A membrane-disruptive action of VBIT-4 challenges its role as a widely used VDAC1 oligomerization inhibitor
- Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS, UMR 7255 – Aix-Marseille Université, France
- Laboratoire de Biologie et Modélisation de la Cellule, CNRS, UMR 5239, Inserm, U1293, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, France
- Centre Blaise Pascal de Simulation et de Modélisation Numérique, Ecole Normale Supérieure de Lyon, France
- Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States
- U1325 Aix-Marseille Université, INSERM, DyNaMo, Turing Centre for Living System, France
- Biomass and Protein Engineering Aix-Marseille Université, CNRS, IMM UAR2044, France
Figures
Figure 1 with 2 supplements
VBIT-4 induces defects in membrane bilayers, both in the presence and in the absence of VDAC1.:7.5.
Upper panels: VDAC1 reconstituted into POPC:POPE:chol (60.5:32.:7.5) liposomes was adsorbed onto mica for atomic force microscopy (AFM) imaging. Representative AFM images and corresponding height profiles before addition (A) and after addition of 1 µM (B) and 10 µM (C) VBIT-4. The purple rectangles in the height profiles indicate the 2.5 nm threshold: values above correspond to VDAC1 pores, whereas values below correspond to defects induced by VBIT-4. Asterisks (*) denote VBIT-4-induced pores. In (C), the left and middle panels show voltage-dependent anion channel (VDAC) clusters, while the right panel presents an overview and a cross-section from a 3D zoom, highlighting the depth of VBIT-4-induced pores in lipid regions devoid of VDAC1. Images acquired at 10 µM VBIT-4 display reduced resolution, consistent with compound-induced alterations of membrane properties. (D) Quantitative pore size distribution derived from pixel depth/height analysis of the overview images shown in A, B, and C. Histograms represent the distributions without VBIT-4 (green, panel A), in the presence of 1 µM (orange, panel B) or 10 µM (purple, panel C) VBIT-4. (E) Monomer and micellar structure of VBIT-4 obtained by molecular dynamics simulations. On the monomer structure, polar and hydrogen-bonding groups are highlighted in red, ionizable/potentially charged groups in blue, neutral aromatic regions in gray, and strongly hydrophobic (lipophilic) substituents in green. Overall, the molecule displays an amphipathic organization, with a charged/polar core (blue/red) flanked by lipophilic aromatic groups (gray/green). (F–H) Models based on AFM topography from panels A, B (right), and C (left), respectively. VDAC1 is shown as a dark solid surface, the first lipid shell in yellow, and surrounding lipids in atom color representation. The red patches in (H) show the position of the VBIT-4 pores, which cannot be modeled by this method. Lower panels: Lipid membranes (without VDAC) were adsorbed onto mica and imaged by AFM. (I) Control membrane before VBIT-4 addition. After addition of 1 µM (J), 5 µM (K), 50 µM (L), and 100 µM (M) VBIT-4. Height profiles are overlaid on the images. The false-color scale is 12 nm in all panels.
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Figure 1—source data 1
Co-ordinates and pixel values of VDAC1 and VBIT-4.
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Figure 1—figure supplement 1
VBIT-4 self-assembles into micelle-like aggregates in aqueous solution.
Molecular dynamics simulations of 1000 VBIT-4 molecules under a charged (A) or neutral state (B) in a 15×15×15 nm3 water box show spontaneous clustering into compact micelle-like structures, consistent with the compound’s amphiphilic and hydrophobic properties.
Figure 1—figure supplement 2
VBIT-4 does not alter the spatial organization of voltage-dependent anion channel (VDAC1) pairs in atomic force microscopy (AFM) micrographs.
