Optogenetic Engineering of BAX to Control Mitochondrial Permeabilization and Attenuate Apoptosis in Cells

Optogenetic Engineering of BAX to Control Mitochondrial Permeabilization and Attenuate Apoptosis in Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Optogenetic Engineering of BAX to Control Mitochondrial Permeabilization and Attenuate Apoptosis in Cells Seok Chung, Dain Lee, Hyunjun Bae, Dongwoo Oh, Jinchul Ahn, Minseop Kim, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6222702/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted 10 You are reading this latest preprint version Abstract Although considerable research has focused on enhancing the apoptotic function of BAX for several decades, inhibition of its functionality remains relatively underexplored, despite intensive BAX activation occurring in various neurodegenerative diseases. Here we present a protein engineering approach to modulate BAX integration into the mitochondrial outer membrane, establishing a tunable strategy for apoptosis inhibition. Utilizing optogenetic methods that employ cryptochrome 2 and its binding partner cryptochrome interacting basic helix loop helix 1, we achieved precise spatial control over BAX localization, a critical determinant of its function. Our results demonstrate that the engineered BAX variant is effectively incapacitated in its apoptotic function while also modulating endogenous BAX activity to enhance cellular resistance to apoptosis. These findings not only advance our understanding of BAX regulation but also offer promising prospects for the development of therapeutic strategies against neurodegenerative and other apoptosis related diseases. Biological sciences/Biological techniques Biological sciences/Biotechnology/Expression systems Optogenetics BAX Anti-apoptosis Cryptochrome Mitochondria MOMP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction BAX belongs to the BCL-2 family, a group of proteins that plays a crucial role in governing apoptosis, a highly regulated process designed to eliminate damaged, unnecessary, or potentially harmful cells in the body. 1 In conjunction with its counterpart BCL-2 and other related proteins, BAX participates in the regulation of mitochondrial outer membrane permeabilization (MOMP), which is a critical step in the apoptotic pathway. 2 BAX is a pro-apoptotic protein that possesses BCL-2 homology (BH) domains, which are critical for protein-protein interactions within the BCL-2 family. 3 – 5 Upon receiving apoptosis signals, BAX undergoes a conformational change that enables its insertion into the mitochondrial outer membrane (MOM). Upon integration, BAX oligomerizes by interacting with other BAX molecules through their BH domains and creates pores in the MOM, facilitating the release of cytochrome c (CytC) and other apoptotic factors from the mitochondria into the cytoplasm. The subsequent release of CytC initiates a series of events resulting in cell death. 6 The delicate equilibrium between pro-apoptotic proteins like BAX and anti-apoptotic proteins like BCL-2 is fundamental for determining whether a cell will undergo apoptosis or survive. In healthy cells, these proteins coexist in a finely tuned balance; however, various signals and stress conditions can disrupt this equilibrium, tilting the scales toward apoptosis. 7 , 8 Dysregulation of the apoptotic pathway, including abnormalities in BAX expression or function, has been associated with various diseases including cancer, autoimmune diseases, cardiovascular diseases, aging, and neurodegenerative disorders. 9 – 15 Owing to its involvement in apoptosis, BAX, along with other BCL-2 family proteins, has been investigated as a potential target for therapeutic interventions, particularly in cancer treatment. 16 – 18 However, most previous research has focused on pro-apoptotic induction using BAX for disease treatment, whereas anti-apoptosis is considered crucial in diseases induced by excessive cell death, such as Alzheimer’s disease or Parkinson’s disease. 19 – 21 This study aimed to explore the anti-apoptotic function of a modified BAX protein, using optogenetics for precise control, to achieve a comprehensive understanding of the structural intricacies and functional manipulation underlying BAX. Optogenetics is a powerful technique that uses light to control cellular and protein activities in living organisms by employing light sensitive proteins often derived from microbial organisms such as algae or bacteria. 22 In the context of studying BAX, optogenetics offers several distinct advantages including spatial precision, reversibility, and a reduction in off target effects. The spatial precision of optogenetic tools allows for the targeted control of BAX activity in specific cellular compartments, providing insights into its localized effects within the cell, particularly in relation to mitochondrial function. Moreover, the reversible nature of optogenetic control enables iterative modulation of BAX activity, which facilitates the investigation of transient changes and helps differentiate between acute and prolonged cellular responses. Unlike traditional genetic manipulation techniques that can accidentally impact multiple cellular processes, optogenetics provides a precise and direct method for protein control that minimizes unintended side effects. In this study, optogenetic control was employed to translocate cytoplasmic BAX into mitochondria using cryptochrome 2 (CRY2) and its binding partner cryptochrome interacting basic helix loop helix 1 (CIB1), which are reversibly associated under blue light and dissociated in the dark. 23 , 24 For genetic modification, CRY2 was fused to BAX, which is normally localized in the cytoplasm in the dark owing to its point mutation (S184E), while CIB1 was fused to TOMM20, a protein resident in the MOM. 25 In detail, CRY2 was inserted between the BAX 𝛼8 and 𝛼9 motifs to deter collective BAX insertion to MOM by weaking the anchor, the hydrophobic motif located on the 𝛼9 helix, and also moving the BH domain binding site away from the membrane. 26 , 27 The final construct consisted of mCherry::BAX 𝛼1-𝛼8::CRY2::𝛼9 S184E (Fig. 1 A, Figure S1 ; Table S1 ). This novel anti-apoptotic optogenetic system was validated by comparing its effects on cellular compartments, such as the mitochondria and nucleus, with those of the pro-apoptotic optogenetic system of BAX, consisting of CRY2::mCherry::BAX S184E . For simplicity, the newly developed anti-apoptotic recombinant protein unit was named deterring-BAX-TOMM20 (DBT), while the pro-apoptotic recombinant protein unit, designed based on previous literature, was named hyperactive-BAX-TOMM20 (HBT). 28 Methods Plasmid constructs. For transient transfection of either the HBT or the DBT system, rTOMM20 plasmid was purchased from Addgene (#226667, Watertown, MA, USA). Information regarding the custom-designed recombinant BAX (rBAX) sequences are provided in Table S1 of Supplementary Information. Light chamber fabrication. A light chamber was 3D-printed using polyethylene filaments and designed with dimensions of 10 cm × 14 cm × 9 cm (width × length × height). The plate entrance was designed to be 9 cm × 4 cm (width × height) with a door that could open or close the chamber entrance. A blue PCB light-emitting diode (LED) (DC 5V, 2.16 W, 60 mA, 120º light angle, #2835, LG Innotek, Seoul, Republic of Korea) was installed on the LED board attached to the ceiling of the light chamber. In addition, a C-type charging socket, switch, and ventilation wickets were installed on the backside of the chamber. The battery could sustain the light chamber for 4 h once fully charged. Detailed illustrations are provided in Figure S2 of Supplementary Information. Cell culture and transient transfection. Human dermal fibroblast-neonatal (NHDF-Neo) cells (#CC-2509, Lonza, Basel, Switzerland) were maintained in a culture medium consisting of RPMI-1640 (#10-040-CV, Cellgro, Corning, NY, USA), 10% fetal bovine serum (FBS, #A2720803, Gibco™, Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin-streptomycin (p/s, #15140163, Invitrogen, Thermo Fisher Scientific) in a 37ºC humidity-controlled incubator with 5% CO 2 . Lipofectamine 3000 (#L3000015; Thermo Fisher Scientific) was used as the transfection reagent. Initially, 2 × 10 5 cells were plated in each well of a 6-well plate (#30006, SPL Life Sciences, Pocheon-si, Gyeonggi-do, Republic of Korea) and incubated for 24 h at 37ºC. The cell medium was then aspirated, and FBS-reduced medium (RPMI-1640, 2% FBS, and 1% P/S) was added an hour before transfection. P3000 reagent (2 µL) was mixed with 1 µg of the HBT or DBT plasmids (500 ng rBAX and 500 ng rTOMM20 (1:1 ratio) in a total volume of 1 µL) in 122 µL of Opti-MEM™ (#31985070, Gibco™, Thermo Fisher Scientific). For the control condition, the reagent solution was mixed with 1 µg rTOMM20 plasmids (1 µL) instead. Separately, 4 µL of Lipofectamine 3000 was added to 121 µL of Opti-MEM™ per well of the 6-well plate. After 5 min incubation at room temperature, the mixtures were combined to a total volume of 250 µL and incubated at room temperature for 30 min. Subsequently, 200 µL of the incubated mixture was added dropwise to each well. The medium was aspirated and replaced with fresh culture medium containing 5 µM Flavin adenine dinucleotide disodium salt hydrate (FAD, #F6625, Sigma-Aldrich, St. Louis, MO, USA) 24 h after transfection. For further experiments, cells were illuminated with blue light 48 h post-transfection in the light chamber for 20 min (if not otherwise mentioned in the text). FRET assay. Cells at 48 h post-transfection with either 50 or 200 ng of HBT or DBT plasmids per 20,000 cells in a 96-well plate were serially irradiated with blue light for 0, 5, or 10 min. The cells were then immediately analyzed using FRET analysis in a fluorescence-detectable multimode microplate reader (Hidex Sense, HIDEX Oy, Turku, Finland). Immunofluorescence microscopy. (Antibody details are provided in Table S2 of the Supplementary Information file) Twenty-four hours post-transfection, the transfected cells were dissociated from the surface using 0.25% trypsin (#24200-072, Thermo Fisher Scientific) and plated on cell culture slide I (#30408, SPL Life Sciences) at a density of 5 × 10 4 cells per well. The cells were incubated in a 37ºC humidity-controlled incubator with 5% CO 2 for 24 h. For immunofluorescence microscopy sample preparation, the cells were irradiated with blue light for 20 min and immediately prepared for the next step in most analyses (1-min incubation in the dark) or incubated in the dark for 10 min. In drug-treated experiments, blue light was applied for 10 min. Following incubation in the dark, the cells were fixed with 4% paraformaldehyde solution (#PC2031-100-00, Biosesang, Yongin-si, Gyeonggi-do, Republic of Korea) and stored at 4ºC overnight. The fixed cells were treated with 0.4% Triton X-100 in PBS for 20 min at room temperature. After aspirating the buffer, 5% bovine serum albumin (BSA) in PBS was treated for 30 min at room temperature. The buffer was then aspirated, and the cells were incubated with primary antibodies in a mixture of 0.2% Tween in PBS (PBST) and 5% BSA (1:1 ratio) overnight at 4ºC. The primary antibodies were aspirated, and the cells were washed thrice with PBST for 5 min at room temperature. The cells were then incubated with secondary antibodies for 1 h at room temperature. After aspirating the secondary antibodies, the cells were washed thrice with PBST for 5 min at room temperature. The mounting medium was applied dropwise to the samples, which were mounted on a 24 × 60 mm microscope cover glass (#HSU-0101242, Marienfeld, Lauda-Königshofen, Germany). The samples were examined under a fluorescence microscope (Axio Imager M1; Carl Zeiss AG, Oberkochen, Germany) or LSM 900 confocal microscope (LSM 900; Carl Zeiss AG, Oberkochen, Germany). Live cell imaging. Transfected cells were treated with 200 µM Cisplatin and maintained in a live cell imaging system (Incubator TS, Live Cell Instrument, Seoul, Republic of Korea) to ensure a humidity-controlled environment at 37ºC with 5% CO 2 . The cells were visualized using the CelenaX imaging system (CELENA® X, Logos Biosystems, Anyang, Gyeonggi-do, Republic of Korea). Time-lapse imaging was conducted by irradiating cells with blue light for 1,000 ms every 5 min during a 3 h recording period. Translocation events of mCherry-tagged rBAX and GFP-tagged rTOMM20 were visualized and traced under a microscope. Protein Structure Simulation. For predicting recombinant protein oligomer structures, the AlphaFold online tool (developed by DeepMind, with data from EMBL-EBI) was used for multimer binding structure prediction and creation of PDB files. RCSB PDB (managed by Rutgers University and UC San Diego, supported by NSF, NIH, and DOE) was utilized for visualizing the multimer structures. 46 , 47 Statistical analysis. Co-localization and morphological analyses were conducted using ImageJ software (NIH, Bethesda, MD, USA). For the co-localization assessment, the color-merged immunostained fluorescence images of the red and green channels were thresholded using ImageJ software (Fig. 4 B; Fig. 5 F,G; Figure S5). The ratio of the co-localized area to the red-thresholded area was analyzed. Alternatively, the plot profile plugin in ImageJ was employed for both colocalization assessment and fluorescence intensity measurement (Fig. 3 B,D; Fig. 5 E,I,J; Figure S12-S13; Figure S16). For morphological analysis, the immunostained fluorescence images were thresholded, and size filtering was performed (Fig. 3 F; Fig. 4 E,G,H,J,K; Figure S6; Figure S7; Figure S9-S11). The morphological analysis of subcellular compartments was conducted by setting subcellular ROIs (randomly selected) in multiple single-cell images. Colocalization, CytC, and CC3 assays were performed on a per-cell basis, whereas APAF1 analysis was conducted for each image obtained from the slide samples. All experiments were performed at least 3 independent experiments. Quantitative data values were analyzed using Prism software (GraphPad Software Inc., San Diego, CA, USA), and bar graphs were presented as mean ± standard error of the mean. Two-tailed tests were conducted for every statistical analysis (95% confidence). Results Characterization of Custom-Built Recombinant Proteins. In this study, we aimed to manipulate the MOMPs induced by endogenous BAX (endo-BAX), which have a distinct structure comprising 𝛼1-𝛼9, with 𝛼9 containing a hydrophobic motif (Fig. 1 A-B). To achieve precise control of optogenetic BAX for pro-apoptotic induction, in comparison with DBT, this study introduced HBT, which demonstrates stable and controllable insertion of CRY2-fused BAX into the MOM upon blue light activation. 