PIGBOS-CLCC1 interaction shapes cellular calcium dynamics and energy metabolism

Abstract

PIGBOS is a recently identified 54-amino acid microprotein localized to the mitochondrial outer membrane and implicated in the endoplasmic reticulum (ER) stress response. Here, we identify a previously unrecognized role for PIGBOS in cellular Ca²⁺ homeostasis. Manipulation of PIGBOS expression in HEK293T cells revealed that PIGBOS enhances Ca²⁺ signaling by promoting ER Ca²⁺ release through inositol 1,4,5-trisphosphate (IP₃) receptors and subsequent mitochondrial Ca²⁺ uptake in response to histamine stimulation. In contrast, siRNA-mediated depletion or genetic ablation of PIGBOS markedly attenuated these responses. PIGBOS influenced Ca²⁺ transfer from the ER to mitochondria without affecting direct mitochondrial Ca²⁺ uptake and also promoted store-operated Ca²⁺ entry. Functional analyses demonstrated that the interaction of PIGBOS with the ER-resident chloride channel CLCC1 via its C-terminal region is required for this activity. Network analysis predicted a direct association between PIGBOS and CLCC1, as well as indirect connections with core Ca²⁺ signaling components, including IP₃ receptors, STIM1, Orai1, and SERCA, whose expression was altered upon modulation of PIGBOS abundance. Loss of PIGBOS impaired mitochondrial respiration, reduced ATP production, and increased reactive oxygen species. Together, these findings establish PIGBOS as a key regulator of ER-mitochondrial Ca²⁺ signaling that couples Ca²⁺ dynamics to mitochondrial bioenergetics and cellular stress responses.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12964-026-03027-3.

Background

Calcium ion (Ca²⁺) regulates a broad spectrum of physiological processes, including muscle contraction, neurotransmitter release, fertilization, cell division, survival, and apoptosis [1–6]. Cytosolic Ca²⁺ concentration is tightly maintained within the nanomolar-to-micromolar range through the coordinated actions of ion channels, receptors, transporters, and calcium-binding proteins [7, 8]. The endoplasmic reticulum (ER) serves as the principal intracellular Ca²⁺ reservoir, releasing or sequestering Ca²⁺ in response to cellular cues [9]. Activation of Gq-class G protein–coupled receptors increases inositol 1,4,5-trisphosphate (IP₃) production, which in turn activates IP₃ receptors (IP₃R) to release ER Ca²⁺ ([Ca²⁺]_ER) stores [10]. The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump gradually replenishes [Ca²⁺]_ER, thereby maintaining steady-state levels essential for ER functions such as protein folding [11]. Disruption of [Ca²⁺]_ER homeostasis impairs Ca²⁺-dependent chaperones, leading to protein misfolding and ER stress; if unresolved, prolonged ER stress can culminate in apoptosis [12, 13]. Dysregulation of intracellular Ca²⁺ homeostasis has been implicated in neurodegenerative diseases, cardiovascular disorders, and cancer [14–17]. The ER also forms specialized contact sites with mitochondria known as the mitochondria-ER contacts (MERCs). These junctions contain regions of juxtaposition between ER-mitochondria membranes and are termed as mitochondria-associated membranes (MAMs) that facilitate Ca²⁺ transfer and lipid exchange between the two organelles [18, 19].

