Annexin A5 controls VDAC1-dependent mitochondrial Ca²⁺ homeostasis and determines cellular susceptibility to apoptosis

The study addresses how Annexin A5 (AnxA5), a Ca2+-dependent phospholipid-binding protein, regulates mitochondrial Ca2+ signaling and influences apoptosis. Prior work shows AnxA5 affects intracellular Ca2+ signaling, binds cardiolipin in mitochondria, and modulates apoptosis, while VDAC1 in the OMM conducts Ca2+ from ER to mitochondria and its oligomerization is linked to apoptosis. The authors hypothesize that AnxA5 modulates mitochondrial Ca2+ homeostasis by regulating Ca2+ flux across the OMM, potentially via VDAC1, thereby shaping intermembrane space (IMS) Ca2+ signals, mitochondrial architecture, and apoptotic susceptibility. The purpose is to define AnxA5’s localization and functional role in mitochondrial Ca2+ transfer during ER Ca2+ release and its impact on VDAC1 behavior and cell fate.

- AnxA5 binds negatively charged phospholipids in a Ca2+-dependent manner and has been implicated in Ca2+ influx across artificial membranes and plasma membranes in response to oxidative stress. It binds cardiolipin and immobilizes cardiolipin microdomains in mitochondrial membranes, impacting apoptosis susceptibility. - VDAC1 mediates Ca2+ passage through the OMM during IP3-induced ER Ca2+ release; its permeability is modulated by proteins (e.g., α-synuclein, Bcl-xL). VDAC1 oligomerization is associated with apoptosis and can be triggered by agents like cisplatin and selenite; inhibitors of VDAC1 oligomerization (e.g., VBIT-4) protect against apoptosis. - Mitochondrial Ca2+ uptake through the MCU complex requires sufficient IMS Ca2+ and is regulated by MICU1/2; mitochondrial Na+/Ca2+ exchange mediates efflux. ER-mitochondria contact sites (MERCs) create local Ca2+ hotspots facilitating mitochondrial uptake. - Despite evidence linking AnxA5 to Ca2+ signaling and apoptosis, its direct role in mitochondrial Ca2+ handling and in regulating VDAC1 Ca2+ permeability in intact cells remained unclear, motivating the present study.

- Cell models: CRISPR/Cas9-generated AnxA5 knockout (AnxA5-KO) in HeLa and EA.hy926 endothelial cells; perivascular cells isolated from AnxA5-KO mice and WT controls. - Ca2+ measurements: Genetically encoded, compartment-targeted biosensors—mitochondrial matrix (4mtD3cpv), IMS (MICU1-140-GEM-GECO1), cristae lumen (ROMO-GEM-GECO1), MICU1 FRET sensor (MICU1-CFP/YFP) to track IMS Ca2+-dependent MICU1 rearrangements; cytosolic Ca2+ by Fura-2 AM or jGCaMP7c; ER Ca2+ by DIER sensor. Agonists: ATP or histamine (IP3-mediated ER release), BHQ (SERCA inhibitor) to assess ER leak; SOCE assayed by Ca2+ re-addition after store depletion. - Mitochondrial membrane potential: TMRM with FCCP depolarization control. - MERCs and morphology: 3D confocal microscopy with Mitotracker and ER marker (ERAT4.03 NA) to compute Pearson’s co-localization; SPLICS split-GFP proximity sensor; mitochondrial 3D morphometrics (volume, surface, branching, elongation) via ImageJ 3D analysis. - Ultrastructure: Transmission EM (chemical fixation) to quantify cristae membrane amount and density; spatial distribution of cristae density analyzed via concentric shell erosion (PCM distribution). Structured illumination microscopy (SIM) to quantify cristae membrane dynamics globally and at MERCs and to assess mitochondrial morphology remodeling. - Protein localization: Subcellular fractionation to cytosol/crude/pure mitochondria; Proteinase K protection assay (TOM20, VDAC1, Cyt c, tubulin markers) to map leaflet exposure; immunogold EM with chemical fixation and high-pressure freezing/freeze substitution to localize AnxA5 at baseline and 20 s post-histamine. - Protein interactions/proximity: Co-immunoprecipitation of AnxA5-FLAG and immunoblotting for VDAC1; in situ proximity ligation assay (PLA) for AnxA5–VDAC1, with VDAC1 knockdown and IP3R–VDAC1 as controls. - Genetic perturbations: siRNA knockdown of VDAC1; overexpression of VDAC2-FLAG or VDAC3-FLAG to test isoform rescue; AnxA5 mutant constructs disrupting Ca2+ binding (AnxA5-2Mt: D144N/E228Q; AnxA5-3Mt: D144N/E228Q/D303N) or self-assembly (AnxA5-5Mt: R18E/R25E/K29E/K58E/K193E) expressed in AnxA5-KO cells. - Electrophysiology: Patch-clamp of intact isolated mitochondria (mitochondria-attached configuration) to record OMM single-channel activity with 10 µM free Ca2+ in pipette; recombinant AnxA5 added to pipette in rescue experiments; assess channel occurrence and NPo at multiple voltages; Ca2+-free control. - Apoptosis assays: Treatments with cisplatin (5–10 µM) or selenite (10 µM), with/without VBIT-4 (20 µM) preincubation; flow cytometry and live-cell imaging with Annexin V-FITC/PI for viability and apoptosis at 24–48 h. - VDAC1 oligomerization: Chemical cross-linking (EGS) followed by immunoblotting to quantify monomer/dimer levels over time (12–48 h) and with VBIT-4. - VDAC1 clustering: Live-cell imaging of VDAC1-TC labeled with FlAsH-EDT2; image analysis (Gaussian blur, watershed, MaxEntropy threshold) to quantify cluster size. - Statistics: Appropriate parametric/nonparametric tests (Student’s t-test, ANOVA with Tukey, Kruskal–Wallis, Kolmogorov–Smirnov), mean ± SEM; n reported as cells/biological replicates; significance threshold p<0.05.