28 In the recombinant protein constructs, BAX was fused to CRY2 and mCherry (mCh) in distinct configurations for HBAX S184E and DBAX S184E (Fig. 1 A,C,D). In HBAX S184E , full-length CRY2 and mCh were positioned upstream of the complete BAX sequence, incorporating a point mutation at residue 184 (S184E). In contrast, DBAX S184E also placed the full mCh sequence upstream but introduced CRY2 between the 𝛼8 and 𝛼9 helices of BAX. As an optogenetic binding partner, TOMM20–CIB1–GFP (recombinant TOMM20; rTOMM20) was engineered to facilitate the translocation of BAX from the cytosol to the mitochondria. Therefore, the novelty of this study is embodied in the engineered DBAX S184E structure, in which the binding of CRY2 and CIB1 immediately above the membrane hinders MOMP because the anchoring function of the hydrophobic motif within the 𝛼9 helices is limited and BH groove binding, which is essential for MOMP, is deterred, thereby modulating pro-apoptotic activity (Fig. 1 E). The sequential process of MOMP induced by endo-BAX is as follows. In the normal state, CytC is retained within the mitochondria by the intact MOM, while BAX remains in the cytoplasm. 6 During apoptosis, BAX translocates to the MOM, accompanied by the exposure of its 𝛼9 helix. Another BAX aligns next to the first on MOM by generating junctions via BH grooves, leading to pore formation due to extensive BAX insertion. 3 – 5 This process results in CytC leakage into the cytosol, triggering cell death via activation of APAF1 and Caspase proteins 29 It is accompanied by morphological changes such as mitochondrial fission, nuclear pyknosis, and cell shrinkage (Fig. 1 F). 30 , 31 Apoptosis by HBT is induced by the binding of CRY2 in recombinant Bax (rBAX) with its binding partner CIB1, which is part of rTOMM20, thereby facilitating vast endo-BAX recruitment to the MOM and inducing MOMP (Fig. 1 G). In comparison, rBAX of DBT, which is limitedly inserted into the MOM along with endo-BAX, hinders the MOMP under blue light irradiation (Fig. 1 H). To verify the anti-apoptotic functionality of the DBT, a blue light chamber was specifically generated to fit into 6-well cell culture plates (Fig. 1 I, Figure S2). For live cell imaging, a 488 nm blue laser equipped with a fluorescence microscope was utilized. For analysis, cells were transfected with plasmids 24 h after seeding and flavin adenine dinucleotide disodium salt hydrate (FAD) was added to the culture medium 24 h post-transfection. Finally, the cells were irradiated with blue light at 48 h post-transfection (Fig. 1 J). Aggregation Simulation and Microscopic Analysis of DBT Reveal Attenuated Pore Formation Impacting endo-BAX In this study, DBAX S184E was engineered to exert controlled effects on endo-BAX. Under blue light irradiation, two distinct binding events occur: one between rBAX and rTOMM20 for locational control of rBAX, and another between rBAX and endo-BAX via the intact BH3 domain, which ultimately hinders BAX-mediated MOMP (Fig. 2 A). To assess the binding pattern of DBAX S184E in cells, protein aggregation events were simulated under three conditions: (1) only endo-BAX is present, (2) endo-BAX and HBAX S184E are present in a 6:4 ratio (endo-HBAX), or (3) endo-BAX and DBAX S184E are present in a 6:4 ratio (endo-DBAX) (Fig. 2 B). The total number of proteins for simulation in each condition was set to 10. According to the simulation results, both the endo-BAX complex and the endo-HBAX complex showed pore generation, but the endo-DBAX complex hardly showed pore formation. DBAX S184E was also proven to interact with endo-BAX via BH grooves. Additionally, the results confirmed normal CRY2 homodimerization events. 32 To further investigate the membrane embedding pattern of the DBT unit in cells, protein aggregation events were simulated under two conditions: (1) endo-BAX, HBAX S184E , and rTOMM20 are present in a 5:3:2 ratio (endo-HBT), or (2) endo-BAX, DBAX S184E , and rTOMM20 are present in a 5:3:2 ratio (endo-DBT) (Fig. 2 C). The total number of proteins for simulation in each condition was set to 10. The condition where only endo-BAX is present was also included as a control for reference. According to the simulation results, both the aggregation of endo-BAX alone and the endo-HBT condition showed deeply embedded residues, while the endo-DBT condition showed minimally embedded residues in the complex. Furthermore, pore formation was still observed in the endo-HBT condition but not in the endo-DBT condition. These simulation results support that the genetic design of DBAX S184E effectively interacts with endo-BAX, impeding pore formation by interacting with endo-BAX. To further evaluate the binding events of the DBT complex in cells irradiated with blue light, 3D-rendered confocal images were analyzed (Fig. 2 D). In the CTRL condition, cells transfected only with rTOMM20, endo-BAX displayed a membrane-embedded conformation, with its terminal region contacting the membrane. From bottom-view images highlighting endo-BAX’s penetration into the MOM, we observed multiple endo-BAX molecules deeply embedded, clearly visible from the underside (Figure S3A). Under the HBT condition, large complexes formed among endo-BAX, rBAX, and rTOMM20. Top-view images revealed extensive endo-BAX conjugation with the HBT complex, generating a sizeable aggregate (1.483 µm) on the mitochondria. In bottom and side views, endo-BAX showed deep penetration alongside rBAX cluster (Figure S3B). By contrast, in the DBT condition, although rBAX and rTOMM20 successfully formed complexes, they were markedly smaller (0.686 µm) than those in HBT. Moreover, unlike HBT, the endo-BAX–rBAX interaction in DBT occurred primarily on the upper region of rBAX, possibly reflecting an altered position of its BH domain. Endo-BAX also exhibited limited membrane penetration under DBT (Figure S3C). Irradiation-Driven rBAX–rTOMM20 Conjugation Regulates Endogenous BAX Assembly To verify the optogenetic controllability of the newly developed recombinant constructs, we examined the subcellular colocalization of rBAX and rTOMM20 (Fig. 3 A–E). For each condition, random subcellular regions of interest (ROIs) were selected to generate fluorescence intensity profiles, with two representative examples shown. Multiple cells were also analyzed independently to confirm reproducibility (Fig. 3 F). Under HBT conditions, blue-light irradiation (Light On) yielded nearly identical fluorescence intensity patterns (gray values) for rBAX and rTOMM20 across all sampled ROIs, indicative of colocalization, whereas similarity was substantially lower under Light Off conditions (Fig. 3 A,B; Figure S4A,B). Notably, endo-BAX intensity also increased under Light On, suggesting that, rather than a rapid upregulation of protein expression (given the ~ 30-min timeframe), the enhanced signal reflects the collective recruitment of endo-BAX triggered by HBT activation. Under DBT conditions, rBAX similarly exhibited a light-dependent colocalization pattern with rTOMM20 (Fig. 3 C,D; Figure S4C,D), although overall endo-BAX levels in DBT cells were lower than those observed under HBT Light On conditions. Correlation analyses with simple linear regression model corroborated these observations. The correlation between rBAX and rTOMM20 intensities strengthened under blue-light stimulation in both HBT Light On (HBT+, R² = 0.874, p < 0.0001) compared to HBT Light Off (HBT-, R² = 0.302, p < 0.0001) and DBT Light On cells (DBT+, R² = 0.852, p < 0.0001) compared to DBT Light Off (DBT-, R² = 0.118, p < 0.0001), with a greater disparity observed under DBT conditions (Fig. 3 E; Figure S4E). When the correlation between endo-BAX and rBAX intensities was similarly evaluated, HBT cells showed a marked increase upon light exposure (R² = 0.508, p < 0.0001) compared to HBT Light Off (R² = 0.197, p < 0.0001), consistent with HBT’s established proapoptotic function. By contrast, DBT cells exhibited similar correlations regardless of light conditions (R² = 0.404, p < 0.0001 under Light Off and R² = 0.419, p < 0.0001 under Light On) (Figure S4F). To further quantify these findings on a per-cell level, mCherry–GFP overlap (Coloc %) was measured across multiple cells (Fig. 3 F). Both HBT and DBT groups exhibited statistically significant differences between Light Off and Light On conditions (p < 0.0001 for both), confirming that blue-light irradiation effectively induces rBAX–rTOMM20 conjugation in both constructs, albeit with distinct outcomes for endo-BAX recruitment. Ultimately, a Förster resonance energy transfer (FRET) assay was employed to demonstrate energy transfer between the two closely bound recombinant proteins under blue-light irradiation (Fig. 3 G). The assay was conducted in cells transfected with either HBT or DBT constructs and subjected to progressively longer blue-light exposures (5 or 10 min). Donor-only conditions were established by expressing rTOMM20 without any acceptor protein. Under these donor-only conditions, no significant FRET increase was detected following either 5 or 10 minutes of irradiation in cells transfected with 50 ng of plasmid per 20,000 cells (p = 0.956 for 0 vs 5 min; p = 0.634 for 0 vs 10 min; p = 0.804 for 5 vs 10 min). Similarly, cells transfected with 200 ng of plasmid per 20,000 cells showed no significant FRET increase (p = 0.961 for 0 vs 5 min; p = 0.657 for 0 vs 10 min; p = 0.816 for 5 vs 10 min) (Fig. 3 H). In HBT-transfected cells, 5 min of blue-light exposure did not significantly elevate FRET signals (p = 0.963), whereas 10 min led to a significant increase (p = 0.02), and the mean difference between 5 and 10 min was also significant (p = 0.0382) in cells transfected with 50 ng. With 200 ng, both 5 and 10 min of irradiation induced increased FRET (p = 0.0402; p = 0.0445), with no significant mean difference (p = 0.999) between these two time points. In DBT-transfected cells, 5 min of light exposure did not produce a significant FRET increase (p = 0.352), but a marked increase occurred at 10 min (p < 0.0001), and the mean difference between 5 and 10 min was significant (p = 0.0003) in cells transfected with 50 ng. When 200 ng was used, both 5 and 10 min of blue-light exposure induced significant FRET increases (p = 0.0022; p = 0.0147), with no significant mean difference between the two time points (p = 0.7785). These data confirm that prolonged blue-light irradiation promotes rBAX–rTOMM20 conjugation in HBT and DBT systems, with a more rapid and extensive response at higher plasmid concentrations. Based on these validated optogenetic modulations of the recombinant proteins, the anti-apoptotic effects of DBT were subsequently evaluated by analyzing multiple cell-death-related indices and comparing cells transfected with rTOMM20 alone (CTRL) or HBT against DBT-transfected cells. Light-Activated DBTs Reduce BAX Mitochondrial Localization and Aggregation, Preserve Nuclear and Mitochondrial Integrity, Enhancing Cell Viability The morphological assessment of apoptosis in cells was based on three key indicators. First, translocation of BAX into the mitochondria was observed, and BAX aggregation, which increases during active apoptosis, was quantified. 33 , 34 Second, the number and morphology of nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) were analyzed as a key indicator of cell death. 35 , 36 Lastly, mitochondrial fission status, determined by measuring mitochondrial length and size, was analyzed to assess the mitochondrial integrity of the cell (Fig. 4 A). 33 , 37 , 38 These assessments were performed by analyzing immunostained images of endo-BAX and endogenous TOMM20 (endo-TOMM20) to verify the effects of recombinant protein manipulation on their endogenous counterparts. The results presented here focus solely on the blue-light–irradiated conditions; comparisons with no-light controls are provided in the supplementary information (Figures S8–S11). First, the colocalization of endo-BAX with mitochondria was evaluated following blue-light irradiation. This evaluation was based on the established property that endo-BAX is translocated to the MOM during active apoptosis. 2 The red and green overlapping region in the images, which indicates mitochondrial BAX, was selectively filtered (white), and its area was quantified (Fig. 4 B; Figure S5). The results showed that endo-BAX recruitment to the mitochondria was significantly enhanced under the pro-apoptotic HBT condition (p = 0.0003 vs. CTRL; p < 0.0001 vs. DBT), whereas it remained attenuated under DBT, with no significant difference from CTRL (p = 0.5015). This indicates that rBAX in the DBT construct does not facilitate rubst BAX translocation to the mitochondria, unlike the HBT construct. To further assess BAX aggregation size, a critical index of MOMP, immunofluorescence images were processed to remove outliers such as debris and highly saturated areas (Fig. 4 D; Figure S6). Given that BAX proteins form larger clusters through redistribution as MOMP progresses, the sizes of BAX aggregates were compared and analyzed across experimental groups (Fig. 4 E). 39 In the DBT condition, the size of BAX clusters within each ROI was markedly smaller than in both the CTRL (p < 0.0001) and HBT (p < 0.0001) conditions. Moreover, the reduction in aggregate size in DBT compared to HBT was significantly greater than that observed between CTRL and HBT (p = 0.0442), highlighting the pronounced attenuation of endo-BAX aggregation in the DBT condition to levels below baseline. Altogether, the mitochondrial colocalization of BAX and aggregate size measurements underscore the anti-apoptotic efficacy of the DBT construct in modulating neighboring endogenous BAX. Second, cellular viability was evaluated by examining the number and morphology of nuclei via DAPI staining under blue-light irradiation in CTRL, HBT, and DBT conditions (Fig. 4 F). As shown in Fig. 4 G, quantitative analysis revealed that the DAPI counts in DBT-transfected cells were identical to those in the CTRL group (p = 0.8750) and significantly higher than those observed in HBT-transfected cells (p < 0.0001). In contrast, the HBT condition exhibited a marked reduction in nuclei number compared to CTRL (p = 0.0002). Furthermore, nuclear solidity, a measure of structural integrity, was well maintained in DBT-transfected cells, with values comparable to CTRL (p = 0.3767) and significantly superior to those in the HBT condition (p = 0.0213) (Fig. 4 H). Notably, the HBT condition showed considerable nuclear distortion relative to CTRL (p < 0.0001). These results underscore that DBT not only preserves nuclear integrity but also maintains overall cellular viability under blue-light irradiation. Third, to confirm the protective effects of DBTs on mitochondrial integrity, mitochondrial morphology was examined. Cells irradiated with blue light under each condition were immunostained with an anti-TOMM20 antibody to visualize mitochondrial structures, which reflect either fission or fusion states (Fig. 4 I; Figure S7). Active mitochondrial fission, commonly associated with cellular stress or damage, is characterized by shorter, and more circular mitochondria that can be quantitatively assessed. 