(A) Distribution of inter-protein distances measured without VBIT-4 (open bars; 829 distances from 2 AFM fields), with 1 μM VBIT-4 (horizontal hatching; 380 distances from 2 fields), and with 10 μM VBIT-4 (oblique hatching; 568 distances from 4 fields). The number of intervening lipid layers between proteins was derived from distances between the proteins: 0 lipids (<46 Å, gray), 1 (46–50 Å, yellow), 2 (50–56 Å, blue), 3 (56–62 Å, green), 4 (62–68 Å, orange), and ≥5 (>68 Å, purple). Error bars represent standard deviations calculated per AFM field. (B) Schematic illustrating a cluster of six proteins separated by various numbers of lipid layers (color-coded as in A). Inter-protein distances were determined using Delaunay triangulation of detected protein positions, which constructs a network of nearest-neighbor connections by linking points such that no additional point lies within the circumcircle of any triangle. This method restricts analysis to local neighbors, minimizing contributions from non-local associations and thereby providing accurate estimates of lipid-mediated spacing within clusters (total N=5331 distances across 8 images, each analyzed in triplicate).
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Figure 1—figure supplement 2—source data 1
Inter-protein distances with and without VBIT-4.
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Figure 2 with 2 supplements
VBIT-4 incorporates into lipid membranes.
(A) Laurdan generalized polarization (GP) analysis of POPC:POPE:chol (60.5:32:7.5) liposomes with VBIT-4. The plot shows a VBIT-4 concentration-dependent decrease in Laurdan generalized polarization (n=5). The inset corresponds to the fluorescence spectra at VBIT-4 concentrations ranging from 0 (light blue) to 100 μM (dark blue); the inset data can be found at a larger scale in Figure 2—figure supplement 1. (B) Liposome leakage test of encapsulated fluorescein sodium salt from POPC:POPE:chol (60.5:32:7.5) liposomes with increasing concentration of VBIT-4 and TX-100 (0.5%) (n=3). The inset represents the kinetics of fluorescein leakage from the liposomes. (C) Representative current traces obtained on the same planar lipid membrane (PLM) made from dioleoyl-phosphatidylcholine (DOPC):dioleoyl-phosphatidylethanolamine (DOPE):chol (60.5:32.:7.5) before and after successive additions of 20 and 30 μM of VBIT-4 to the cis compartment of the chamber at 80 mV of applied voltage. Large fast fluctuations of the membrane conductance at 30 μM of VBIT-4 preceded the membrane rupture shown by the upward red arrow. The inset shows the current-voltage (I/V) curves obtained in the experiments at different VBIT-4 concentrations. Gray lines are linear regressions, indicating the nearly Ohmic behavior of VBIT-4-induced conductances. Dashed gray lines indicate zero current. The membrane-bathing solutions consisted of 150 mM KCl buffered with 5 mM HEPES at pH 7.4. The current was digitally filtered using a 500 Hz Bessel (8-pole) filter for presentation. (D) Conductance of planar membranes increases with VBIT-4 concentration. PLMs were made from DOPC/DOPE/chol as in (C) and from polar lipid extract (PLE) as in Figure 2—figure supplement 2A. Membrane conductance was calculated from the corresponding I/V curves. The dashed lines are an exponential fit to guide the eye. Error bars are ± SD from measured conductances at different voltages.
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Figure 2—source data 1
Laurdan fluorescence readings.
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Figure 2—source data 2
Liposome fluorescein release assay readings.
- https://cdn.elifesciences.org/articles/111980/elife-111980-fig2-data2-v1.xlsx
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Figure 2—source data 3
Current leakages induced by VBIT-4 in planar lipid bilayers.
- https://cdn.elifesciences.org/articles/111980/elife-111980-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
Laurdan fluorescence emission spectra at different concentrations of VBIT-4 (excitation wavelength 375 nm).
Laurdan fluorescence is sensitive to the polarity and hydration of the lipid bilayer, allowing detection of changes in lipid packing and water penetration within the membrane. Shifts in Laurdan’s generalized polarization (GP), therefore, provide a quantitative measure of membrane perturbation. A clear VBIT-4-induced shift in Laurdan emission is visible, characteristic of a change in the environment around the dye. The DMSO concentration was constant for all the conditions.
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Figure 2—figure supplement 1—source data 1
Laurdan emission spectra of VBIT-4 in liposomes.