- AnxA5 is essential for mitochondrial Ca2+ uptake upon IP3-mediated ER Ca2+ release: • In HeLa and EA.hy926 cells, AnxA5-KO reduced mitochondrial [Ca2+]Matrix responses to ATP/histamine, while basal mitochondrial Ca2+ and cytosolic and ER Ca2+ signals were unchanged (or slightly higher cytosolic peaks in EA.hy926) (e.g., HeLa: rescue by AnxA5 restored [Ca2+]Matrix; Fig. 1A–C; p=0.0034, p=0.0404). • Perivascular cells from AnxA5-KO mice also showed diminished ATP-induced [Ca2+]Matrix rises (p=0.0062) with unchanged cytosolic responses. • Mitochondrial membrane potential (TMRM) was unaffected by AnxA5 loss; expression of VDAC1, MICU1/2, UCP2, MCU, EMRE unchanged (Fig. 2A–D). - MERCs integrity preserved, but mitochondrial architecture altered by AnxA5 loss: • ER–mitochondria colocalization (Pearson’s R) and SPLICS signals were similar in WT and KO, indicating intact MERCs. • AnxA5-KO increased mitochondrial volume (p=0.0092) and branching (lower Nmito/Nellipse; p=0.0036). Cristae membrane amount/density were similar overall, but cristae density was redistributed, increased toward the mitochondrial center. - AnxA5 localizes on and within mitochondria and accumulates at the OMM upon ER Ca2+ release: • Fractionation and PK protection: substantial AnxA5 in pure mitochondria; partial PK digestion indicates majority at cytosolic leaflet of OMM with a fraction protected within mitochondria. • Immunogold EM detects AnxA5 in cytosol, OMM, and intramitochondrial regions; after histamine, gold particles accumulate within ~20 nm of the OMM on both cytosolic and IMS sides. - AnxA5 specifically promotes IMS Ca2+ entry during ER Ca2+ release: • IMS [Ca2+] elevations upon histamine were significantly reduced in AnxA5-KO (HeLa and EA.hy926), while BHQ-induced ER leak and SOCE-driven IMS Ca2+ rises were comparable between genotypes. • Dose–response: histamine EC50 for IMS Ca2+ shifted from 1.3 µM (WT) to 4.8 µM (KO), with cytosolic EC50 unchanged (~2 µM), indicating an OMM-specific defect. • Functional rescue: WT AnxA5 and the self-assembly–deficient AnxA5-5Mt restored IMS signaling; Ca2+-binding mutants (AnxA5-2Mt, -3Mt) failed to rescue, demonstrating necessity of AnxA5 Ca2+ binding. - AnxA5-mediated IMS Ca2+ controls IMM dynamics and MCU positioning: • MICU1-FRET decrease upon ER Ca2+ release was attenuated in KO (p<0.0001), indicating reduced MICU1 de-oligomerization. • Cristae membrane dynamics at MERCs decreased upon histamine in WT and rescue, but not in KO; cristae [Ca2+] responses were reduced in KO (p<0.0001). • MCU translocation from cristae to IBM occurred in WT after histamine but was impaired in KO; ER Ca2+ release induced mitochondrial remodeling (area and aspect ratio changes) in WT but not KO. - AnxA5 is proximal to VDAC1 and is required for VDAC1/2-mediated Ca2+ permeability: • PLA revealed AnxA5–VDAC1 proximity (<30 nm), lost in AnxA5-KO and reduced by VDAC1 knockdown; co-IP did not detect stable interaction. • VDAC1 knockdown reduced mitochondrial Ca2+ responses in WT, with no further reduction in AnxA5-KO, indicating interdependence. • VDAC2 (but not VDAC3) overexpression rescued VDAC1-KD mitochondrial Ca2+ responses in WT; rescue required AnxA5 presence, suggesting AnxA5 also supports VDAC2 Ca2+ function. - OMM single-channel recordings revealed a 35 pS Ca2+-permeable channel whose activity depends on AnxA5: • With 10 µM Ca2+ in pipette, a 35 pS inward channel was observed between −60 to −120 mV; no activity without Ca2+. • AnxA5-KO mitochondria exhibited reduced channel occurrence and lower NPo at −80 mV; addition of recombinant AnxA5 restored occurrence and NPo. VDAC1 knockdown reduced occurrence and NPo; no further reduction by AnxA5-KO in VDAC1-KD background. - AnxA5 protects against apoptosis by limiting VDAC1 oligomerization: • Cisplatin (12 h) elevated mitochondrial matrix and IMS Ca2+ in WT but less so in KO; cytosolic increases were modest and similar. Despite lower mitochondrial Ca2+ elevations, AnxA5-KO cells showed higher apoptosis after 24–48 h cisplatin in a dose-dependent manner. • VBIT-4 co-treatment reduced cisplatin-induced cell death and late apoptosis in both genotypes. • Cross-linking blots: cisplatin increased VDAC1 dimerization more in KO than WT at 24–48 h; VBIT-4 suppressed dimerization. Selenite similarly caused greater dimerization and apoptosis in KO, both reduced by VBIT-4. VDAC1 clustering (VDAC1-TC) increased with cisplatin, especially in KO; reduced by VBIT-4.