38 Mitochondrial morphology was evaluated at two time points: immediately after blue-light irradiation (1 min post-irradiation) and 10 min post-irradiation (following 10 min of dark incubation) (Fig. 4 J,K). In samples prepared immediately post-irradiation, DBT-transfected cells exhibited a significantly elongated and continuous mitochondrial perimeter compared to both CTRL (p = 0.0241) and HBT (p < 0.0001) groups, whereas the HBT condition showed a markedly shorter perimeter relative to CTRL (p < 0.0001). Similarly, the aspect ratio (AR) was significantly higher in the DBT condition than in CTRL (p < 0.0001) and HBT (p < 0.0001), while HBT exhibited a lower AR compared to CTRL (p = 0.0008). Moreover, the index of circularity, a marker of intensive, dot-like mitochondrial fission, was significantly elevated in the HBT condition relative to CTRL (p = 0.0002) but was markedly reduced in DBT-transfected cells compared to both CTRL (p < 0.0001) and HBT (p < 0.0001) conditions. After 10 min of dark incubation following blue-light irradiation, these trends were somewhat attenuated. In the DBT condition, the mitochondrial perimeter was comparable with the CTRL condition (p = 0.1217), and no significant differences were observed in either AR or circularity between DBT and CTRL (p = 0.4185 and p > 0.9999, respectively). However, both the mitochondrial perimeter and AR of DBT-transfected cells remained significantly higher than those in the HBT condition (p < 0.0001 and p = 0.0001, respectively). In contrast, while the perimeter and AR in the HBT condition continued to be lower than those in the CTRL condition (p 0.9999) during the dark incubation. This recovery may be attributed to rapid mitochondrial dynamics that promote the fusion of highly circular, fissioned mitochondria. 40 , 41 These results indicate that the anti-apoptotic effect conferred by DBT, evidenced by sustained mitochondrial fusion, is remarkably pronounced even surpassing baseline levels observed in the CTRL group. This effect is rapidly reversed by dark incubation, likely due to the intrinsic rapid reversibility of optogenetic systems. DBT Preserves Mitochondrial Integrity and Suppresses APAF1/Caspase-3–Dependent Apoptotic Signaling Under Apoptotic Drug Treatment Finally, the anti-apoptotic effects of DBTs were assessed by exposing cells to 200 µM cisplatin for 10 min with or without irradiation, a compound known to induce MOMP. 42 Upon cisplatin treatment, mitochondrial CytC is released into the cytosol, where it binds to APAF1 to form the apoptosome, triggering downstream signaling cascades that result in the accumulation of cleaved caspase-3 (CC3) (Fig. 5 A). To evaluate this process, the spatial distribution of key apoptotic markers including CytC, APAF1, and CC3 were investigated after cisplatin treatment. Firstly, CytC distribution was examined to assess the preservation of mitochondrial integrity (Fig. 5 B,C; Figure S12,13). Immunofluorescence images revealed that apoptotic cells exhibit diffuse, smudged CytC signals indicative of leakage, alongside visible mitochondrial structures. In HBT-transfected cells, the integrated fluorescence density of CytC was significantly attenuated (p = 0.0048 vs. CTRL; p < 0.0034 vs. DBT), indicating a dispersed, non-contained distribution of CytC. In contrast, no significant difference was observed between DBT-transfected cells and control cells (p = 0.9881). Moreover, when mitochondrial integrity was evaluated by measuring the AR of CytC distribution, DBT-transfected cells demonstrated superior preservation relative to both the CTRL (p = 0.0391; one-way ANOVA, Tukey’s post hoc test, n = 300 for all) and HBT conditions (p = 0.0093, n = 300), whereas no significant difference was observed between CTRL and HBT conditions (p = 0.8716) (Figure S12D). these effects were not observed under no-light control conditions (Figure S13B-C). Secondly, APAF1 accumulation was assessed. Given that baseline APAF1 expression is broad and variable, a high dose of cisplatin (1 mM) was used to elicit a robust apoptotic response, ensuring drug-specific APAF1 accumulation for apoptosome assembly (Fig. 5 D-G). Immunofluorescence microscopy revealed extensive cell death following drug treatment across all conditions, with a significant reduction observed in DBT-transfected cells under blue-light irradiation. Quantitative analysis demonstrated that APAF1 intensity in DBT-transfected cells under irradiation was significantly lower than that in both CTRL (p = 0.0023) and HBT (p = 0.0023) groups (Fig. 5 E). In contrast, no significant difference in APAF1 accumulation was detected between CTRL and HBT groups, likely due to the harsh drug treatment obscuring subtle differences (p > 0.9999). Furthermore, in DBT-transfected cells, APAF1-rBAX colocalization was markedly attenuated under blue-light conditions compared to no-light conditions (p = 0.0014), whereas HBT-transfected cells showed a significant increase of that under blue-light irradiation (p = 0.0130) (Fig. 5 F,G). Thirdly, CC3 accumulation and its colocalization with rBAX were evaluated by immunofluorescence microscopy that highlighted the localized distribution of CC3 around mitochondria (Fig. 5 H-J; Figure S14-S16). The 3D reconstructions revealed pronounced colocalization of CC3 with the HBT complex, whereas DBT-transfected cells displayed less colocalization (Fig. 5 H). Quantitative analysis of immunofluorescence images indicated that overall CC3 concentrations did not differ significantly between groups under dark (p = 0.0032) (Figure S16A,C). Under blue-light irradiation, DBT-transfected cells exhibited markedly lower CC3 levels compared to HBT-transfected cells (p = 0.0446), highlighting DBT’s capacity to attenuate apoptotic signaling (Fig. 5 I; Figure S15A). By contrast, CC3 accumulation in HBT-transfected cells was significantly higher than in CTRL (p = 0.0129), whereas DBT-transfected cells remained comparable to CTRL (p = 0.7456). To further elucidate the relationship between rBAX and CC3 distributions, simple linear regression analysis was performed. Under blue-light conditions, HBT-transfected cells displayed a strong positive correlation between rBAX and CC3 (R² = 0.9804; p < 0.0001), whereas DBT-transfected cells showed no significant correlation (R² = 0.1371; p = 0.3267), with a negative regression slope. Under no-light conditions, no significant correlations were observed in either condition (Figure S16B). These findings indicate that DBT-transfected cells exhibit attenuated CC3 accumulation, reinforcing the anti-apoptotic efficacy of the DBT construct. Finally, to observe the anti-apoptotic function of DBTs in real time, a live-cell imaging assay was performed with 1,000 ms pulses every 5 min over a 3-hr period under drug condition (Fig. 5 K). The results showed that HBT and DBT recombinant proteins gradually interacted over time. Notably, DBT-transfected cells retained their morphological integrity, whereas HBT-transfected cells exhibited progressive structural disruption. Integrating these observations with data obtained across a range of apoptotic inducer experiments highlights the robust anti-apoptotic functionality of DBTs. This protective effect is attributed to the unique structural features of DBTs, which deter pore formation. Collectively, these findings highlight the potential of the structural modifications proposed in this study to modulate BAX activity and bolster anti-apoptotic responses, offering promising prospects for future drug development. Discussion Recognizing the importance of modulating apoptosis in cells, this study proposes BAX as a promising therapeutic target for apoptosis-associated diseases. This potential is attributed not only to its organelle-specific assembly but also to its intrinsic interaction mechanism, whereby a single dysfunctional BAX molecule can influence neighboring BAX proteins, thereby altering the dynamics of apoptotic signaling. In our work, we employed an optogenetic approach to direct BAX to the mitochondria, the primary site of its apoptotic activity, and to elicit anti-apoptotic effects within BAX assemblies. Consequently, apoptosis was attenuated through targeted alterations that prevented pore formation in the mitochondrial outer membrane (MOM). Notably, this system was activated simply by exposure to blue light. Mitochondria, which serve as the primary platform for BAX function, undergo dynamic cycles of fission, fusion, and transport on timescales ranging from seconds to hours, thus playing a vital role in cellular quality control. Under certain stimuli or stress conditions, they can rapidly eliminate damaged components or quickly compensate for functional deficits, enabling swift adaptation. 37 , 38 , 43 Typically, upon receiving cell death signals, mitochondria undergo pronounced fission, prompting CytC release and accelerating proapoptotic processes, thus mitochondrial dynamics is important in regulating cell death. 6 During this process, multiple BAX proteins respond to apoptotic cues by undergoing structural rearrangements and integrating into the MOM within minutes, where they form pores that compromise mitochondrial integrity. 3 , 4 BAX is also thought to interact directly with mitochondrial fission mediators like Drp1, thereby exerting a substantial influence on mitochondrial morphology. 44 , 45 Accordingly, we aimed to regulate BAX's mitochondrial binding event by hindering the interaction of its hydrophobic motif, which serves as a mitochondrial anchor. To achieve this, we engineered an optogenetic system that strategically positions CRY2 over the hydrophobic motif, thereby promoting its binding to CIB1-fused TOMM20, a mitochondrial membrane protein, and effectively impeding BAX-mediated pore formation. Additionally, this design repositions the BH groove away from the MOM, further disrupting collective BAX interactions (Fig. 1 – 2 ). In our study, we confirmed that CRY2 and CIB1 bind tightly under blue light and exhibit more rapid interactions at higher expression levels in cells (Fig. 2 – 3 ). This light-induced interaction drove the translocation of recombinant BAX (rBAX) to the mitochondria and influenced the recruitment of endogenous BAX (endo-BAX) (Fig. 4 ). These effects were validated using a combination of simulation, as well as 2D and 3D immunofluorescence image analyses for precise localization. Throughout this study, we compared HBT, a system previously demonstrated to be reliable and that preserves the native BAX structure, with DBT, as well as with a rTOMM20-only condition (CTRL) that exhibits stable transient expression of recombinant proteins without facilitating apoptosis. 28 Our findings reveal that DBT not only diminishes the mitochondrial localization of endo-BAX but also suppresses its aggregation (Fig. 3 D; Fig. 4 B-E). This dual effect mitigates cell death, enhances cell viability, and promotes sustained mitochondrial fusion (Figs. 4 F-K). In essence, DBT functions as a covert modulator among BAX molecules, effectively inactivating their proapoptotic activity and reducing apoptosis to levels below those observed in control cells. We further conducted a comparative analysis of HBT and DBT under drug-induced stress by exposing cells to high concentrations of cisplatin, followed by light illumination. Under these conditions, we tested mitochondrial CytC release, APAF1 aggregation, and subsequent CC3 accumulation (Fig. 5 ). Overall, the DBT construct markedly mitigated these effects. The maintenance of CytC distribution and mitochondrial integrity in DBT-transfected cells, as evidenced by integrated fluorescence density and aspect ratio measurements, suggests that DBT effectively preserves mitochondrial function under stress (Fig. 5 B-C; Figure S12). Moreover, the reduced APAF1 accumulation and diminished CC3 levels in DBT-transfected cells, coupled with the lack of significant rBAX–CC3 correlation, indicate that DBT not only disrupts BAX’s ability to promote apoptotic pore formation but also attenuates downstream apoptotic signaling (Fig. 5 D-J). Live-cell imaging further corroborated these findings by demonstrating sustained cellular integrity over time in DBT-transfected cells, in stark contrast to the progressive morphological deterioration observed in HBT-transfected cells (Fig. 5 K). Collectively, these results support the hypothesis that the novel optogenetic modification of BAX structure can effectively suppress its proapoptotic activity, thereby offering a promising strategy for modulating apoptosis in therapeutic contexts. Even though this novel technology offers significant future potential for addressing disease-related challenges, there remain limitations that must be overcome to enhance its utility. One limitation of this study is the inherent toxicity associated with transfection reagents, which complicates the accurate evaluation of recombinant proteins in apoptosis-related research. Future studies should consider integrating advanced transfection methodologies to remove these effects and enable more precise assessments. Additionally, further validation across a broader range of cell types is necessary, with particular emphasis on neuronal cells, where preventing unwarranted apoptosis is critical. It is also important to note that inhibiting cell death does not inherently equate to improved health. Indeed, excessive suppression of apoptosis may lead to inefficient energy utilization, accelerated aging, or compromised quality control mechanisms. Therefore, careful deliberation is required in the development and application of this technology. Lastly, a comprehensive evaluation of the interplay between apoptosis and regeneration is indispensable, as the therapeutic potential of such interventions is likely to be maximized when these processes are balanced. Given the prevalence of age-related diseases associated with apoptosis, including dementia, Parkinson’s disease, arthritis, and diabetes, this study elucidates critical mechanisms for precisely targeting BAX by examining how a single genetically engineered variant, designed to hinder its mitochondrial permeabilization, disrupts the cell's ability to mediate and trigger apoptosis. By overcoming several of the noted limitations, we expect that our results will establish a solid foundation for evaluating anti-apoptotic interventions in cells and facilitate the advancement of novel therapies designed to reduce apoptosis in forthcoming disease treatments. Declarations Data availability Data are available upon request. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT)(RS-2024-00455532), and the Nano & Material Technology Development Program through the National Research Foundation of Korea(NRF) funded by Ministry of Science and ICT(RS-2024-00450828) Author contributions D.L. developed the concepts, methodology, validation, formal analysis, and investigation, administered the project, and wrote the original draft. H.B. curated the data and reviewed the original draft. D.O. conducted the investigation and data curation. J.A., M.K., and S.K. reviewed the original draft. J.K. supported the investigation. D.H.K., H.O., and W.D.H. contributed to the visualization and review of the original draft. W.D.H also provided supervision. S.C. supported conceptualization and methodology development, supervised the project, reviewed and edited the original draft, and secured funding. Competing interests The authors declare no competing interests. Additional information Supplementary information Detailed information is provided in the Supporting Information. Video S1 Comparison of Apoptosis Under Drug Conditions in HBT- and DBT-Transfected Cells Correspondence and requests for materials should be addressed to Seok Chung. References Cory, S. & Adams, J. M. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2, 647–656 (2002). Cosentino, K. & García-Sáez, A. J. Bax and Bak Pores: Are We Closing the Circle? Trends Cell Biol 27, 266–275 (2017). Gavathiotis, E., Reyna, D. E., Davis, M. L., Bird, G. H. & Walensky, L. D. BH3-Triggered Structural Reorganization Drives the Activation of Proapoptotic BAX. Mol Cell 40, 481–492 (2010). Annis, M. G. et al. Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J 24, 2096–2103 (2005). Suzuki, M., Youle, R. J. & Tjandra, N. Structure of Bax. 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Additional Declarations There is no conflict of interest Supplementary Files VideoS1.mp4 Comparison of Apoptosis Under Drug Conditions in HBT- and DBT-Transfected Cells SI.pdf Supplementary Information File Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 24 Apr, 2025 Review # 2 received at journal 23 Apr, 2025 Review # 1 received at journal 16 Apr, 2025 Reviewer # 2 agreed at journal 11 Apr, 2025 Reviewer # 1 agreed at journal 02 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Submission checks completed at journal 26 Mar, 2025 First submitted to journal 26 Mar, 2025 Unknown event 24 Mar, 2025 Editor assigned by journal 24 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6222702","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":436907825,"identity":"fc144b3e-d16f-44e3-b9eb-589ce32bcc12","order_by":0,"name":"Seok Chung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACZgYDZhDNzwMXYiNSi2QP0VoYoFoMzhCrRb6deePnwrY7dpvPHD724UMNgzx/A1vaB7xWHGYrlp7Z9ix529m25JkzjjEYzjjAdngGflfxmDHzth1ONjvPY8zMw8bAuIGBvRm/w5qhWoz7gVr+/GOwJ6iF4TBEi50Bb48xM2MbQ+IGBrbDeHWA/cJz7nCCxJljyYy9fRLJMw6zJeN3WP/hjZ95yg7b8/ckH2b48c3Gtr+9zRi/w6AgsQFCS4AilzhgT6S6UTAKRsEoGIkAAOygQAWDqCm5AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1735-8338","institution":"Korea University","correspondingAuthor":true,"prefix":"","firstName":"Seok","middleName":"","lastName":"Chung","suffix":""},{"id":436907826,"identity":"1ce03856-9af3-4235-b41d-2e8d8f01c88b","order_by":1,"name":"Dain Lee","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Dain","middleName":"","lastName":"Lee","suffix":""},{"id":436907827,"identity":"b6546ca1-e075-417d-948b-67c22f3beb26","order_by":2,"name":"Hyunjun Bae","email":"","orcid":"","institution":"Absology","correspondingAuthor":false,"prefix":"","firstName":"Hyunjun","middleName":"","lastName":"Bae","suffix":""},{"id":436907828,"identity":"48a7b46e-1e2f-46b8-be95-e179485438cd","order_by":3,"name":"Dongwoo Oh","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Dongwoo","middleName":"","lastName":"Oh","suffix":""},{"id":436907829,"identity":"af156f89-cd74-48d5-a959-41010763f1cd","order_by":4,"name":"Jinchul Ahn","email":"","orcid":"https://orcid.org/0009-0007-7493-4219","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Jinchul","middleName":"","lastName":"Ahn","suffix":""},{"id":436907830,"identity":"d78559e3-4978-4ecf-b106-578cbe96d2a9","order_by":5,"name":"Minseop Kim","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Minseop","middleName":"","lastName":"Kim","suffix":""},{"id":436907831,"identity":"72614b9f-ac34-4a64-bc8e-c4546181dd19","order_by":6,"name":"Seok-Hyeon Kang","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Seok-Hyeon","middleName":"","lastName":"Kang","suffix":""},{"id":436907832,"identity":"67c29c59-8148-47c7-a813-7f0093e195bb","order_by":7,"name":"Ju-Hee Kim","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Ju-Hee","middleName":"","lastName":"Kim","suffix":""},{"id":436907833,"identity":"61fcafc1-61ac-4259-8d6a-6d1e0a587342","order_by":8,"name":"Dong-Hwee Kim","email":"","orcid":"https://orcid.org/0000-0003-0625-0660","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Dong-Hwee","middleName":"","lastName":"Kim","suffix":""},{"id":436907834,"identity":"12309902-5f7f-40a6-af86-e5921697349c","order_by":9,"name":"Hyunjeong Oh","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Hyunjeong","middleName":"","lastName":"Oh","suffix":""},{"id":436907835,"identity":"c524ecb8-9084-44e6-8543-87903f94bd69","order_by":10,"name":"Won Do Heo","email":"","orcid":"https://orcid.org/0000-0001-7541-7319","institution":"Korea Advanced Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Won","middleName":"Do","lastName":"Heo","suffix":""}],"badges":[],"createdAt":"2025-03-13 22:10:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6222702/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6222702/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-025-01605-y","type":"published","date":"2025-12-26T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81133072,"identity":"625dbd27-fb78-48c3-9289-621c1786776c","added_by":"auto","created_at":"2025-04-22 15:05:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1073344,"visible":true,"origin":"","legend":"\u003cp\u003eTheoretical demonstration of endo-BAX and engineered BAX variants (see also Figure S1,2 and Table S1). (A) Schematic depiction of the functional domains in endogenous BAX (endo-BAX), recombinant BAX (rBAX), and recombinant TOMM20 (rTOMM20). In hyperactive BAX (HBAX\u003csup\u003eS184E\u003c/sup\u003e), the residue at position 184 in the 𝛼9 domain is mutated to glutamic acid, and the resulting construct is fused to Cryptochrome 2 (CRY2) and mCherry (mCh). Deterring BAX (DBAX\u003csup\u003eS184E\u003c/sup\u003e) is a novel BAX variant developed in this study; it also carries the S184E mutation but differs by having CRY2 inserted between the 𝛼8 and 𝛼9 domains, in addition to an mCh fusion. rTOMM20 is fused to Cryptochrome-interacting basic-helix-loop-helix 1 (CIB1), as well as to GFP. All recombinant proteins contain nonfunctional linker sequences. (B) Illustration of endo-BAX with its nine 𝛼-helical segments. The C-terminal 𝛼9 helix contains a hydrophobic motif. (C) Optogenetically modified constructs in the HBT system. HBAX\u003csup\u003eS184E\u003c/sup\u003e and rTOMM20 are shown along with their proposed interaction. (D) Optogenetically modified constructs in the DBT system. DBAX\u003csup\u003eS184E\u003c/sup\u003e and rTOMM20 are illustrated, including their predicted interaction. (E) Predicted PDB structure of the DBAX\u003csup\u003eS184E\u003c/sup\u003e. (F) Model of mitochondrial membranes (outer, MOM; inner, MIM) with CytC in the intermembrane space, and endo-BAX in the cytoplasm. Upon receiving apoptotic signals, the 𝛼9 helix of BAX is exposed, allowing BAX insertion into the MOM, aggregation, and subsequent release of CytC into the cytosol, which triggers apoptotic signaling. (G) Mechanism of HBT complex formation under blue-light stimulation. HBAX\u003csup\u003eS184E\u003c/sup\u003e translocates to the MOM, where CRY2 binds to CIB1 in rTOMM20. The exposed 𝛼9 helix of HBAX\u003csup\u003eS184E\u003c/sup\u003e inserts into the MOM, recruiting endo-BAX and inducing MOMP. (H) Mechanism of DBT complex formation upon optogenetic stimulation. DBAX\u003csup\u003eS184E\u003c/sup\u003e also translocates to the MOM and binds rTOMM20 via CRY2–CIB1 interaction. While its 𝛼9 helix is inserted into the MOM, the binding of CRY2-CIB1 above the hydrophobic motif in DBAX\u003csup\u003eS184E \u003c/sup\u003edisrupts BAX oligomerization, limiting MOMP. (I) Schematic of the blue-light irradiation chamber and calibration. The light chamber is depicted with a 4 × 9 cm² entrance, accommodating a standard 6-well cell culture plate. (J) Workflow for recombinant protein expression in cells. FAD=flavin adenine dinucleotide\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/eca59169aa2a5da1182aa951.jpg"},{"id":81133074,"identity":"81b34485-4721-4327-a122-a8d34fcf2308","added_by":"auto","created_at":"2025-04-22 15:05:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1525052,"visible":true,"origin":"","legend":"\u003cp\u003eSimulations of protein aggregation and z-stack confocal microscopy with 3D-rendered images to confirm the functionality of the DBAX\u003csup\u003eS184E\u003c/sup\u003e (see also Figure S3). (A) Schematic description of rBAX, endo-BAX, and rTOMM20 interactions. Under dark conditions, the three components remain dissociated. Upon blue-light exposure, however, they bind together at the MOM. In this system, rBAX and rTOMM20 interact via CRY2-CIB1 binding, while rBAX and endo-BAX associate through their BH grooves. (B) To model optogenetically induced BAX aggregation, we performed AlphaFold Multimer simulations with either HBAX\u003csup\u003eS184E\u003c/sup\u003e or DBAX\u003csup\u003eS184E\u003c/sup\u003e at a 6:4 ratio to endo-BAX (total of 10 proteins). Each protein is rendered in a different color, with BH grooves, 𝛼9 helices, endo-BAX, or rBAX highlighted in green. In contrast to endo-BAX and HBAX\u003csup\u003eS184E\u003c/sup\u003e, the DBAX\u003csup\u003eS184E\u003c/sup\u003e complex barely shows pore formation. (C) To simulate conditions in which rTOMM20 associates with rBAX under blue-light irradiation, we combined endo-BAX, rBAX, and rTOMM20 in a 5:3:2 ratio. Membrane-embedded residues are highlighted in pink, and the remnants are shown in gray. Both top and side views are presented. The DBAX\u003csup\u003eS184E\u003c/sup\u003e–rTOMM20 (DBT) complex displays limited hydrophobic motif insertion into the MOM compared to the HBAX\u003csup\u003eS184E\u003c/sup\u003e–rTOMM20 (HBT) complex. (D) For experimental co-localization studies, cells expressing only rTOMM20 (CTRL), the HBT complex, or the DBT complex were irradiated with blue light, then immunostained and analyzed by confocal microscopy. Three-dimensional reconstructions from z-stack images, viewed at multiple angles, confirm protein co-localization and approximate molecular dimensions.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/263f49341f1dd2df5069ffc3.jpg"},{"id":81133073,"identity":"51fd40a4-6a80-4b13-ae12-27ef26cf8fa4","added_by":"auto","created_at":"2025-04-22 15:05:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4903326,"visible":true,"origin":"","legend":"\u003cp\u003eOptogenetic control of DBT and its impact on endo-BAX (See also Figure S4). (A, C) Representative immunofluorescence images illustrating the co-localization of rTOMM20, rBAX, and endo-BAX in cells transfected with either the HBT or DBT complex and subjected to blue-light irradiation. Images are shown under conditions of continuous blue light exposure (Light On) or in the absence of light (Light Off). (B, D) Intensity profiles corresponding to the boxed regions in the microscopy images. Red line represents endo-BAX intensity, cyan line denotes rTOMM20 intensity, and yellow line indicates rBAX intensity. (E) R² values (coefficient of determination) from simple linear regression analyses of rTOMM20–rBAX and rBAX–endo-BAX interactions in HBT or DBT conditions under both blue-light and dark conditions (n = 5 for all). (F) Bar graphs showing the co-localization ratio (Coloc %) between rTOMM20 and rBAX in cells transfected with HBT or DBT (n = 6 for all). HBT- refers to non-irradiated HBT-transfected cells; HBT+ to blue-light-irradiated HBT-transfected cells; DBT- to non-irradiated DBT-transfected cells; and DBT+ to blue-light-irradiated DBT-transfected cells. ****p \u0026lt; 0.0001 (unpaired t-test). (G) Illustration of rBAX within the DBT unit. Upon blue-light irradiation, DBT units associate, facilitating interactions between mCherry (mCh) and GFP on each component that enable Förster resonance energy transfer (FRET). (H) Bar graphs of FRET between the GFP donor and mCherry acceptor, indicating an increase in FRET efficiency under prolonged blue-light irradiation (n = 6 for all). Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001; ns, not significant; mean ± SEM for all\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/a09730b8760c657b44bb123f.jpg"},{"id":81133075,"identity":"496d4988-6c51-4d1f-8201-f624dae1caae","added_by":"auto","created_at":"2025-04-22 15:05:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6559885,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of BAX translocation to mitochondria, nuclear aberrations, and mitochondrial morphological changes (See also Figures S5-S11) (A) Schematic representation of the sequential events during apoptosis versus anti-apoptosis. In the apoptotic scenario, BAX translocates to the mitochondria and aggregates with neighboring BAX molecules, which triggers mitochondrial fission and aberrant nuclear morphology. In contrast, under anti-apoptotic conditions, the DBT unit limits mitochondrial BAX aggregation, thereby preserving mitochondrial fusion and maintaining normal nuclear morphology. (B) Representative immunofluorescence images of endo-BAX (red) and endo-TOMM20 (green). The co-localized area (white) highlights BAX associated with mitochondria under each experimental condition. (C) Bar graph comparing BAX–mitochondria co-localization in each group (n = 10 for all). Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s multiple comparisons. ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001. (D) Immunofluorescence microscopy of endo-BAX with size-filtered BAX particles indicating aggregation under each condition. (E) Bar graph comparing the size of BAX aggregates (n = 300 for all), analyzed by the Kruskal–Wallis test with Dunn’s multiple comparisons. *p \u0026lt; 0.05; ****p \u0026lt; 0.0001; ns, non-significant. (F) Representative DAPI-stained nuclei under each condition. (G) Bar graph showing the number of DAPI-stained nuclei per region of interest (n = 10 for all), analyzed by one-way ANOVA with Tukey’s post hoc test. (H) Bar graph of nuclear solidity, where solidity is the ratio of the nucleus area to its convex hull area. Data were analyzed by the Kruskal–Wallis test with Dunn’s multiple comparisons (n = 40 for all). *p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001; ns, non-significant. (I) Representative images of mitochondrial morphology in cells labeled with anti-TOMM20, captured either immediately after 20 min of blue-light irradiation or following an additional 10 min of dark incubation post-irradiation. (J, K) Bar graphs of mitochondrial perimeter, aspect ratio, and circularity under the indicated conditions (n = 300 for all), analyzed by the Kruskal–Wallis test with Dunn’s multiple comparisons. *p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001; ns, non-significant; mean ± SEM for all\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/caa9073c400b031edcd65f76.jpg"},{"id":81133077,"identity":"5247c280-bf67-4351-9743-ca9fb27300ab","added_by":"auto","created_at":"2025-04-22 15:05:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2294383,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of DBT anti-apoptotic functionality following cisplatin-induced apoptosis was performed using immunofluorescence and live-cell imaging (see also Figure S12-16; Video S1). (A) Experimental scheme. Cells were treated with cisplatin to induce apoptosis, followed by 10 min of blue-light irradiation. Cisplatin triggers the release of CytC from mitochondria into the cytosol, where it binds APAF1 to form the apoptosome. This cascade leads to the accumulation of cleaved caspase-3 (CC3). (B) CytC localization and rBAX Distribution. Immunofluorescence analysis revealed that intact cells retain CytC within mitochondria, whereas apoptotic cells exhibit a diffuse, smudged CytC pattern indicative of leakage. Since integrated fluorescence density is higher when CytC remains mitochondrial, a decrease in CytC integrated density indicates leakage. (C) A bar graph illustrates the decrease in CytC integrated density in HBT condition, with statistical significance determined by one-way ANOVA and Tukey’s post hoc test (n = 9 for all) **p \u0026lt; 0.01; ns, non-significant (D–G) APAF1 accumulation in each condition. Immunofluorescence microscopy was used to analyse APAF1 accumulation using the Kruskal–Wallis test with Dunn’s multiple comparisons (n = 8 for all), while rBAX–APAF1 co-localization was assessed using an unpaired t-test for HBT (n = 8) and the Mann–Whitney test for DBT (n = 11). *p \u0026lt; 0.05; **p \u0026lt; 0.01; ns, non-significant. (H–J) CC3 accumulation and its colocalization with rBAX–rTOMM20 unit. Representative z-stack immunofluorescence images (rendered in 3D) illustrate CC3 co-localizing with rBAX–rTOMM20 following drug treatment and blue-light irradiation. Additional analysis of normalized CC3 levels per cell (with blue light) was performed using one-way ANOVA with Tukey’s post hoc test (n = 8 for all). To further evaluate rBAX–CC3 co-localization, a correlation analysis was conducted under blue-light conditions, with the R², slope, and p-value reported (n = 8 for all). (K) Live-cell imaging. Time-lapse recordings over 3 h demonstrate the spatial overlap of recombinant proteins and progressive changes in cell morphology. mean ± SEM for all\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/bc14b84c9e1ad2fc23410d60.jpg"},{"id":99028630,"identity":"e2ddf29c-6b6c-43da-9d71-895d859d759f","added_by":"auto","created_at":"2025-12-26 08:05:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17213787,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/ccd138e5-494f-424c-838b-89ea585c501c.pdf"},{"id":81134587,"identity":"a45c9d8a-f340-49cd-b750-f0cde00ae2e6","added_by":"auto","created_at":"2025-04-22 15:21:42","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4496622,"visible":true,"origin":"","legend":"Comparison of Apoptosis Under Drug Conditions in HBT- and DBT-Transfected Cells","description":"","filename":"VideoS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/ebf2e4b434dadce4fb6bb5ce.mp4"},{"id":81133086,"identity":"e31e5f28-7fc5-4f03-8222-d2bd84f24598","added_by":"auto","created_at":"2025-04-22 15:05:42","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13825042,"visible":true,"origin":"","legend":"Supplementary Information File","description":"","filename":"SI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6222702/v1/c32450eec624df52f1610197.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Optogenetic Engineering of BAX to Control Mitochondrial Permeabilization and Attenuate Apoptosis in Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBAX belongs to the BCL-2 family, a group of proteins that plays a crucial role in governing apoptosis, a highly regulated process designed to eliminate damaged, unnecessary, or potentially harmful cells in the body.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e In conjunction with its counterpart BCL-2 and other related proteins, BAX participates in the regulation of mitochondrial outer membrane permeabilization (MOMP), which is a critical step in the apoptotic pathway.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBAX is a pro-apoptotic protein that possesses BCL-2 homology (BH) domains, which are critical for protein-protein interactions within the BCL-2 family.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Upon receiving apoptosis signals, BAX undergoes a conformational change that enables its insertion into the mitochondrial outer membrane (MOM). Upon integration, BAX oligomerizes by interacting with other BAX molecules through their BH domains and creates pores in the MOM, facilitating the release of cytochrome c (CytC) and other apoptotic factors from the mitochondria into the cytoplasm. The subsequent release of CytC initiates a series of events resulting in cell death.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e The delicate equilibrium between pro-apoptotic proteins like BAX and anti-apoptotic proteins like BCL-2 is fundamental for determining whether a cell will undergo apoptosis or survive. In healthy cells, these proteins coexist in a finely tuned balance; however, various signals and stress conditions can disrupt this equilibrium, tilting the scales toward apoptosis.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Dysregulation of the apoptotic pathway, including abnormalities in BAX expression or function, has been associated with various diseases including cancer, autoimmune diseases, cardiovascular diseases, aging, and neurodegenerative disorders.\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOwing to its involvement in apoptosis, BAX, along with other BCL-2 family proteins, has been investigated as a potential target for therapeutic interventions, particularly in cancer treatment.\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, most previous research has focused on pro-apoptotic induction using BAX for disease treatment, whereas anti-apoptosis is considered crucial in diseases induced by excessive cell death, such as Alzheimer\u0026rsquo;s disease or Parkinson\u0026rsquo;s disease.\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e This study aimed to explore the anti-apoptotic function of a modified BAX protein, using optogenetics for precise control, to achieve a comprehensive understanding of the structural intricacies and functional manipulation underlying BAX.\u003c/p\u003e \u003cp\u003eOptogenetics is a powerful technique that uses light to control cellular and protein activities in living organisms by employing light sensitive proteins often derived from microbial organisms such as algae or bacteria.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e In the context of studying BAX, optogenetics offers several distinct advantages including spatial precision, reversibility, and a reduction in off target effects. The spatial precision of optogenetic tools allows for the targeted control of BAX activity in specific cellular compartments, providing insights into its localized effects within the cell, particularly in relation to mitochondrial function. Moreover, the reversible nature of optogenetic control enables iterative modulation of BAX activity, which facilitates the investigation of transient changes and helps differentiate between acute and prolonged cellular responses. Unlike traditional genetic manipulation techniques that can accidentally impact multiple cellular processes, optogenetics provides a precise and direct method for protein control that minimizes unintended side effects.\u003c/p\u003e \u003cp\u003eIn this study, optogenetic control was employed to translocate cytoplasmic BAX into mitochondria using cryptochrome 2 (CRY2) and its binding partner cryptochrome interacting basic helix loop helix 1 (CIB1), which are reversibly associated under blue light and dissociated in the dark.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e For genetic modification, CRY2 was fused to BAX, which is normally localized in the cytoplasm in the dark owing to its point mutation (S184E), while CIB1 was fused to TOMM20, a protein resident in the MOM.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In detail, CRY2 was inserted between the BAX \u0026#120572;8 and \u0026#120572;9 motifs to deter collective BAX insertion to MOM by weaking the anchor, the hydrophobic motif located on the \u0026#120572;9 helix, and also moving the BH domain binding site away from the membrane.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The final construct consisted of mCherry::BAX \u0026#120572;1-\u0026#120572;8::CRY2::\u0026#120572;9\u003csup\u003eS184E\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This novel anti-apoptotic optogenetic system was validated by comparing its effects on cellular compartments, such as the mitochondria and nucleus, with those of the pro-apoptotic optogenetic system of BAX, consisting of CRY2::mCherry::BAX\u003csup\u003eS184E\u003c/sup\u003e. For simplicity, the newly developed anti-apoptotic recombinant protein unit was named deterring-BAX-TOMM20 (DBT), while the pro-apoptotic recombinant protein unit, designed based on previous literature, was named hyperactive-BAX-TOMM20 (HBT).\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePlasmid constructs.\u003c/b\u003e For transient transfection of either the HBT or the DBT system, rTOMM20 plasmid was purchased from Addgene (#226667, Watertown, MA, USA). Information regarding the custom-designed recombinant BAX (rBAX) sequences are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e of Supplementary Information.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLight chamber fabrication.\u003c/b\u003e A light chamber was 3D-printed using polyethylene filaments and designed with dimensions of 10 cm \u0026times; 14 cm \u0026times; 9 cm (width \u0026times; length \u0026times; height). The plate entrance was designed to be 9 cm \u0026times; 4 cm (width \u0026times; height) with a door that could open or close the chamber entrance. A blue PCB light-emitting diode (LED) (DC 5V, 2.16 W, 60 mA, 120\u0026ordm; light angle, #2835, LG Innotek, Seoul, Republic of Korea) was installed on the LED board attached to the ceiling of the light chamber. In addition, a C-type charging socket, switch, and ventilation wickets were installed on the backside of the chamber. The battery could sustain the light chamber for 4 h once fully charged. Detailed illustrations are provided in Figure S2 of Supplementary Information.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and transient transfection.\u003c/b\u003e Human dermal fibroblast-neonatal (NHDF-Neo) cells (#CC-2509, Lonza, Basel, Switzerland) were maintained in a culture medium consisting of RPMI-1640 (#10-040-CV, Cellgro, Corning, NY, USA), 10% fetal bovine serum (FBS, #A2720803, Gibco\u0026trade;, Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin-streptomycin (p/s, #15140163, Invitrogen, Thermo Fisher Scientific) in a 37\u0026ordm;C humidity-controlled incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Lipofectamine 3000 (#L3000015; Thermo Fisher Scientific) was used as the transfection reagent. Initially, 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were plated in each well of a 6-well plate (#30006, SPL Life Sciences, Pocheon-si, Gyeonggi-do, Republic of Korea) and incubated for 24 h at 37\u0026ordm;C. The cell medium was then aspirated, and FBS-reduced medium (RPMI-1640, 2% FBS, and 1% P/S) was added an hour before transfection. P3000 reagent (2 \u0026micro;L) was mixed with 1 \u0026micro;g of the HBT or DBT plasmids (500 ng rBAX and 500 ng rTOMM20 (1:1 ratio) in a total volume of 1 \u0026micro;L) in 122 \u0026micro;L of Opti-MEM\u0026trade; (#31985070, Gibco\u0026trade;, Thermo Fisher Scientific). For the control condition, the reagent solution was mixed with 1 \u0026micro;g rTOMM20 plasmids (1 \u0026micro;L) instead. Separately, 4 \u0026micro;L of Lipofectamine 3000 was added to 121 \u0026micro;L of Opti-MEM\u0026trade; per well of the 6-well plate. After 5 min incubation at room temperature, the mixtures were combined to a total volume of 250 \u0026micro;L and incubated at room temperature for 30 min. Subsequently, 200 \u0026micro;L of the incubated mixture was added dropwise to each well. The medium was aspirated and replaced with fresh culture medium containing 5 \u0026micro;M Flavin adenine dinucleotide disodium salt hydrate (FAD, #F6625, Sigma-Aldrich, St. Louis, MO, USA) 24 h after transfection. For further experiments, cells were illuminated with blue light 48 h post-transfection in the light chamber for 20 min (if not otherwise mentioned in the text).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFRET assay.\u003c/b\u003e Cells at 48 h post-transfection with either 50 or 200 ng of HBT or DBT plasmids per 20,000 cells in a 96-well plate were serially irradiated with blue light for 0, 5, or 10 min. The cells were then immediately analyzed using FRET analysis in a fluorescence-detectable multimode microplate reader (Hidex Sense, HIDEX Oy, Turku, Finland).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence microscopy.\u003c/b\u003e (Antibody details are provided in Table S2 of the Supplementary Information file) Twenty-four hours post-transfection, the transfected cells were dissociated from the surface using 0.25% trypsin (#24200-072, Thermo Fisher Scientific) and plated on cell culture slide I (#30408, SPL Life Sciences) at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. The cells were incubated in a 37\u0026ordm;C humidity-controlled incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. For immunofluorescence microscopy sample preparation, the cells were irradiated with blue light for 20 min and immediately prepared for the next step in most analyses (1-min incubation in the dark) or incubated in the dark for 10 min. In drug-treated experiments, blue light was applied for 10 min. Following incubation in the dark, the cells were fixed with 4% paraformaldehyde solution (#PC2031-100-00, Biosesang, Yongin-si, Gyeonggi-do, Republic of Korea) and stored at 4\u0026ordm;C overnight. The fixed cells were treated with 0.4% Triton X-100 in PBS for 20 min at room temperature. After aspirating the buffer, 5% bovine serum albumin (BSA) in PBS was treated for 30 min at room temperature. The buffer was then aspirated, and the cells were incubated with primary antibodies in a mixture of 0.2% Tween in PBS (PBST) and 5% BSA (1:1 ratio) overnight at 4\u0026ordm;C. The primary antibodies were aspirated, and the cells were washed thrice with PBST for 5 min at room temperature. The cells were then incubated with secondary antibodies for 1 h at room temperature. After aspirating the secondary antibodies, the cells were washed thrice with PBST for 5 min at room temperature. The mounting medium was applied dropwise to the samples, which were mounted on a 24 \u0026times; 60 mm microscope cover glass (#HSU-0101242, Marienfeld, Lauda-K\u0026ouml;nigshofen, Germany). The samples were examined under a fluorescence microscope (Axio Imager M1; Carl Zeiss AG, Oberkochen, Germany) or LSM 900 confocal microscope (LSM 900; Carl Zeiss AG, Oberkochen, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLive cell imaging.