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Figure 2—figure supplement 2
VBIT-4 increases membrane conductance across all lipid compositions.
(A) Representative current records obtained on the planar lipid membrane (PLM) made from polar lipid extract (PLE) before and after the addition of 15, 20, and 25 μM of VBIT-4 to both sides of the membrane at 50 mV applied voltage. Large fast fluctuations of the conductance at 25 μM of VBIT-4 (inset (i) shows the current trace at 25 μM VBIT-4 at a finer current scale) preceded the membrane rupture shown by the upward red arrow. Dashed gray lines indicate a zero current. The membrane bathing solutions consisted of 150 mM KCl buffered with 5 mM HEPES at pH 7.4. The current was digitally filtered using a 500 Hz Bessel (8-pole) filter for presentation. (B) The current-voltage (I/V) curves obtained in experiments shown in (A) at different VBIT-4 concentrations. Gray lines arei linear regressions, indicating the Ohmic behavior of VBIT-4-induced conductances. (C) To test whether the differences in membrane preparation account for the variation in reported VBIT-4 effects, we compared solvent-free PLMs with ‘painted’ membranes formed by Mueller’s method, which incorporates organic solvent (decane). Painted membranes were prepared using the Orbit Mini system (Nanion Technologies, Munich, Germany; see Materials and methods). Shown are representative I/V curves from the same painted planar membrane composed of diphytanoyl-phosphatidylcholine (DPhPC) in decane at increasing VBIT-4 concentrations added to the cis (grounded) compartment. The medium was 150 mM KCl and 5 mM HEPES (pH 7.4). Starting at 5 μM, VBIT-4 reproducibly induced leakage in both painted and ‘ solvent-free membranes composed of DPhPC or PLE, confirming that its permeabilizing activity is independent of the bilayer formation method.
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Figure 2—figure supplement 2—source data 1
Current leakages induced by VBIT-4 in different membranes formed by different methods.
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Figure 3 with 2 supplements
VBIT-4 destabilizes lipid membranes independently of VDAC1.
(A) Representative single-channel current traces obtained with VDAC1 reconstituted in planar lipid membrane (PLM) formed from polar lipid extract (PLE) before (control) and after the addition of 5 and 30 μM VBIT-4 to the cis compartment (n=8). Addition of VBIT-4 and the time of the recordings after additions are indicated by upward arrows. The jumps in the current correspond to the application of 10 mV voltage following the application of 0 mV. The record was obtained on the membrane with the same single channel of 4 nS conductance as in the control. The gray dash line indicates zero current (I=0). The dashed blue lines indicate the current through the open single channel. Addition of 30 μM VBIT-4 induced a monotonic increase in membrane conductance, leading to membrane rupture. Current records were digitally filtered using an averaging time of 0.2 ms. (B) Characteristic bell-shaped plots of open probability as a function of the applied voltage obtained in a multichannel experiment with VDAC1 in a PLE membrane in control and after addition of 5 and 10 μM VBIT-4. In all panels, the membrane-bathing solutions consisted of 1 M KCl buffered with 5 mM HEPES at pH 7.4. (C) Binding of VBIT-4 to fluorescently labeled VDAC1 reconstituted into nanodiscs (blue) or to empty nanodiscs, with MSP1D1 labeled (yellow), monitored by microscale thermophoresis. The plot shows normalized fluorescence at 650 nm measured 2.5 s after IR-laser activation across increasing VBIT-4 concentrations. An average apparent kD of 70 µM with a confidence interval from 20 to 167 µM (n=4) was obtained for VBIT-4 binding to empty nanodiscs and 260 µM with a confidence interval from 105 to 331 µM (n=2) for VDAC1-containing nanodiscs. Figure 3—figure supplement 1B shows the control with MSP1D1 protein only.
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Figure 3—source data 1
VDAC1 open probability values with VBIT-4.
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Figure 3—source data 2
MST titrations for VBIT-4 with empty nanodiscs and VDAC1 nanodiscs.