Recently, a 54-amino-acid microprotein, PIGB opposite strand 1 (PIGBOS), was identified on the mitochondrial outer membrane, where it interacts with the ER-localized chloride channel CLIC-like 1 (CLCC1) [20]. Deletion of PIGBOS sensitized HEK293 cells to chemically induced ER stress, suggesting a role in ER homeostasis [20]. Although PIGBOS does not influence the physical juxtaposition of the ER and mitochondria and is not a structural tethering component of MAMs, its interaction with CLCC1 [20] likely contributes to ER-mitochondria functional coupling. Notably, CLCC1 knockdown also induces ER stress, characterized by increased chaperone protein GRP78 expression and impaired protein folding [21]. Moreover, amyotrophic lateral sclerosis (ALS) associated CLCC1 mutations disrupt ER luminal ion homeostasis (K⁺, Ca²⁺, Cl⁻), thereby exacerbating ER stress, possibly through ionic imbalance [22]. However, the precise role of PIGBOS in ER function and the cellular stress response remains poorly understood. Given the involvement of CLCC1 in [Ca²⁺]_ER regulation and its interaction with PIGBOS, this complex may represent a critical modulator of Ca²⁺ signaling, particularly in mediating ER-to-mitochondria Ca²⁺ transfer.

In this study, we manipulated PIGBOS expression to investigate its role in Ca²⁺ signaling and mitochondrial function in HEK293T cells. Both overexpression and knockdown or knockout of PIGBOS significantly altered intracellular Ca²⁺ signaling dynamics. These changes were accompanied by differential expression of multiple Ca²⁺ signaling-related genes and alterations in mitochondrial energy production. The interaction between PIGBOS and CLCC1 proved essential, as deletion of PIGBOS’s C-terminal CLCC1-interacting domain attenuated these effects. Notably, CLCC1 knockdown recapitulated the phenotypes observed upon PIGBOS depletion. Collectively, our findings identify PIGBOS as an integral component of the cellular Ca²⁺ signaling machinery, regulating Ca²⁺ homeostasis and mitochondrial metabolism through its association with CLCC1. Communication between organelles via the PIGBOS-CLCC1 axis governs cellular sensitivity to ER stress and, when dysregulated, may contribute to disease pathogenesis.

Results

PIGBOS regulates ER, cytosolic, and mitochondrial Ca²⁺ dynamics

To investigate the role of PIGBOS in cellular Ca²⁺ homeostasis, we expressed FLAG-tagged PIGBOS in HEK293T cells and confirmed its expression by western blotting (Fig. 1A). The mitochondrial localization of the overexpressed protein was verified by confocal microscopy (Fig. S1A). Ca²⁺ signaling was induced using histamine, which activates histamine receptors and triggers the generation of IP₃. The resulting IP₃ binds to IP₃ receptors on the ER, leading to Ca²⁺ release from ER stores. The histamine-induced increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto), measured using jGCaMP7s, was significantly higher in PIGBOS-overexpressing cells than in mock-transfected controls (Fig. 1B). One possible explanation for this enhanced response is an increased [Ca²⁺]_ER pool. To test this, we measured basal [Ca²⁺]_ER in unstimulated cells using GCEPIA1-SNAP_ER, as described in the methods section. Steady-state [Ca²⁺]_ER was significantly higher in PIGBOS-overexpressing cells, indicating that PIGBOS can modulate basal [Ca²⁺]_ER content despite its predominant mitochondrial localization (Fig. 1C).

Fig. 1.

Effects of PIGBOS overexpression on Ca²⁺ dynamics. A Western blot from isolated mitochondrial fractions, confirm elevated PIGBOS protein levels in PIGBOS-transfected HEK293T cells. PIGBOS expression, normalized to VDAC (loading control), is quantified from four independent experiments (bar graph). B PIGBOS overexpression enhances the histamine-induced rise in cytosolic Ca²⁺ relative to mock-transfected controls (N = 4). C Representative confocal images show elevated basal ER Ca²⁺ levels ([Ca²⁺]_ER) in PIGBOS-overexpressing cells. Normalized GCEPIA1 intensity relative to SNAP signal is shown in the adjacent bar graph (N = 5). D Histamine-induced ER Ca²⁺ release is augmented in PIGBOS-overexpressing cells (N = 4). E PIGBOS overexpression increases histamine-stimulated mitochondrial Ca²⁺ uptake, measured with GCEPIA2-mt (N = 4). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (A: Mann-Whitney U test; B-E: unpaired Student’s t-test)