\u003c/b\u003e Transfected cells were treated with 200 \u0026micro;M Cisplatin and maintained in a live cell imaging system (Incubator TS, Live Cell Instrument, Seoul, Republic of Korea) to ensure a humidity-controlled environment at 37\u0026ordm;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were visualized using the CelenaX imaging system (CELENA\u0026reg; X, Logos Biosystems, Anyang, Gyeonggi-do, Republic of Korea). Time-lapse imaging was conducted by irradiating cells with blue light for 1,000 ms every 5 min during a 3 h recording period. Translocation events of mCherry-tagged rBAX and GFP-tagged rTOMM20 were visualized and traced under a microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein Structure Simulation.\u003c/b\u003e For predicting recombinant protein oligomer structures, the AlphaFold online tool (developed by DeepMind, with data from EMBL-EBI) was used for multimer binding structure prediction and creation of PDB files. RCSB PDB (managed by Rutgers University and UC San Diego, supported by NSF, NIH, and DOE) was utilized for visualizing the multimer structures.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Co-localization and morphological analyses were conducted using ImageJ software (NIH, Bethesda, MD, USA). For the co-localization assessment, the color-merged immunostained fluorescence images of the red and green channels were thresholded using ImageJ software (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF,G; Figure S5). The ratio of the co-localized area to the red-thresholded area was analyzed. Alternatively, the plot profile plugin in ImageJ was employed for both colocalization assessment and fluorescence intensity measurement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB,D; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE,I,J; Figure S12-S13; Figure S16). For morphological analysis, the immunostained fluorescence images were thresholded, and size filtering was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE,G,H,J,K; Figure S6; Figure S7; Figure S9-S11). The morphological analysis of subcellular compartments was conducted by setting subcellular ROIs (randomly selected) in multiple single-cell images. Colocalization, CytC, and CC3 assays were performed on a per-cell basis, whereas APAF1 analysis was conducted for each image obtained from the slide samples. All experiments were performed at least 3 independent experiments. Quantitative data values were analyzed using Prism software (GraphPad Software Inc., San Diego, CA, USA), and bar graphs were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Two-tailed tests were conducted for every statistical analysis (95% confidence).\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of Custom-Built Recombinant Proteins.\u003c/b\u003e In this study, we aimed to manipulate the MOMPs induced by endogenous BAX (endo-BAX), which have a distinct structure comprising \u0026#120572;1-\u0026#120572;9, with \u0026#120572;9 containing a hydrophobic motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). To achieve precise control of optogenetic BAX for pro-apoptotic induction, in comparison with DBT, this study introduced HBT, which demonstrates stable and controllable insertion of CRY2-fused BAX into the MOM upon blue light activation.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e In the recombinant protein constructs, BAX was fused to CRY2 and mCherry (mCh) in distinct configurations for HBAX\u003csup\u003eS184E\u003c/sup\u003e and DBAX\u003csup\u003eS184E\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,C,D). In HBAX\u003csup\u003eS184E\u003c/sup\u003e, full-length CRY2 and mCh were positioned upstream of the complete BAX sequence, incorporating a point mutation at residue 184 (S184E). In contrast, DBAX\u003csup\u003eS184E\u003c/sup\u003e also placed the full mCh sequence upstream but introduced CRY2 between the \u0026#120572;8 and \u0026#120572;9 helices of BAX. As an optogenetic binding partner, TOMM20\u0026ndash;CIB1\u0026ndash;GFP (recombinant TOMM20; rTOMM20) was engineered to facilitate the translocation of BAX from the cytosol to the mitochondria. Therefore, the novelty of this study is embodied in the engineered DBAX\u003csup\u003eS184E\u003c/sup\u003e structure, in which the binding of CRY2 and CIB1 immediately above the membrane hinders MOMP because the anchoring function of the hydrophobic motif within the \u0026#120572;9 helices is limited and BH groove binding, which is essential for MOMP, is deterred, thereby modulating pro-apoptotic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThe sequential process of MOMP induced by endo-BAX is as follows. In the normal state, CytC is retained within the mitochondria by the intact MOM, while BAX remains in the cytoplasm.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e During apoptosis, BAX translocates to the MOM, accompanied by the exposure of its \u0026#120572;9 helix. Another BAX aligns next to the first on MOM by generating junctions via BH grooves, leading to pore formation due to extensive BAX insertion.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This process results in CytC leakage into the cytosol, triggering cell death via activation of APAF1 and Caspase proteins\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e It is accompanied by morphological changes such as mitochondrial fission, nuclear pyknosis, and cell shrinkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eApoptosis by HBT is induced by the binding of CRY2 in recombinant Bax (rBAX) with its binding partner CIB1, which is part of rTOMM20, thereby facilitating vast endo-BAX recruitment to the MOM and inducing MOMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In comparison, rBAX of DBT, which is limitedly inserted into the MOM along with endo-BAX, hinders the MOMP under blue light irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eTo verify the anti-apoptotic functionality of the DBT, a blue light chamber was specifically generated to fit into 6-well cell culture plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, Figure S2). For live cell imaging, a 488 nm blue laser equipped with a fluorescence microscope was utilized. For analysis, cells were transfected with plasmids 24 h after seeding and flavin adenine dinucleotide disodium salt hydrate (FAD) was added to the culture medium 24 h post-transfection. Finally, the cells were irradiated with blue light at 48 h post-transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAggregation Simulation and Microscopic Analysis of DBT Reveal Attenuated Pore Formation Impacting endo-BAX\u003c/h2\u003e \u003cp\u003eIn this study, DBAX\u003csup\u003eS184E\u003c/sup\u003e was engineered to exert controlled effects on endo-BAX. Under blue light irradiation, two distinct binding events occur: one between rBAX and rTOMM20 for locational control of rBAX, and another between rBAX and endo-BAX via the intact BH3 domain, which ultimately hinders BAX-mediated MOMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To assess the binding pattern of DBAX\u003csup\u003eS184E\u003c/sup\u003e in cells, protein aggregation events were simulated under three conditions: (1) only endo-BAX is present, (2) endo-BAX and HBAX\u003csup\u003eS184E\u003c/sup\u003e are present in a 6:4 ratio (endo-HBAX), or (3) endo-BAX and DBAX\u003csup\u003eS184E\u003c/sup\u003e are present in a 6:4 ratio (endo-DBAX) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The total number of proteins for simulation in each condition was set to 10. According to the simulation results, both the endo-BAX complex and the endo-HBAX complex showed pore generation, but the endo-DBAX complex hardly showed pore formation. DBAX\u003csup\u003eS184E\u003c/sup\u003e was also proven to interact with endo-BAX via BH grooves. Additionally, the results confirmed normal CRY2 homodimerization events.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e To further investigate the membrane embedding pattern of the DBT unit in cells, protein aggregation events were simulated under two conditions: (1) endo-BAX, HBAX\u003csup\u003eS184E\u003c/sup\u003e, and rTOMM20 are present in a 5:3:2 ratio (endo-HBT), or (2) endo-BAX, DBAX\u003csup\u003eS184E\u003c/sup\u003e, and rTOMM20 are present in a 5:3:2 ratio (endo-DBT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The total number of proteins for simulation in each condition was set to 10. The condition where only endo-BAX is present was also included as a control for reference. According to the simulation results, both the aggregation of endo-BAX alone and the endo-HBT condition showed deeply embedded residues, while the endo-DBT condition showed minimally embedded residues in the complex. Furthermore, pore formation was still observed in the endo-HBT condition but not in the endo-DBT condition. These simulation results support that the genetic design of DBAX\u003csup\u003eS184E\u003c/sup\u003e effectively interacts with endo-BAX, impeding pore formation by interacting with endo-BAX.\u003c/p\u003e \u003cp\u003eTo further evaluate the binding events of the DBT complex in cells irradiated with blue light, 3D-rendered confocal images were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In the CTRL condition, cells transfected only with rTOMM20, endo-BAX displayed a membrane-embedded conformation, with its terminal region contacting the membrane. From bottom-view images highlighting endo-BAX\u0026rsquo;s penetration into the MOM, we observed multiple endo-BAX molecules deeply embedded, clearly visible from the underside (Figure S3A).\u003c/p\u003e \u003cp\u003eUnder the HBT condition, large complexes formed among endo-BAX, rBAX, and rTOMM20. Top-view images revealed extensive endo-BAX conjugation with the HBT complex, generating a sizeable aggregate (1.483 \u0026micro;m) on the mitochondria. In bottom and side views, endo-BAX showed deep penetration alongside rBAX cluster (Figure S3B).\u003c/p\u003e \u003cp\u003eBy contrast, in the DBT condition, although rBAX and rTOMM20 successfully formed complexes, they were markedly smaller (0.686 \u0026micro;m) than those in HBT. Moreover, unlike HBT, the endo-BAX\u0026ndash;rBAX interaction in DBT occurred primarily on the upper region of rBAX, possibly reflecting an altered position of its BH domain. Endo-BAX also exhibited limited membrane penetration under DBT (Figure S3C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIrradiation-Driven rBAX–rTOMM20 Conjugation Regulates Endogenous BAX Assembly\u003c/h3\u003e\n\u003cp\u003eTo verify the optogenetic controllability of the newly developed recombinant constructs, we examined the subcellular colocalization of rBAX and rTOMM20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;E). For each condition, random subcellular regions of interest (ROIs) were selected to generate fluorescence intensity profiles, with two representative examples shown. Multiple cells were also analyzed independently to confirm reproducibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eUnder HBT conditions, blue-light irradiation (Light On) yielded nearly identical fluorescence intensity patterns (gray values) for rBAX and rTOMM20 across all sampled ROIs, indicative of colocalization, whereas similarity was substantially lower under Light Off conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B; Figure S4A,B). Notably, endo-BAX intensity also increased under Light On, suggesting that, rather than a rapid upregulation of protein expression (given the ~\u0026thinsp;30-min timeframe), the enhanced signal reflects the collective recruitment of endo-BAX triggered by HBT activation. Under DBT conditions, rBAX similarly exhibited a light-dependent colocalization pattern with rTOMM20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D; Figure S4C,D), although overall endo-BAX levels in DBT cells were lower than those observed under HBT Light On conditions.\u003c/p\u003e \u003cp\u003eCorrelation analyses with simple linear regression model corroborated these observations. The correlation between rBAX and rTOMM20 intensities strengthened under blue-light stimulation in both HBT Light On (HBT+, R\u0026sup2; = 0.874, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to HBT Light Off (HBT-, R\u0026sup2; = 0.302, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and DBT Light On cells (DBT+, R\u0026sup2; = 0.852, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to DBT Light Off (DBT-, R\u0026sup2; = 0.118, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with a greater disparity observed under DBT conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; Figure S4E). When the correlation between endo-BAX and rBAX intensities was similarly evaluated, HBT cells showed a marked increase upon light exposure (R\u0026sup2; = 0.508, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to HBT Light Off (R\u0026sup2; = 0.197, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), consistent with HBT\u0026rsquo;s established proapoptotic function. By contrast, DBT cells exhibited similar correlations regardless of light conditions (R\u0026sup2; = 0.404, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 under Light Off and R\u0026sup2; = 0.419, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 under Light On) (Figure S4F).\u003c/p\u003e \u003cp\u003eTo further quantify these findings on a per-cell level, mCherry\u0026ndash;GFP overlap (Coloc %) was measured across multiple cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Both HBT and DBT groups exhibited statistically significant differences between Light Off and Light On conditions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both), confirming that blue-light irradiation effectively induces rBAX\u0026ndash;rTOMM20 conjugation in both constructs, albeit with distinct outcomes for endo-BAX recruitment.