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Figure 3—figure supplement 1
Additional microscale thermophoresis (MST) data.
(A) Spectral shift at 670/650 nm of VDAC1 nanodiscs and lipid nanodiscs does not show any difference, indicating the absence of a specific binding site for VBIT-4 on VDAC1. (B) Titration of VBIT-4 into labeled MSP1D1 without lipids. The MSP-only binding check showed no aggregations and a measurable fluorescent count. Upon addition of VBIT4, there was aggregation in capillaries 1, 4, 7, 9, 11 (data points marked in red). The response amplitude and signal-to-noise ratio (SNR) were too low to analyze (≈ 0.8). Data were plotted as fraction bound = (F–Fmin)/(Fmax–Fmin).
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Figure 3—figure supplement 1—source data 1
MST titrations monitoring spectral shift for VBIT-4 with empty nanodiscs and VDAC1 nanodiscs.
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Figure 3—figure supplement 1—source data 2
MST titrations for VBIT-4 with fluorescently labelled MSP1D1.
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Figure 3—figure supplement 2
Fourier-transform infrared spectroscopy (FTIR) analysis reveals poor solubility and strong aggregation of VBIT-4.
To assess VBIT-4 solubility, we used FTIR, which detects molecular vibrations and distinguishes dissolved from aggregated species through characteristic spectral shifts—making it well suited for amphiphilic molecules like VBIT-4. Measurements were performed in attenuated total reflectance (ATR) mode, with the sample deposited directly on a diamond crystal serving as an infrared-transparent support. (A) FTIR spectra of VBIT-4 showing its characteristic absorption bands at 1470–1570 cm–1 and 1125–1300 cm–1. From the upper to lower traces, six successive washings of the adsorbed material were performed. Despite repeated buffer exchanges (4 μL each), the spectra remained unchanged, indicating that VBIT-4 remained bound to the crystal surface, consistent with aggregation rather than solubilization. This behavior correlates with the turbid appearance of VBIT-4 suspensions in buffer, whereas clear concentrated solutions can be obtained by adding excess acid, even with moderate DMSO (20%). (B) Relative absorbance of VBIT-4, computed by integrating the 1125–1300 cm–1 region in (A) after successive washes with phosphate buffer. The dashed line represents a desorption model, indicating that only ~0.44% of the adsorbed VBIT-4 was resolubilized per microliter of wash, confirming its strong aggregation and limited aqueous solubility.
Figure 4 with 4 supplements
VBIT-4 partitions into lipid bilayers and disrupts membrane integrity through pore-like structures.
(A) VBIT-4 chemical structure and Martini 3 model mapping. Atom-to-bead mappings are indicated by the colored shapes. Assigned Martini 3 bead names are indicated for each bead as overlaid bold text, with the corresponding bead types in italic text. The protonable group is highlighted (⋆). (B) VBIT-4 Martini 3 coarse-grained (CG) model. (C) Potential of mean force (PMF) of VBIT-4 insertion into CG mitochondrial outer membrane (MOM) membranes. Free energy profiles are shown for both the neutral (blue) and protonated (+1 charge, red) forms of VBIT-4. (D) Snapshots of the insertion of neutral VBIT-4 at different distances from the bilayer center (ξ); lipid PO4 beads are represented in orange and VBIT-4 in gray/red/blue. (E) Predicted octanol-water partition coefficient (logP) values for VBIT-4. VBIT-4 hydrophobicity was estimated using several computational predictors (gray) and the Martini 3 CG model developed in this study (blue). Higher logP values indicate greater lipid solubility. The dashed black line shows the consensus value across predictors, while the orange dashed line marks the logP of ibuprofen (~3.9), a well-known hydrophobic drug used here for comparison. (F) Representative snapshots showing the impact of increasing VBIT-4 concentrations on MOM membrane mimics after 10 μs of CG molecular dynamics (MD) simulation. (Lipid phosphate beads are shown in orange; VBIT in gray, with chemically distinct beads highlighted in red