We next analyzed [Ca²⁺]_ER release following histamine stimulation. Direct measurement of [Ca²⁺]_ER with GCEPIA1-SNAP_ER showed that histamine-induced depletion was greater in magnitude in PIGBOS-overexpressing cells, compared with the control cells. Within 30 s of histamine application, normalized fluorescence intensity (F/F₀) decreased by approximately 45% in PIGBOS-overexpressing cells versus ~ 35% in controls, consistent with the enhanced [Ca²⁺]_cyto rise (Fig. 1D). A substantial fraction of the released Ca²⁺ is rapidly taken up by mitochondria at mitochondria-ER contact sites (Ca²⁺- MERCs) to restore the cellular Ca²⁺ homeostasis. Mitochondrial Ca²⁺ uptake, measured using GCEPIA2-mt, indicated markedly increased levels in PIGBOS-overexpressing cells upon histamine stimulation in the presence of extracellular Ca²⁺ (Fig. 1E).

This increase is not attributable to extracellular Ca²⁺ influx, as demonstrated in our previous study, in which cytoplasmic Ca²⁺ uptake was measured in a Ca²⁺-free extracellular buffer using ionomycin as the agonist. The same dataset presented in Fig. S1B shows elevated cytoplasmic Ca²⁺ uptake in PIGBOS-overexpressing cells [23]. Mitochondrial Ca²⁺ uptake in Ca²⁺-free extracellular buffer using histamine as the agonist also showed the same trend of higher mitochondrial Ca²⁺ uptake in PIGBOS-overexpressing cells (Fig. S1C). To verify that the FLAG epitope did not affect the observed results, the experiments were repeated using untagged PIGBOS, cloned into the IRES-mCherry vector (pICherryNeo). Comparable results were obtained with untagged PIGBOS, confirming that the findings were not influenced by the presence of the FLAG tag (Fig. S1D).

To address potential non-physiological effects of overexpression, we examined the effects after siRNA-mediated PIGBOS knockdown and CRISPR-Cas9 knockout (KO) in HEK293T cells. Efficient knockdown of PIGBOS was verified by western blotting. siRNA transfection reduced PIGBOS protein levels by approximately 70% (Fig. 2A). The generation of the KO line is described in the methods and summarized in Fig. S1E. Upon histamine stimulation, PIGBOS-deficient cells displayed the opposite phenotype of overexpressing cells: a smaller [Ca²⁺]_cyto transient (Fig. 2B), lower basal [Ca²⁺]_ER (Fig. 2C), reduced [Ca²⁺]_ER release (Fig. 2D), and diminished mitochondrial Ca²⁺ uptake (Fig. 2E). Similarly, PIGBOS-KO cells exhibited attenuated [Ca²⁺]_cyto elevations and reduced mitochondrial Ca²⁺ uptake following histamine stimulation (Fig. S2A-B; The data shown in Fig. S2A has also been used in Fig. 5C for supporting our hypothesis).

Fig. 2.

PIGBOS knockdown impairs cellular Ca²⁺ dynamics. A Western blot from isolated mitochondrial fraction showing reduced PIGBOS expression following siRNA-mediated knockdown in HEK293T cells. VDAC was used as a loading control. Quantification from independent experiments indicates an approximately 70% decrease in PIGBOS protein levels (N= 4). B Knockdown of PIGBOS attenuates the cytosolic Ca²⁺ response to histamine stimulation. Bar graph shows the mean fluorescence ratio (F/F₀) from four independent biological replicates, measured using jGCaMP7s. C Representative confocal images showing reduced basal ER Ca²⁺ levels in PIGBOS knockdown cells compared with scramble siRNA controls. The bar graph (right) summarizes data from five biological replicates measured using G-CEPIA1-SNAP_ER. D Histamine-induced ER Ca²⁺ release is diminished in PIGBOS knockdown cells relative to scramble siRNA controls (N = 4). E Mitochondrial Ca²⁺ uptake following histamine stimulation is reduced in PIGBOS knockdown cells compared with scramble control (N = 4), measured using GCEPIA-2mt. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (A, E: Mann-Whitney U test; B-D: unpaired Student’s t-test).

