Boris Macek3 and Ana J Garcia-Saez *,1,2 1Institute for Genetics, CECAD, University of Cologne, Cologne, Germany 2Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany 3Interfaculty Institute of Cell Biology, University of Tübingen, Tübingen, Germany 4Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany 5University of Colorado, Boulder, CO, USA † These authors contributed equally to this work *Corresponding author. Tel: +49 221 478 84263; E-mail: [email protected] The EMBO Journal (2022)41:e108587https://doi.org/10.15252/embj.2021108587 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region. Synopsis Apoptotic executioner BAX and dynamin-like protein DRP1 colocalize at mitochondria during apoptosis, but their interplay in mediating mitochondrial permeabilization and fragmentation remains incompletely understood. Here, DRP1 is found as direct interactor and noncanonical activator of BAX promoting apoptosis. BAX and DRP1 interact directly and their association is enhanced during apoptosis. BAX/DRP1 interaction takes place in the membrane environment and requires the N-terminal region of BAX. BAX and DRP1 mutually enhance each other's membrane remodeling activity. Forced dimerization of BAX and DRP1 triggers their translocation to mitochondria and induces apoptosis in the absence of apoptotic triggers. Introduction Apoptosis is a form of programmed cell death that plays a key role in fundamental biological processes like embryo development, tissue homeostasis, and the correct functioning of the immune system. Dysregulation of apoptosis has been related with human pathology, including neurodegenerative diseases and cancer (Strasser et al, 2011). Apoptosis execution is mediated by the apoptotic caspases, which accelerate cell death by cleaving a defined set of target proteins that leads to the organized dismantling of the cellular components. In most cells, activation of the caspase cascade requires mitochondrial outer membrane permeabilization (MOMP), which releases cytochrome c (cyt c) and Smac/DIABLO into the cytosol (Bock & Tait, 2020). MOMP is considered the point of no return in the cell's commitment to death, as cells where all mitochondria underwent MOMP ultimately die also in the absence of caspase activity (Tait et al, 2010). Besides MOMP, mitochondria undergo multiple alterations during apoptosis including cristae remodeling, extensive fragmentation, changes in lipid composition and calcium signaling, loss of mitochondrial potential, and swelling (Cosentino & Garcia-Saez, 2014). Furthermore, recent studies reported the release of mitochondrial DNA (mtDNA) into the cytosol during apoptosis, which happens via extrusion of the mitochondrial inner membrane (MIM) through the mitochondrial outer membrane (MOM) and initiates type I interferon inflammatory responses normally blocked by caspase activity (McArthur et al, 2018; Riley et al, 2018). BAX is a pro-apoptotic member of the BCL-2 family of proteins that, together with its homolog protein BAK, is necessary for the execution of MOMP and the additional mitochondrial alterations in apoptosis (Pena-Blanco & Garcia-Saez, 2018). BAX is kept in an inactive form that constantly retrotranslocates between cytosol and mitochondria in healthy cells (Edlich et al, 2011; Schellenberg et al, 2013). Upon apoptosis induction, BAX is activated by interaction with the BH3 domain of the direct activator BH3-only proteins, like tBID and BIM, which induce BAX accumulation at discrete puncta at the MOM, called apoptotic foci (Karbowski et al, 2002). This is accompanied by conformational changes that allow extensive membrane interactions, dimerization, and further self-assembly of BAX into multiple oligomeric species (Subburaj et al, 2015). BAX oligomers form supramolecular structures shaped as lines, arcs, and rings, and both arcs and rings have been associated with growing membrane pores at the MOM that reach sizes in the order of hundreds of nanometers in diameter (Große et al, 2016; Salvador-Gallego et al, 2016). These pores are responsible for the release of mitochondrial contents to the cytosol, ranging from cyt c and Smac to mtDNA (McArthur et al, 2018; Riley et al, 2018). Despite the clear paramount role of BAX and BAK in MOMP, the contribution of additional mitochondrial proteins to this process remains an open question. Mitochondrial fragmentation is conserved in apoptotic cell death, even in organisms that do not involve MOMP (Martinou & Youle, 2011), yet its relevance for cell death is poorly understood. The dynamin-like protein DRP1 promotes mitochondrial fission in healthy human cells to maintain cellular homeostasis, and it mediates mitochondrial fragmentation and participates in cristae remodeling in apoptotic human cells to facilitate cyt c release (Frank et al, 2001; Otera et al, 2016). DRP1 is SUMOylated during apoptosis, which stabilizes its oligomeric form at mitochondria to stabilize membrane contacts