and blue.) Top and side snapshots are shown for a 50:50 mix of charged and neutral VBIT-4 at increasing VBIT-4:lipid ratios (0, 1:16, 1:8, 1:3, 1:1.5). Lipid biophysical properties of MOM mimic membranes were evaluated in the presence of increasing VBIT-4 concentrations, either in the charged (red) or neutral (blue) form, as well as a 50:50 mixture of both states (orange). We monitored several indicators of membrane integrity: (G) area per lipid, (H) bilayer thickness, (I) POPC acyl-chain order (membrane packing and organization), (J) lipid tail protrusion (packing defects), (K) bilayer water crossing (membrane permeability), and (L) lipid flip-flop (bilayer asymmetry). Each system was simulated for 10 µs in triplicate, and error bars represent the standard deviation across replicates. (M) PMF profiles for polar defect formation in CG MOM membrane systems, shown as a function of the pore formation reaction coordinate ξ, for membranes without VBIT-4 (black), with charged VBIT-4 (red), and with neutral VBIT-4 (blue). Curve uncertainty (under the single-digit kJ/mol range) is represented by shading in the same colors along the corresponding curve. (N) Representative snapshots of the membrane systems in the presence of neutral VBIT along the reaction coordinate; lipid PO4 beads are represented in orange, cholesterol ROH in dark orange, water beads in blue (only those in the vicinity of the lipid headgroups or the established defect are shown), and VBIT-4 in gray.
Figure 4—figure supplement 1
VBIT-4 forms pore-like structures through phase separation.
Representative coarse-grained molecular dynamics (MD) snapshots showing the effect of increasing VBIT-4 concentrations on mitochondrial outer membrane (MOM) mimics after 10 μs of simulation. Lipid phosphate beads are shown in orange, and VBIT-4 molecules in gray, with chemically distinct beads highlighted in red and blue. Top and side views are displayed for (A) charged, (B) neutral, and (C) 50:50 charged-to-neutral mixtures of VBIT-4 at increasing VBIT-4:lipid ratios (0, 1:16, 1:8, 1:3, 1:1.5). Given that the pKa of the piperazine group is close to physiological pH, VBIT-4 is unlikely to exist exclusively in a charged or neutral form. Its protonation state likely depends on the local environment—being predominantly neutral when deeply inserted in the bilayer and charged near the phosphodiester/glycerol region. Consequently, pore formation at high concentrations likely involves both species: neutral VBIT-4 forming the membrane core, while charged molecules accumulate near the lipid headgroups or within the pore lumen, promoting water penetration and defect stabilization. To approximate this mixed state, simulations of a 50:50 charged-to-neutral mixture were performed, which showed aggregation and pore-like organization similar to neutral VBIT-4 but with intermediate membrane perturbation behavior.
Figure 4—figure supplement 2
VBIT-4 does not prevent VDAC1 oligomerization, but slows it down.
Representative simulation snapshots at 10 μs for charged (A) and neutral (B) VBIT-4 protonation states at increasing VBIT:VDAC ratios (left to right: VDAC only, 20:1, 55:1). VDAC proteins are shown in gray; lipid phosphate beads are shown in orange; VBIT in gray, with chemically distinct beads highlighted in red and blue; the blue box indicates the periodic boundary unit cell. Mean aggregation kinetics (± SD, shaded) for charged (C) and neutral (D) VBIT4 conditions. The fraction of proteins in the largest cluster is shown over 10 μs. The mean and standard deviation values are obtained from 10 independent simulations for each condition. Black: voltage-dependent anion channel (VDAC) only; light/dark color: VBIT 1:20 and 1:55, respectively. Observed oligomerization extent at 10 μs (final frame value) for charged (E) and neutral (F) conditions. Predicted oligomerization plateau estimated by hyperbolic curve fitting to each replicate trajectory, for charged (G) and neutral (H) conditions. Violins show the distribution across 10 replicates; the red line indicates the median. Statistical comparisons by Mann-Whitney U test with Holm correction; **p<0.01, n.s. not significant.