\u003c/p\u003e \u003cp\u003eUltimately, a F\u0026ouml;rster resonance energy transfer (FRET) assay was employed to demonstrate energy transfer between the two closely bound recombinant proteins under blue-light irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The assay was conducted in cells transfected with either HBT or DBT constructs and subjected to progressively longer blue-light exposures (5 or 10 min). Donor-only conditions were established by expressing rTOMM20 without any acceptor protein. Under these donor-only conditions, no significant FRET increase was detected following either 5 or 10 minutes of irradiation in cells transfected with 50 ng of plasmid per 20,000 cells (p\u0026thinsp;=\u0026thinsp;0.956 for 0 vs 5 min; p\u0026thinsp;=\u0026thinsp;0.634 for 0 vs 10 min; p\u0026thinsp;=\u0026thinsp;0.804 for 5 vs 10 min). Similarly, cells transfected with 200 ng of plasmid per 20,000 cells showed no significant FRET increase (p\u0026thinsp;=\u0026thinsp;0.961 for 0 vs 5 min; p\u0026thinsp;=\u0026thinsp;0.657 for 0 vs 10 min; p\u0026thinsp;=\u0026thinsp;0.816 for 5 vs 10 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eIn HBT-transfected cells, 5 min of blue-light exposure did not significantly elevate FRET signals (p\u0026thinsp;=\u0026thinsp;0.963), whereas 10 min led to a significant increase (p\u0026thinsp;=\u0026thinsp;0.02), and the mean difference between 5 and 10 min was also significant (p\u0026thinsp;=\u0026thinsp;0.0382) in cells transfected with 50 ng. With 200 ng, both 5 and 10 min of irradiation induced increased FRET (p\u0026thinsp;=\u0026thinsp;0.0402; p\u0026thinsp;=\u0026thinsp;0.0445), with no significant mean difference (p\u0026thinsp;=\u0026thinsp;0.999) between these two time points.\u003c/p\u003e \u003cp\u003eIn DBT-transfected cells, 5 min of light exposure did not produce a significant FRET increase (p\u0026thinsp;=\u0026thinsp;0.352), but a marked increase occurred at 10 min (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the mean difference between 5 and 10 min was significant (p\u0026thinsp;=\u0026thinsp;0.0003) in cells transfected with 50 ng. When 200 ng was used, both 5 and 10 min of blue-light exposure induced significant FRET increases (p\u0026thinsp;=\u0026thinsp;0.0022; p\u0026thinsp;=\u0026thinsp;0.0147), with no significant mean difference between the two time points (p\u0026thinsp;=\u0026thinsp;0.7785). These data confirm that prolonged blue-light irradiation promotes rBAX\u0026ndash;rTOMM20 conjugation in HBT and DBT systems, with a more rapid and extensive response at higher plasmid concentrations.\u003c/p\u003e \u003cp\u003eBased on these validated optogenetic modulations of the recombinant proteins, the anti-apoptotic effects of DBT were subsequently evaluated by analyzing multiple cell-death-related indices and comparing cells transfected with rTOMM20 alone (CTRL) or HBT against DBT-transfected cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLight-Activated DBTs Reduce BAX Mitochondrial Localization and Aggregation, Preserve Nuclear and Mitochondrial Integrity, Enhancing Cell Viability\u003c/b\u003e The morphological assessment of apoptosis in cells was based on three key indicators. First, translocation of BAX into the mitochondria was observed, and BAX aggregation, which increases during active apoptosis, was quantified.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Second, the number and morphology of nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) were analyzed as a key indicator of cell death.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Lastly, mitochondrial fission status, determined by measuring mitochondrial length and size, was analyzed to assess the mitochondrial integrity of the cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e These assessments were performed by analyzing immunostained images of endo-BAX and endogenous TOMM20 (endo-TOMM20) to verify the effects of recombinant protein manipulation on their endogenous counterparts. The results presented here focus solely on the blue-light\u0026ndash;irradiated conditions; comparisons with no-light controls are provided in the supplementary information (Figures S8\u0026ndash;S11).\u003c/p\u003e \u003cp\u003eFirst, the colocalization of endo-BAX with mitochondria was evaluated following blue-light irradiation. This evaluation was based on the established property that endo-BAX is translocated to the MOM during active apoptosis.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The red and green overlapping region in the images, which indicates mitochondrial BAX, was selectively filtered (white), and its area was quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Figure S5). The results showed that endo-BAX recruitment to the mitochondria was significantly enhanced under the pro-apoptotic HBT condition (p\u0026thinsp;=\u0026thinsp;0.0003 vs. CTRL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 vs. DBT), whereas it remained attenuated under DBT, with no significant difference from CTRL (p\u0026thinsp;=\u0026thinsp;0.5015). This indicates that rBAX in the DBT construct does not facilitate rubst BAX translocation to the mitochondria, unlike the HBT construct.\u003c/p\u003e \u003cp\u003eTo further assess BAX aggregation size, a critical index of MOMP, immunofluorescence images were processed to remove outliers such as debris and highly saturated areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD; Figure S6). Given that BAX proteins form larger clusters through redistribution as MOMP progresses, the sizes of BAX aggregates were compared and analyzed across experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e In the DBT condition, the size of BAX clusters within each ROI was markedly smaller than in both the CTRL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and HBT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) conditions. Moreover, the reduction in aggregate size in DBT compared to HBT was significantly greater than that observed between CTRL and HBT (p\u0026thinsp;=\u0026thinsp;0.0442), highlighting the pronounced attenuation of endo-BAX aggregation in the DBT condition to levels below baseline. Altogether, the mitochondrial colocalization of BAX and aggregate size measurements underscore the anti-apoptotic efficacy of the DBT construct in modulating neighboring endogenous BAX.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecond, cellular viability was evaluated by examining the number and morphology of nuclei via DAPI staining under blue-light irradiation in CTRL, HBT, and DBT conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, quantitative analysis revealed that the DAPI counts in DBT-transfected cells were identical to those in the CTRL group (p\u0026thinsp;=\u0026thinsp;0.8750) and significantly higher than those observed in HBT-transfected cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, the HBT condition exhibited a marked reduction in nuclei number compared to CTRL (p\u0026thinsp;=\u0026thinsp;0.0002). Furthermore, nuclear solidity, a measure of structural integrity, was well maintained in DBT-transfected cells, with values comparable to CTRL (p\u0026thinsp;=\u0026thinsp;0.3767) and significantly superior to those in the HBT condition (p\u0026thinsp;=\u0026thinsp;0.0213) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Notably, the HBT condition showed considerable nuclear distortion relative to CTRL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These results underscore that DBT not only preserves nuclear integrity but also maintains overall cellular viability under blue-light irradiation.\u003c/p\u003e \u003cp\u003eThird, to confirm the protective effects of DBTs on mitochondrial integrity, mitochondrial morphology was examined. Cells irradiated with blue light under each condition were immunostained with an anti-TOMM20 antibody to visualize mitochondrial structures, which reflect either fission or fusion states (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI; Figure S7). Active mitochondrial fission, commonly associated with cellular stress or damage, is characterized by shorter, and more circular mitochondria that can be quantitatively assessed.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Mitochondrial morphology was evaluated at two time points: immediately after blue-light irradiation (1 min post-irradiation) and 10 min post-irradiation (following 10 min of dark incubation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ,K). In samples prepared immediately post-irradiation, DBT-transfected cells exhibited a significantly elongated and continuous mitochondrial perimeter compared to both CTRL (p\u0026thinsp;=\u0026thinsp;0.0241) and HBT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) groups, whereas the HBT condition showed a markedly shorter perimeter relative to CTRL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similarly, the aspect ratio (AR) was significantly higher in the DBT condition than in CTRL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and HBT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while HBT exhibited a lower AR compared to CTRL (p\u0026thinsp;=\u0026thinsp;0.0008). Moreover, the index of circularity, a marker of intensive, dot-like mitochondrial fission, was significantly elevated in the HBT condition relative to CTRL (p\u0026thinsp;=\u0026thinsp;0.0002) but was markedly reduced in DBT-transfected cells compared to both CTRL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and HBT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) conditions.\u003c/p\u003e \u003cp\u003eAfter 10 min of dark incubation following blue-light irradiation, these trends were somewhat attenuated. In the DBT condition, the mitochondrial perimeter was comparable with the CTRL condition (p\u0026thinsp;=\u0026thinsp;0.1217), and no significant differences were observed in either AR or circularity between DBT and CTRL (p\u0026thinsp;=\u0026thinsp;0.4185 and p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999, respectively). However, both the mitochondrial perimeter and AR of DBT-transfected cells remained significantly higher than those in the HBT condition (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;=\u0026thinsp;0.0001, respectively). In contrast, while the perimeter and AR in the HBT condition continued to be lower than those in the CTRL condition (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both), circularity was restored to levels comparable to both CTRL (p\u0026thinsp;=\u0026thinsp;0.6749) and DBT (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999) during the dark incubation. This recovery may be attributed to rapid mitochondrial dynamics that promote the fusion of highly circular, fissioned mitochondria.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e These results indicate that the anti-apoptotic effect conferred by DBT, evidenced by sustained mitochondrial fusion, is remarkably pronounced even surpassing baseline levels observed in the CTRL group. This effect is rapidly reversed by dark incubation, likely due to the intrinsic rapid reversibility of optogenetic systems.\u003c/p\u003e\n\u003ch3\u003eDBT Preserves Mitochondrial Integrity and Suppresses APAF1/Caspase-3–Dependent Apoptotic Signaling Under Apoptotic Drug Treatment\u003c/h3\u003e\n\u003cp\u003eFinally, the anti-apoptotic effects of DBTs were assessed by exposing cells to 200 \u0026micro;M cisplatin for 10 min with or without irradiation, a compound known to induce MOMP.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Upon cisplatin treatment, mitochondrial CytC is released into the cytosol, where it binds to APAF1 to form the apoptosome, triggering downstream signaling cascades that result in the accumulation of cleaved caspase-3 (CC3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To evaluate this process, the spatial distribution of key apoptotic markers including CytC, APAF1, and CC3 were investigated after cisplatin treatment.\u003c/p\u003e \u003cp\u003eFirstly, CytC distribution was examined to assess the preservation of mitochondrial integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C; Figure S12,13). Immunofluorescence images revealed that apoptotic cells exhibit diffuse, smudged CytC signals indicative of leakage, alongside visible mitochondrial structures. In HBT-transfected cells, the integrated fluorescence density of CytC was significantly attenuated (p\u0026thinsp;=\u0026thinsp;0.0048 vs. CTRL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0034 vs. DBT), indicating a dispersed, non-contained distribution of CytC. In contrast, no significant difference was observed between DBT-transfected cells and control cells (p\u0026thinsp;=\u0026thinsp;0.9881). Moreover, when mitochondrial integrity was evaluated by measuring the AR of CytC distribution, DBT-transfected cells demonstrated superior preservation relative to both the CTRL (p\u0026thinsp;=\u0026thinsp;0.0391; one-way ANOVA, Tukey\u0026rsquo;s post hoc test, n\u0026thinsp;=\u0026thinsp;300 for all) and HBT conditions (p\u0026thinsp;=\u0026thinsp;0.0093, n\u0026thinsp;=\u0026thinsp;300), whereas no significant difference was observed between CTRL and HBT conditions (p\u0026thinsp;=\u0026thinsp;0.8716) (Figure S12D). these effects were not observed under no-light control conditions (Figure S13B-C).\u003c/p\u003e \u003cp\u003eSecondly, APAF1 accumulation was assessed. Given that baseline APAF1 expression is broad and variable, a high dose of cisplatin (1 mM) was used to elicit a robust apoptotic response, ensuring drug-specific APAF1 accumulation for apoptosome assembly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G). Immunofluorescence microscopy revealed extensive cell death following drug treatment across all conditions, with a significant reduction observed in DBT-transfected cells under blue-light irradiation. Quantitative analysis demonstrated that APAF1 intensity in DBT-transfected cells under irradiation was significantly lower than that in both CTRL (p\u0026thinsp;=\u0026thinsp;0.0023) and HBT (p\u0026thinsp;=\u0026thinsp;0.0023) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In contrast, no significant difference in APAF1 accumulation was detected between CTRL and HBT groups, likely due to the harsh drug treatment obscuring subtle differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999). Furthermore, in DBT-transfected cells, APAF1-rBAX colocalization was markedly attenuated under blue-light conditions compared to no-light conditions (p\u0026thinsp;=\u0026thinsp;0.0014), whereas HBT-transfected cells showed a significant increase of that under blue-light irradiation (p\u0026thinsp;=\u0026thinsp;0.0130) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF,G).\u003c/p\u003e \u003cp\u003eThirdly, CC3 accumulation and its colocalization with rBAX were evaluated by immunofluorescence microscopy that highlighted the localized distribution of CC3 around mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-J; Figure S14-S16). The 3D reconstructions revealed pronounced colocalization of CC3 with the HBT complex, whereas DBT-transfected cells displayed less colocalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Quantitative analysis of immunofluorescence images indicated that overall CC3 concentrations did not differ significantly between groups under dark (p\u0026thinsp;=\u0026thinsp;0.0032) (Figure S16A,C). Under blue-light irradiation, DBT-transfected cells exhibited markedly lower CC3 levels compared to HBT-transfected cells (p\u0026thinsp;=\u0026thinsp;0.0446), highlighting DBT\u0026rsquo;s capacity to attenuate apoptotic signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI; Figure S15A). By contrast, CC3 accumulation in HBT-transfected cells was significantly higher than in CTRL (p\u0026thinsp;=\u0026thinsp;0.0129), whereas DBT-transfected cells remained comparable to CTRL (p\u0026thinsp;=\u0026thinsp;0.7456).