sites between ER and mitochondria (Prudent et al, 2015). DRP1 co-localizes with BAX at apoptotic foci in mitochondria (Karbowski et al, 2002) and several lines of evidence support an interplay between the two proteins in apoptosis. DRP1 has been shown to promote the oligomerization of BAX in in vitro reconstituted systems by promoting negative membrane curvature (Montessuit et al, 2010). However, the contribution of DRP1 to apoptosis is controversial, because mitochondrial fission can be uncoupled from cyt c release and cells deficient in DRP1 also undergo cell death albeit with altered kinetics (Parone et al, 2006; Estaquier & Arnoult, 2007; Sheridan et al, 2008). As a result, the molecular mechanisms and functional significance of the connection between DRP1 and BAX in apoptosis remain unclear. Here we show that direct interaction between BAX and DRP1 is induced at apoptotic foci in correlation with MOMP and maintained until the death of the cell. The association between BAX and DRP1 requires the lipid environment and affects the membrane activity of both proteins. We identify several surfaces of BAX involved in the interaction with DRP1 and determine that the N-terminal region of the protein is required for the association with DRP1 in cells. Interestingly, forced interaction between BAX and DRP1 induces their translocation to mitochondria, accumulation in apoptotic foci, as well as their activation for MOMP, and mitochondrial remodeling leading to apoptosis. Our findings provide a molecular basis for the functional link between the machineries for apoptosis execution and for mitochondrial dynamics and reveal that DRP1 can act as a non-BH3 activator of BAX to promote apoptosis. Results Apoptosis induction brings BAX and DRP1 in close proximity in correlation with MOMP While we and others have so far not been able to detect direct interaction between endogenous BAX and DRP1 (Montessuit et al, 2010), these two proteins clearly co-localize at discrete foci in apoptotic cells visualized with confocal microscopy (Karbowski et al, 2002). However, the spatial resolution limit of around 200 nm does not allow to discern whether BAX and DRP1 are connected by direct physical interactions. To gain further insight into the structural organization of BAX and DRP1 during apoptosis, we took advantage of the high spatial resolution offered by single-molecule localization microscopy (SMLM) (Salvador-Gallego et al, 2016). We imaged fixed HeLa cells transiently transfected with GFP-BAX 3 h after staurosporine (STS) treatment for apoptosis induction and stained them with anti-GFP nanobodies labeled with Alexa Fluor (AF)647. Endogenous DRP1 was immunostained using a secondary antibody labeled with the cyanine-based fluorescent (CF) dye CF680. Using spectral unmixing based on a ratiometric classification of the individual localizations in the individual emission channels (Winterflood et al, 2015), we built two-color super-resolved images that revealed a close apposition between the fluorescent signals corresponding to BAX and DRP1, which appeared in discrete foci (Fig 1A). The degree of overlap was comparable to that of the positive co-localization control based on DRP1 immunostaining with two secondary antibodies labeled with AF647 and CF680 dyes, indicating that in apoptotic cells BAX and DRP1 co-localize up to a resolution of 30 nm (Fig 1B and C). In agreement with this, quantification of the distance between BAX and DRP1 in the foci revealed that the distance between them was < 30 nm in > 80% of the cases (Fig 1D). Figure 1. BAX and DRP1 specifically interact at mitochondria during apoptosis A–D. Dual-color SMLM shows colocalization of BAX and DRP1 at the nanoscale. (A) Overview SMLM image of a HeLa cell transfected with GFP-BAX stained with anti-GFP nanobody-AF647 and anti-DRP1 antibody probed with a CF680-labeled secondary antibody, 3 h after apoptosis induction. Scale bar 2 μm. (B) Magnified SMLM images of GFP-BAX and DRP1 assemblies colocalizing up to 30 nm during apoptosis. Scale bar 100 nm. (C) Colocalization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. (D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n = 4 independent experiments with a total of 720 BAX structure. E, F. Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm. G. BAX/DRP1 interaction compared to BAX/BAX and BCL-xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n = 3 independent experiments (with n = 100 cells each). Levels of significance were determined by paired two-tailed Student's t-test (*P < 0.05, ***P < 0.001) compared to BAX/DRP1 after apoptotic induction (STS). Download figure Download PowerPoint Since both BAX and DRP1 are known to form large oligomers with sizes in the order of the measured distances between them by SMLM, we reasoned that they were likely to interact physically at the apoptotic foci. To test this hypothesis, we used the dimerization-dependent fluorescent protein (ddFP) technique (Ding et al, 2015). In this method, the proteins of interest are tagged with the fusion proteins RA and GB, respectively, which emit significant measurable fluorescence only when they are part of the same complex (< 10 nm apart). Compared to similar approaches, the ddFP pair offers the advantage that it does not enhance association between the tagged proteins and can be used to follow the dynamics of reversible interactions (Ding et al, 2015). We transiently expressed RA-BAX and GB-DRP1 in HeLa cells and imaged cells before and after apoptosis induction with STS for 3 h (optimal treatment for BAX translocation to mitochondrial foci) (Salvador-Gallego et al, 2016). As shown in Fig 1E, the fluorescence of RA-BAX/GB-DRP1 complexes was negligible in untreated cells, but became apparent as discrete foci in apoptotic cells with a distribution similar to that of colocalized BAX and DRP1 in Fig 1A and B. These results were consistent when we used different cell lines (U2OS BAX/BAK DKO and MEFs DRP1 KO) or different apoptotic triggers (paclitaxel and etoposide) (Fig EV1A and B). As a positive control, RA-BAX/GB-BAX interaction signal was also induced in apoptosis (Fig 1F). In contrast, the combination of the anti-apoptotic BCL-2 protein BCL-xL tagged with RA and GB-DRP1 did not give any fluorescent signal neither in untreated nor in treated cells (Fig EV1C). Quantification of the % cells with positive RA/GB signal revealed that the interaction between BAX and DRP1 was enhanced in apoptotic cells compared to untreated cells (Fig 1G). Click here to expand this figure. Figure EV1. Interaction of BAX and DRP1 is independent of cell type and apoptotic stimuli Representative confocal microscopy images of ddFP of RA-BAX and GB-DFP1 (green) in untreated or apoptotic (STS) HeLa (same images as used in Fig 1E), U2OS BAX/BAK DKO, and MEF DRP1 KO cells. Mitochondria are visualized using mito-BFP (magenta). Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm. Representative confocal microscopy images of RA-BAX/GB-DRP1 (green) and mito-BFP (magenta) overexpressed in U2OS BAX/BAK DKO cells after apoptosis induction using STS, Paclitaxel or Etoposide as indicated. Scale bar 10 μm. Representative confocal microscopy image of RA-BCL-xL/GB-DRP1 (green) and mito-BFP (magenta) overexpressed in U2OS BAX/BAK DKO cells after apoptosis induction using STS. Scale bar 10 μm. Images are representative for n = 3 independent experiments. Data information: Images are representative for n = 3 independent experiments. Download figure Download PowerPoint To examine the dynamics of association between BAX and DRP1 during apoptosis progression, we performed live cell confocal imaging of HeLa cells transiently expressing RA-BAX, GB-DRP1, and Smac-GFP following STS treatment. Smac is a mitochondrial pro-apoptotic factor that is released to the cytosol upon MOMP. As shown in Fig 2A and B, the interaction between RA-BAX and GB-DRP1 was enhanced upon apoptosis induction and correlated in time with Smac-GFP release into the cytosol, persisting until cell death. Of note, we observed a delay between the redistribution of Smac-GFP and the appearance of BAX/DRP1 complexes, which was of about 10 min at the 50% increase in both signals. This is comparable to that observed between the formation of GFP-BAX foci and Smac release during apoptosis (Salvador-Gallego et al, 2016) and likely related to their relative efficiency of detection. Due to this technical limitation, and although we observe a clear temporal correlation, we cannot exclude that one event happens before the other from these experiments. Figure 2. Dynamics of BAX and DRP1 interaction during apoptosis Confocal microscopy time-series images of the increase in cytosolic Smac-GFP (magenta) in relation to the detection of RA-BAX/GB-DRP1 complexes (green) during apoptosis induction in MEF DRP1 KO cells. Scale bar 10 μm. Images are representative for n = 3 independent experiments. Normalized fluorescence intensity of Smac-GFP in a region of interest in the cytosol and of RA-BAX/GB-DRP1 foci for individual cells (thin lines) and the average of all cells (thick lines, n = 5). Time 0 min corresponds to the time point when RA-BAX/GB-DRP1 foci were detected. Quantification of BAX/DRP1 interaction (% cells with RA-BAX/GB-DRP1 foci normalized to the mito-BFP positive cells, n = 100 cells per experiment) during inhibition of effector caspases (Q-VD-OPh), the mitochondrial permeability transition pore (CsA) versus control (DMSO), with or without apoptosis induction (+/− STS). Box plots represent the interquartile (box), median (line) and SD (whiskers). Significance was determined from n = 3 independent experiments (with n = 100 cells each) by paired two-tailed Student's t-test (n.s. indicating P > 0.05) compared to DMSO control after apoptotic induction (STS). Download figure Download PowerPoint To discard that the interaction between BAX and DRP1 was not a product