Figure 4—figure supplement 3
MBAR overlap matrices for the water (top) and octanol-water 74:26 (bottom) alchemical legs across three independent replicates.
Each cell reports the overlap between lambda windows i and j; values closer to 1 indicate greater phase-space overlap and more reliable free energy estimates between those states. Bold borders highlight the diagonal and first off-diagonal elements, representing the nearest-neighbor overlaps most critical for convergence. All adjacent lambda pairs exceed the minimal overlap threshold of 0.03, confirming adequate phase-space sampling across the full alchemical pathway.
Figure 4—figure supplement 4
Forward and reverse MBAR convergence of the solvation free energy for water (top) and octanol-water 74:26 (bottom) across three independent replicates.
The forward estimate is computed using increasing fractions of the trajectory from the beginning, while the reverse estimate uses increasing fractions from the end. Rapid convergence of both estimates and their agreement within error at the full trajectory (shaded bands) indicates well-equilibrated simulations. All six legs show stable free energy estimates.
Figure 5
VBIT-4 decreases cell viability, mitochondrial respiration, and mitochondrial membrane potential in HeLa cells.
(A) The graph shows a decrease in viability (black and teal) and an increase in cytotoxicity (pink and purple) with increasing concentration of VBIT-4 in wild-type (WT) and VDAC1 knockout (V1KO) HeLa cells. The data represent the average from three independent experiments, and the error bars represent the ± SD. The data were fitted using nonlinear regression (GraphPad Prism 11.0.0). (B) Bar graphs represent changes in the mitochondrial oxygen consumption rate (OCR) after 6 hr of treatment with 1.25–10 µM VBIT-4 (red square symbols) compared to DMSO control (●) normalized to OCR before drug treatment. HeLa cells were treated with 0.5 µM oligomycin (▲), 2 µM (♦), and 4 µM FCCP (◊) as controls. Symbols represent 8 replicates, and error bars represent the ± SD from the mean. Flow cytometry measurements of mitochondrial membrane potential using tetramethylrhodamine methyl ester (TMRM) (C) and mitochondrial calcium using Rhod2 (D), upon the addition of 5 µM (■) and 10 µM (▲) VBIT-4 compared to vehicle control (●). Data from 6 independent experiments are represented in the box plots. The symbols represent data from independent experiments. The borders of the boxes define the 25th and 75th percentiles, with the median displayed as lines and error bars indicating the ± SD from the mean. Significance was tested using one-way ANOVA followed by the Dunnett post hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
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Figure 5—source data 1
Cell viability, OCR, TMRM and Rhod2 measurements at different concentrations of VBIT-4.
- https://cdn.elifesciences.org/articles/111980/elife-111980-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
Storage conditions affect the cellular activity of VBIT-4.
Tetramethylrhodamine methyl ester (TMRM)/MitoTracker Green (MTG) ratio comparing control (●) and 10 µM VBIT-4 (■) stored in different conditions. The 1st dataset was collected within 2 weeks of preparation of fresh stock solution in DMSO. The 2nd dataset was using the same sample stored at –20°C for 2 months, and the 3rd dataset was using a sample stored at –80°C for less than 1 month. Data from 5 to 6 independent experiments are represented in the box plots. The symbols represent data from independent experiments. The borders of the boxes define the 25th and 75th percentiles, with the median displayed as black lines and error bars indicating the ± SD from the mean. Significance was tested using one-way ANOVA followed by the Dunnett post hoc test (*p<0.05, ****p<0.0001).
Figure 6—figure supplement 1
VBIT-4 decreases mitochondrial membrane potential but does not effect the mitochondria mass.
(A) Flow cytometry measurements of mitochondrial mass using MitoTracker Green (MTG) upon the addition of 5 μM (■) and 10 μM (▲) VBIT-4 compared to vehicle control. (B) Tetramethylrhodamine methyl ester (TMRM) fluorescence normalized to MTG fluorescence (TMRM/MTG). Data from 6 independent experiments are represented in the box plots. The symbols represent data from independent experiments. The borders of the boxes define the 25th and 75th percentiles, with the median displayed as black lines and error bars indicating the standard deviation from the mean. Significance was tested using one-way ANOVA followed by the Dunnett post hoc test (*p<0.05).