Fig. 5.

The C-terminus of PIGBOS is essential for modulating ER Ca²⁺ release and subsequent mitochondrial uptake. A Expression of full-length PIGBOS (WT), but not C-terminally truncated PIGBOS (ΔC-PIGBOS), significantly enhanced histamine-induced cytosolic Ca²⁺ elevations (N = 4). B WT PIGBOS expression also augmented histamine-evoked mitochondrial Ca²⁺ responses, whereas ΔC-PIGBOS expression attenuated them (N = 3). C Re-expression of WT PIGBOS in PIGBOS-KO HEK293T cells restored the histamine-induced cytosolic Ca²⁺ rise (third bar from left), whereas ΔC-PIGBOS failed to rescue the phenotype (fourth bar). Untransfected PIGBOS-KO cells exhibited significantly reduced Ca²⁺ transients (second bar) compared to cells with endogenous PIGBOS (first bar, control WT) (N = 3), as also shown in Fig. S2A. Data represent mean ± SD. n.s., not significant, *P < 0.05, ***P < 0.001 (A-C: one-way ANOVA and Tukey’s test). Note that part of the data set presented in Fig. 5C has also been used to derive Fig. S2A.

Further, a key question came up through our findings was whether the Ca²⁺ signaling imbalance observed in PIGBOS-KO (and knockdown) cells is a cause or a consequence of heightened ER stress sensitivity. To address this, we pharmacologically alleviated ER stress in PIGBOS-KO cells using 4-phenylbutyric acid (4-PBA), a well-established chemical chaperone and ER stress inhibitor, and assessed its effect on both ER stress markers and Ca²⁺ signaling. We first confirmed the efficacy of 4-PBA treatment by measuring levels of Activating Transcription Factor 6 (ATF6), a canonical ER stress marker. ATF6 protein levels were significantly elevated in PIGBOS-KO cells compared to wild-type controls, consistent with heightened ER stress upon PIGBOS depletion. Treatment with 4-PBA markedly reduced ATF6 levels in PIGBOS-KO cells, confirming successful attenuation of ER stress (Fig. S2C). Strikingly, histamine-induced cytosolic [Ca²⁺]_cyto transients in 4-PBA-treated PIGBOS-KO cells were significantly elevated compared to untreated PIGBOS-KO cells and were restored to levels comparable to wild-type control cells (Fig. S2D).

These observations suggest that ER stress is a likely upstream driver of the Ca²⁺ handling defect observed in PIGBOS-KO cells, rather than merely co-occurring with it. We note that formally establishing full causality would ideally require the reverse experiment: rescuing Ca²⁺ signaling and determining whether that alleviates ER stress. However, since PIGBOS depletion leads to broad alterations across multiple Ca²⁺ signaling components at several regulatory levels, selectively and specifically restoring Ca²⁺ flux without confounding other cellular processes is technically not straightforward, and therefore was not pursued here in this study. Taken together, our data support a model in which PIGBOS depletion triggers ER stress, which in turn disrupts intracellular Ca²⁺ dynamics, thereby linking the established role of PIGBOS in ER stress regulation to the Ca²⁺ signaling phenotypes reported here.