of downstream caspase activity or of the permeability transition pore (PTP), we confirmed that the signal was maintained when cells were treated with a pan-caspase inhibitor (Q-VD-OPh) or with the PTP blocker cyclosporine A (CsA) (Fig 2C). Together, these findings demonstrate that, specifically during apoptosis, BAX and DRP1 associate into complexes that accumulate at discrete foci, correlate in time with MOMP, and persist until the death of the cell. Direct interaction between BAX and DRP1 requires the membrane and affects the activity of both proteins To investigate whether BAX and DRP1 interact directly or whether additional components are required for their association, we quantified their interaction in minimal systems of chemically controlled composition. We performed Fluorescence Cross-Correlation Spectroscopy (FCCS) measurements (Garcia-Saez et al, 2009; Bleicken et al, 2017) using recombinant, fluorescently labeled BAX (BAX-AF633) and DRP1 (DRP1-AF488) (Fig 3A–C). FCCS is a technique with single molecule sensitivity that calculates the temporal auto-correlation of diffusing BAX-AF633 and DRP1-AF488 particles, from which the diffusion coefficient is calculated (Fig EV2A and B). It also quantifies the cross-correlation (CC) signal due to the codiffusion of BAX-AF633/DRP1-AF488 complexes, which is directly proportional to the extent of association between the two proteins. As shown in Fig 3A and C, we could not detect any CC between BAX-AF633 and DRP1-AF488 in solution. In contrast, when we measured FCCS on BAX-AF633 and DRP1-AF488 bound to Giant Unilamellar Vesicles (GUVs), we clearly detected a positive CC indicative of direct interaction between the proteins (Fig 3B and C). Of note, DRP1 bound spontaneously to GUVs with a simple lipid composition containing the mitochondrial lipid cardiolipin, while association of BAX with the membrane was not induced by DRP1 and was promoted by incubation at 42°C (heat activation). As a control to confirm that the interaction between BAX and DRP1 is specific and not an artifact due to accumulation of both proteins in the membrane, an excess of unlabeled cBID (a known BAX interactor (Czabotar et al, 2013)) successfully competed for the association between BAX-AF633 and DRP1-AF488 and decreased the %CC (Figs 3C and EV2C). These results demonstrate that BAX and DRP1 directly interact in vitro and that the membrane environment is the only additional component required for such interaction. Figure 3. Direct interaction of BAX and DRP1 in the membrane affects their respective activities A, B. Representative auto- (green and violet curves) and cross-correlation (CC, BAX-DRP1, grey curves) curves of DRP1-AF488 and BAX-AF633 measured by FCCS in solution (A) and in the membrane of GUVs (B). Dash gray line depicts raw data and solid lines correspond to data fitting. C. Quantification of %CC between DRP1-AF488 and BAX-AF633 in solution (grey), in the membrane (violet), and in the membrane in presence of excess unlabeled cBID (beige). Box plots represent the interquartile (outer box), mean (inner box), median (line) and range (whiskers). Levels of significance were determined by paired two-tailed Student's t-test (*P < 0.05, **P < 0.01) from n = 9 measurements in solution, n = 46 individually measured GUVs in the membrane or n = 17 GUVs in presence of cBID. D. Effect of DRP1 on BAX-induced LUV permeabilization. BAX was activated by cBID (blue lines), or mild heat (42°C, grey lines). Data are presented as mean ± SD of n = 3 individual experiments. Significance was determined at the end-point of the kinetic measurement (180 min) by paired two-tailed Student's t-test (*P < 0.05, ***P < 0.001). E. Effect of DRP1 on BAX-induced GUV permeabilization. Left: % GUVs permeabilized to Cytochrome c488 (Cyt c, 12 kDa, beige) and allophycocianin (APC, 104 kDa, blue) in the absence or presence of cBID, BAX and DRP1 combined as indicated. Data are presented as mean ± SD of n = 4 independent experiments. *P < 0.05 (paired two-tailed Student's t-test). Right: representative confocal microscopy images showing GUVs (grey) in a solution of Cyt c (green) and APC (magenta). Scale bar 10 µm. F, G. Effect of BAX on DRP1 membrane density and DRP1-induced shape alterations of liposomes measured by flow cytometry. (F) Representative flow cytometry plots outlining DRP1 (Alexa Fluor 488 signal) binding to LUVs (Rhodamine signal) in the absence or presence of BAX. % DRP1-positive LUVs indicated in green. (G) Membrane density of DRP1 (corrected fluorescence units, cFU, left graph) and DRP1-induced membrane tethering (indicated by a shape index > 1, right graph) in LUVs in the absence or presence of different concentrations of BAX. Data are presented as mean ± SD of n = 3 independent experiments. *P < 0.05 (paired two-tailed Student's t-test) vs. DRP1 without BAX. Download figure Download PowerPoint Click here to
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