The findings demonstrate that AnxA5 is a key regulator of Ca2+ transfer across the OMM during ER-derived Ca2+ release, acting at the level of VDAC1/VDAC2. AnxA5 localizes to mitochondria and rapidly accumulates at the OMM in response to high local Ca2+ microdomains at MERCs. Functional assays show AnxA5 is required for robust IMS Ca2+ rises without altering cytosolic or ER Ca2+ signaling, mitochondrial membrane potential, or MERC abundance. Mechanistically, AnxA5’s Ca2+-binding (but not self-assembly) is necessary, suggesting a membrane-associated regulatory role rather than pore formation. Electrophysiology supports that AnxA5 enhances the occurrence and open probability of a 35 pS Ca2+-permeable OMM channel, with VDAC1 influencing channel behavior; together with PLA data, this points to AnxA5 stabilizing the Ca2+-permeable state of VDAC1 (and VDAC2), likely via effects on local lipid microdomains enriched in negatively charged phospholipids. Diminished IMS Ca2+ signaling in AnxA5-KO reduces MICU1 rearrangement, cristae junction opening, MCU relocation, and cristae Ca2+, linking OMM Ca2+ flux regulation to IMM dynamics and mitochondrial morphology remodeling. In pathological contexts, AnxA5’s presence in the VDAC1 microenvironment limits apoptotic VDAC1 oligomerization induced by cisplatin/selenite, thereby reducing apoptosis; VBIT-4 phenocopies this protection by inhibiting VDAC1 oligomerization. Overall, AnxA5 emerges as a crucial modulator of VDAC-dependent Ca2+ transport and mitochondrial function with implications for cell survival under stress.

This study identifies Annexin A5 as an integral modulator of VDAC-dependent Ca2+ permeation across the outer mitochondrial membrane during ER Ca2+ release, thereby shaping IMS and matrix Ca2+ signaling, IMM dynamics (MICU1 state, cristae junction opening, MCU redistribution), and mitochondrial morphology. AnxA5’s Ca2+ binding is required for this function, and its proximity to VDAC1/2 supports a model in which AnxA5 stabilizes a Ca2+-permeable VDAC state within specific lipid microdomains. In stress conditions, AnxA5 attenuates VDAC1 oligomerization and protects against cisplatin/selenite-induced apoptosis. Future work should identify the specific OMM phospholipids and microdomain features that recruit AnxA5, define the precise molecular mechanism by which AnxA5 modulates VDAC gating/oligomerization, and clarify the relationship between the observed 35 pS OMM channel and VDAC1 in vivo.

- Interaction evidence: Co-immunoprecipitation did not detect a stable AnxA5–VDAC1 interaction; proximity (PLA) suggests colocalization within <30 nm but not direct binding, leaving the mechanism indirect or transient. - Channel identity: The 35 pS OMM Ca2+ channel’s molecular identity cannot be conclusively assigned to VDAC1; VDAC1 knockdown effects were modest, potentially due to incomplete knockdown efficiency. High-conductance VDAC-like activity observed in artificial systems was not detected here, reflecting model differences. - Lipid specificity unknown: The exact negatively charged phospholipids and membrane microdomains in the OMM that recruit AnxA5 remain unidentified. - Imaging constraints: Strong cytosolic AnxA5 signal limited conventional fluorescence localization, necessitating cryo-fixation and immunogold EM; temporal resolution of AnxA5 dynamics is limited by EM methodology. - Generalizability: Most functional data are from HeLa and EA.hy926 cell lines; in vivo validation beyond perivascular cells is limited. - Ca2+ source specificity: AnxA5 effects were specific to IP3-mediated ER release and did not alter SOCE-driven IMS Ca2+ increases, which may limit applicability to other Ca2+ entry routes.