PIGBOS influences ER-to-mitochondria Ca²⁺ transfer

Both overexpression and depletion experiments identified PIGBOS as a positive regulator of mitochondrial Ca²⁺ uptake. To assess whether this effect depends on ER-derived Ca²⁺, cells were permeabilized with digitonin in Ca²⁺-free buffer, and ER stores were depleted with thapsigargin (TG) in the presence of ethylene glycol tetraacetic acid (EGTA) (Fig. 3A). Subsequent reintroduction of extracellular Ca²⁺ triggered robust mitochondrial Ca²⁺ uptake in both control and PIGBOS-overexpressing cells, with no significant difference in amplitude (Fig. 3B). These results suggest that PIGBOS does not directly influence mitochondrial Ca²⁺ uptake machinery. Rather, since PIGBOS positively regulates ER Ca²⁺ release (Fig. 1C and D), the observed changes in mitochondrial Ca²⁺ uptake are likely a consequence of altered ER Ca²⁺ availability. Consistent with this, mitochondrial Ca²⁺ uptake was selectively affected when ER-released Ca²⁺ served as the source, but remained unaltered when Ca²⁺ was supplied from extracellular sources. Similar results were observed in PIGBOS-knockdown cells (Fig. S3A).

Fig. 3.

PIGBOS modulates ER–mitochondria Ca²⁺ transfer and alters organellar membrane potentials. A Schematic of the experimental protocol for assessing ER-independent mitochondrial Ca²⁺ uptake in digitonin-permeabilized cells. Sequential steps (1–6)]: (1) thapsigargin (TG) inhibits SERCA, triggering ER Ca²⁺ release through leak channels (2); (3) plasma membrane permeabilization allows EGTA to chelate residual cytosolic Ca²⁺(4); (5) reintroduction of Ca²⁺-containing buffer enables mitochondrial uptake; (6) mitochondrial Ca²⁺ rise is measured using the targeted indicator GCEPIA2-mt. B Mitochondrial Ca²⁺ uptake from the extracellular buffer is unchanged upon PIGBOS overexpression (N = 3). C ER membrane depolarizes more rapidly after histamine stimulation in PIGBOS-overexpressing cells. t_1/2 indicates the time to 50% reduction in fluorescence. Right, quantification of ER membrane depolarization (N = 3). D Histamine-induced ER membrane depolarization is attenuated in PIGBOS-depleted cells relative to scrambled controls (N = 4). E FCCP-induced mitochondrial depolarization monitored by TMRE fluorescence. PIGBOS-overexpressing cells show greater FCCP-induced decay than controls (N = 4), indicating higher basal mitochondrial membrane potential. F PIGBOS knockdown decreases mitochondrial depolarization compared with scrambled siRNA controls (N = 4), suggesting reduced mitochondrial membrane potential. Data are shown as mean ± SD. n.s., not significant, ***P < 0.001 (B-F: unpaired Student’s t-test)

PIGBOS modulates ER and mitochondrial membrane potentials

Given the influence of PIGBOS on ER-mitochondria Ca²⁺ flux, we next examined whether PIGBOS influences the ER and mitochondrial membrane potentials. Using the ER-targeted voltage indicator ASAP3_ER, we measured changes in ER membrane potential (ΔΨ_ER) following histamine stimulation. PIGBOS overexpression led to a faster decrease in fluorescence, indicating accelerated ER depolarization. In control cells, a 50% fluorescence reduction occurred within ~ 20 s, whereas in PIGBOS-overexpressing cells, it occurred within ~ 12 s (Fig. 3C). Conversely, PIGBOS knockdown cells exhibited smaller depolarization responses, consistent with reduced [Ca²⁺]_ER release (Fig. 3D). This implies that higher release of ER Ca²⁺ in PIGBOS-overexpressing cells creates more ER depolarization, and vice versa during PIGBOS knockdown.

Mitochondrial membrane potential (ΔΨ_m) was measured using tetramethylrhodamine ethyl ester (TMRE). Application of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) caused a loss of ΔΨ_m, reflected by a decrease in TMRE fluorescence. PIGBOS-overexpressing cells exhibited a larger fluorescence drop, suggesting an elevated basal ΔΨ_m - a hallmark of enhanced mitochondrial function (Fig. 3E). In contrast, PIGBOS knockdown and KO cells showed a smaller decrease in TMRE fluorescence, indicative of lower basal ΔΨ_m, implying that these cells suffer from compromised mitochondrial health (Fig. 3F, Fig. S3B).