\u003c/p\u003e \u003cp\u003eTo further elucidate the relationship between rBAX and CC3 distributions, simple linear regression analysis was performed. Under blue-light conditions, HBT-transfected cells displayed a strong positive correlation between rBAX and CC3 (R\u0026sup2; = 0.9804; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas DBT-transfected cells showed no significant correlation (R\u0026sup2; = 0.1371; p\u0026thinsp;=\u0026thinsp;0.3267), with a negative regression slope. Under no-light conditions, no significant correlations were observed in either condition (Figure S16B). These findings indicate that DBT-transfected cells exhibit attenuated CC3 accumulation, reinforcing the anti-apoptotic efficacy of the DBT construct.\u003c/p\u003e \u003cp\u003eFinally, to observe the anti-apoptotic function of DBTs in real time, a live-cell imaging assay was performed with 1,000 ms pulses every 5 min over a 3-hr period under drug condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). The results showed that HBT and DBT recombinant proteins gradually interacted over time. Notably, DBT-transfected cells retained their morphological integrity, whereas HBT-transfected cells exhibited progressive structural disruption.\u003c/p\u003e \u003cp\u003eIntegrating these observations with data obtained across a range of apoptotic inducer experiments highlights the robust anti-apoptotic functionality of DBTs. This protective effect is attributed to the unique structural features of DBTs, which deter pore formation. Collectively, these findings highlight the potential of the structural modifications proposed in this study to modulate BAX activity and bolster anti-apoptotic responses, offering promising prospects for future drug development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecognizing the importance of modulating apoptosis in cells, this study proposes BAX as a promising therapeutic target for apoptosis-associated diseases. This potential is attributed not only to its organelle-specific assembly but also to its intrinsic interaction mechanism, whereby a single dysfunctional BAX molecule can influence neighboring BAX proteins, thereby altering the dynamics of apoptotic signaling. In our work, we employed an optogenetic approach to direct BAX to the mitochondria, the primary site of its apoptotic activity, and to elicit anti-apoptotic effects within BAX assemblies. Consequently, apoptosis was attenuated through targeted alterations that prevented pore formation in the mitochondrial outer membrane (MOM). Notably, this system was activated simply by exposure to blue light.\u003c/p\u003e \u003cp\u003eMitochondria, which serve as the primary platform for BAX function, undergo dynamic cycles of fission, fusion, and transport on timescales ranging from seconds to hours, thus playing a vital role in cellular quality control. Under certain stimuli or stress conditions, they can rapidly eliminate damaged components or quickly compensate for functional deficits, enabling swift adaptation.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Typically, upon receiving cell death signals, mitochondria undergo pronounced fission, prompting CytC release and accelerating proapoptotic processes, thus mitochondrial dynamics is important in regulating cell death.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e During this process, multiple BAX proteins respond to apoptotic cues by undergoing structural rearrangements and integrating into the MOM within minutes, where they form pores that compromise mitochondrial integrity.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e BAX is also thought to interact directly with mitochondrial fission mediators like Drp1, thereby exerting a substantial influence on mitochondrial morphology.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAccordingly, we aimed to regulate BAX's mitochondrial binding event by hindering the interaction of its hydrophobic motif, which serves as a mitochondrial anchor. To achieve this, we engineered an optogenetic system that strategically positions CRY2 over the hydrophobic motif, thereby promoting its binding to CIB1-fused TOMM20, a mitochondrial membrane protein, and effectively impeding BAX-mediated pore formation. Additionally, this design repositions the BH groove away from the MOM, further disrupting collective BAX interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, we confirmed that CRY2 and CIB1 bind tightly under blue light and exhibit more rapid interactions at higher expression levels in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This light-induced interaction drove the translocation of recombinant BAX (rBAX) to the mitochondria and influenced the recruitment of endogenous BAX (endo-BAX) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These effects were validated using a combination of simulation, as well as 2D and 3D immunofluorescence image analyses for precise localization.\u003c/p\u003e \u003cp\u003eThroughout this study, we compared HBT, a system previously demonstrated to be reliable and that preserves the native BAX structure, with DBT, as well as with a rTOMM20-only condition (CTRL) that exhibits stable transient expression of recombinant proteins without facilitating apoptosis.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Our findings reveal that DBT not only diminishes the mitochondrial localization of endo-BAX but also suppresses its aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). This dual effect mitigates cell death, enhances cell viability, and promotes sustained mitochondrial fusion (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-K). In essence, DBT functions as a covert modulator among BAX molecules, effectively inactivating their proapoptotic activity and reducing apoptosis to levels below those observed in control cells.\u003c/p\u003e \u003cp\u003eWe further conducted a comparative analysis of HBT and DBT under drug-induced stress by exposing cells to high concentrations of cisplatin, followed by light illumination. Under these conditions, we tested mitochondrial CytC release, APAF1 aggregation, and subsequent CC3 accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Overall, the DBT construct markedly mitigated these effects. The maintenance of CytC distribution and mitochondrial integrity in DBT-transfected cells, as evidenced by integrated fluorescence density and aspect ratio measurements, suggests that DBT effectively preserves mitochondrial function under stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C; Figure S12). Moreover, the reduced APAF1 accumulation and diminished CC3 levels in DBT-transfected cells, coupled with the lack of significant rBAX\u0026ndash;CC3 correlation, indicate that DBT not only disrupts BAX\u0026rsquo;s ability to promote apoptotic pore formation but also attenuates downstream apoptotic signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-J). Live-cell imaging further corroborated these findings by demonstrating sustained cellular integrity over time in DBT-transfected cells, in stark contrast to the progressive morphological deterioration observed in HBT-transfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Collectively, these results support the hypothesis that the novel optogenetic modification of BAX structure can effectively suppress its proapoptotic activity, thereby offering a promising strategy for modulating apoptosis in therapeutic contexts.\u003c/p\u003e \u003cp\u003eEven though this novel technology offers significant future potential for addressing disease-related challenges, there remain limitations that must be overcome to enhance its utility. One limitation of this study is the inherent toxicity associated with transfection reagents, which complicates the accurate evaluation of recombinant proteins in apoptosis-related research. Future studies should consider integrating advanced transfection methodologies to remove these effects and enable more precise assessments. Additionally, further validation across a broader range of cell types is necessary, with particular emphasis on neuronal cells, where preventing unwarranted apoptosis is critical. It is also important to note that inhibiting cell death does not inherently equate to improved health. Indeed, excessive suppression of apoptosis may lead to inefficient energy utilization, accelerated aging, or compromised quality control mechanisms. Therefore, careful deliberation is required in the development and application of this technology. Lastly, a comprehensive evaluation of the interplay between apoptosis and regeneration is indispensable, as the therapeutic potential of such interventions is likely to be maximized when these processes are balanced.\u003c/p\u003e \u003cp\u003eGiven the prevalence of age-related diseases associated with apoptosis, including dementia, Parkinson\u0026rsquo;s disease, arthritis, and diabetes, this study elucidates critical mechanisms for precisely targeting BAX by examining how a single genetically engineered variant, designed to hinder its mitochondrial permeabilization, disrupts the cell's ability to mediate and trigger apoptosis. By overcoming several of the noted limitations, we expect that our results will establish a solid foundation for evaluating anti-apoptotic interventions in cells and facilitate the advancement of novel therapies designed to reduce apoptosis in forthcoming disease treatments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT)(RS-2024-00455532), and the Nano \u0026amp; Material Technology Development Program through the National Research Foundation of Korea(NRF) funded by Ministry of Science and ICT(RS-2024-00450828)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.L. developed the concepts, methodology, validation, formal analysis, and investigation, administered the project, and wrote the original draft. H.B. curated the data and reviewed the original draft. D.O. conducted the investigation and data curation. J.A., M.K., and S.K. reviewed the original draft. J.K. supported the investigation. D.H.K., H.O., and W.D.H. contributed to the visualization and review of the original draft. W.D.H also provided supervision. S.C. supported conceptualization and methodology development, supervised the project, reviewed and edited the original draft, and secured funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003eDetailed information is provided in the Supporting Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVideo S1\u0026nbsp;\u003c/strong\u003eComparison of Apoptosis Under Drug Conditions in HBT- and DBT-Transfected Cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to Seok Chung.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCory, S. \u0026amp; Adams, J. M. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2, 647\u0026ndash;656 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosentino, K. \u0026amp; Garc\u0026iacute;a-S\u0026aacute;ez, A. J. Bax and Bak Pores: Are We Closing the Circle? Trends Cell Biol 27, 266\u0026ndash;275 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGavathiotis, E., Reyna, D. E., Davis, M. L., Bird, G. H. \u0026amp; Walensky, L. D. BH3-Triggered Structural Reorganization Drives the Activation of Proapoptotic BAX. Mol Cell 40, 481\u0026ndash;492 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnnis, M. G. \u003cem\u003eet al.\u003c/em\u003e Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J 24, 2096\u0026ndash;2103 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki, M., Youle, R. J. \u0026amp; Tjandra, N. Structure of Bax. 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Nature 596, 583\u0026ndash;589 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Optogenetics, BAX, Anti-apoptosis, Cryptochrome, Mitochondria, MOMP","lastPublishedDoi":"10.21203/rs.3.rs-6222702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6222702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough considerable research has focused on enhancing the apoptotic function of BAX for several decades, inhibition of its functionality remains relatively underexplored, despite intensive BAX activation occurring in various neurodegenerative diseases. Here we present a protein engineering approach to modulate BAX integration into the mitochondrial outer membrane, establishing a tunable strategy for apoptosis inhibition. Utilizing optogenetic methods that employ cryptochrome 2 and its binding partner cryptochrome interacting basic helix loop helix 1, we achieved precise spatial control over BAX localization, a critical determinant of its function. Our results demonstrate that the engineered BAX variant is effectively incapacitated in its apoptotic function while also modulating endogenous BAX activity to enhance cellular resistance to apoptosis. These findings not only advance our understanding of BAX regulation but also offer promising prospects for the development of therapeutic strategies against neurodegenerative and other apoptosis related diseases.\u003c/p\u003e","manuscriptTitle":"Optogenetic Engineering of BAX to Control Mitochondrial Permeabilization and Attenuate Apoptosis in Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 15:05:37","doi":"10.21203/rs.3.rs-6222702/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-04-24T07:38:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-23T06:59:48+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-16T12:38:52+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-04-11T14:58:09+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-04-02T07:38:48+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-04-01T13:06:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-27T00:08:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-03-26T04:35:07+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-03-24T23:38:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T08:52:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0356d2cd-b350-43e4-ae6c-2a65b2c7f195","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46522155,"name":"Biological sciences/Biological techniques"},{"id":46522156,"name":"Biological sciences/Biotechnology/Expression systems"}],"tags":[],"updatedAt":"2025-12-26T08:05:17+00:00","versionOfRecord":{"articleIdentity":"rs-6222702","link":"https://doi.org/10.1038/s12276-025-01605-y","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2025-12-26 05:00:00","publishedOnDateReadable":"December 26th, 2025"},"versionCreatedAt":"2025-04-22 15:05:37","video":"","vorDoi":"10.1038/s12276-025-01605-y","vorDoiUrl":"https://doi.org/10.1038/s12276-025-01605-y","workflowStages":[]},"version":"v1","identity":"rs-6222702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6222702","identity":"rs-6222702","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}