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Figure 6—figure supplement 1—source data 1
Mitochondrial mass readings.
- https://cdn.elifesciences.org/articles/111980/elife-111980-fig6-figsupp1-data1-v1.xlsx
Figure 7 with 1 supplement
Putative model of VBIT-4 partitioning and pore formation in a VDAC1-containing membrane.
(A) VDAC1 forms clusters of different sizes and compactions with both direct and lipid-mediated contacts between VDAC1 β-barrels. (B) At low concentrations (<10 µM), VBIT-4 partitions into the membrane, destabilizing it, without measurably altering VDAC1 cluster compaction. (C) At high concentrations (>10 µM), VBIT-4 forms water-permeable pores in the membrane, inducing membrane permeabilization, further affecting membrane integrity. In all three scenarios, VDAC1 monomers maintain their transport channel properties, conducting the water-soluble metabolites, such as ATP, ADP, and NADH, and small ions, including calcium.
Figure 7—figure supplement 1
Changing the membrane composition affects the size and compaction of VDAC1 assemblies.
Figure adapted from Lafargue et al., 2025. This figure illustrates the cluster assignment for VDAC1 assemblies within a mitochondria outer membrane mimicking membrane, characterized by varying cholesterol concentrations, ranging from 5% to 10% cholesterol. VDACs forming direct contacts with another VDAC are represented in red. Isolated VDAC1 monomers are depicted in black. It is clearly evident that the number of VDAC1 with direct contacts with other VDACs, and available for cross-linking (all color dots), are highly variable in the different conditions.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | pQE9 6xHis-mVDAC1-WT | Dearden et al., 2024 | N/A | |
| Gene (Homo sapiens) | pET28a 7xHis-TEV-MSP1D1 | Denisov et al., 2004 | RRID:Addgene_20061 | |
| Cell line (Escherichia coli) | M15 (pREP4) | Creative Biolabs, MOFY-0822-FY509 | ||
| Cell line (HeLa) | N/A | EditCo Bio. Inc | N/A | |
| Commercial assay or kit | Lysine-reactive labeling kit | Nanotemper | NT-L021 | |
| Chemical compound, drug | VBIT-4 | Selleck Chemicals | S3544 | |
| Commercial assay or kit | MitoProbe TMRM Assay Kit for Flow Cytometry | Thermo Fisher | M20036 | |
| Commercial assay or kit | Live/Dead fixable Near-IR viability kit | Thermo Fisher | L34994 | |
| Chemical compound, drug | MitoTracker Green Dye (MTG) | Thermo Fisher | M46750 | |
| Chemical compound, drug | Rhod-2,AM, cell permeant | Thermo Fisher | R1245MP | |
| Chemical compound, drug | Pluronic F-127, 10% in water | Thermo Fisher | P6866 | |
| Chemical compound, drug | DMEM | Gibco | 15607 | |
| Chemical compound, drug | FBS | Gibco | 10437 | |
| Commercial assay or kit | MultiTox-Fluor Multiplex Cytotoxicity Assay kit | Promega | G9201 | |
| Chemical compound, drug | Oligomycin A | Tocris | 4110 | |
| Chemical compound, drug | FCCP | Tocris | 453 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/111980/elife-111980-mdarchecklist1-v1.pdf
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Source code 1
Python script for plotting the inter-VDAC1 distances.
- https://cdn.elifesciences.org/articles/111980/elife-111980-code1-v1.zip
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Supplementary file 1
VBIT-4 induces conductance in planar lipid membranes (PLMs) of different lipid compositions.
- https://cdn.elifesciences.org/articles/111980/elife-111980-supp1-v1.docx
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A membrane-disruptive action of VBIT-4 challenges its role as a widely used VDAC1 oligomerization inhibitor
eLife 15:RP111980.
https://doi.org/10.7554/eLife.111980.3