PIGBOS influences store-operated Ca²⁺ entry

Since we observed that manipulation of PIGBOS levels alters ER Ca²⁺ stores and their homeostasis, we next examined whether PIGBOS influences store-operated Ca²⁺ entry (SOCE), which is activated upon depletion of ER Ca²⁺ stores. To assess SOCE, the SERCA pump was inhibited with TG in Ca²⁺-free extracellular solution to deplete ER Ca²⁺ stores, followed by re-addition of 1.8 mM Ca²⁺ to trigger SOCE. In PIGBOS-overexpressing cells, TG elicited a smaller increase in cytosolic Ca²⁺ under Ca²⁺-free conditions, indicating reduced ER Ca²⁺ leak/depletion, but resulted in a significantly enhanced SOCE response upon Ca²⁺ re-addition (Fig. 4A). In contrast, PIGBOS knockdown cells showed no significant change in TG-induced ER Ca²⁺ release, yet exhibited markedly reduced SOCE amplitudes (Fig. 4B). Notably, PIGBOS KO cells displayed increased TG-induced ER Ca²⁺ leak, which was followed by a diminished SOCE response (Fig. S4A).

Fig. 4.

PIGBOS modulates store-operated Ca²⁺ entry (SOCE). A TG-induced ER Ca²⁺ leak and SOCE recordings in HEK293T cells show reduced ER Ca²⁺ leak and enhanced SOCE in PIGBOS-overexpressing cells compared with mock controls (N = 4). B PIGBOS knockdown leaves ER Ca²⁺ leak unchanged but markedly reduces SOCE relative to control cells (N = 4). C Confocal images showing STIM1 and Orai1 puncta formation following TG-induced ER Ca²⁺ store depletion in control and PIGBOS KO cells. D and E Quantification of STIM1 (N = 7–18 cells) and Orai1 (N = 6–18 cells) puncta before and after TG treatment. F Combined plasma membrane fluorescence intensity of STIM1 and Orai1 before and after TG stimulation. Each data point represents a manually defined ROI along the plasma membrane. Data represent mean ± SD. n.s., not significant, *P < 0.05, ***P < 0.001 (A-B: unpaired Student’s t-test; D: one-way ANOVA and Tukey’s test; E-F: Kruskal-Wallis and Dunn’s multiple comparison test)

One possible explanation for the altered SOCE is an alteration in STIM1-Orai1 interactions. To test this, YFP-tagged Orai1 and mCherry-tagged STIM1 were expressed in PIGBOS KO and control cells. The number of STIM1 and Orai1 puncta, reflecting their interaction at ER-plasma membrane junctions, was quantified before and after TG treatment. Notably, upon TG stimulation, PIGBOS KO cells exhibited significantly fewer STIM1 and Orai1 puncta compared to control cells (Fig. 4C-E). Consistently, the combined plasma membrane fluorescence intensity of STIM1 and Orai1 was reduced in PIGBOS KO cells (Fig. 4F), indicating weakened STIM1-Orai1 coupling in the absence of PIGBOS. To determine whether PIGBOS also affects STIM1 and Orai1 expression, we performed western blot analysis. Both STIM1 and Orai1 protein levels increased upon PIGBOS overexpression and decreased following PIGBOS knockdown or knockout (Fig. S4B). Collectively, these findings suggest that PIGBOS positively regulates both the expression and interaction of STIM1 and Orai1, thereby modulating SOCE.

To further test if STIM1/Orai1 overexpression recapitulates the PIGBOS overexpression phenotypes, we measured the [Ca²⁺]_ER release following histamine stimulation. STIM1 overexpressed cells show higher [Ca²⁺]_ER release as compared to mock-transfected controls, thus following the trend of PIGBOS overexpressed cells (Fig. S4C). However, Orai1 overexpressed cells show no significant effect on the [Ca²⁺]_ER release (Fig. S4D). This could be because Orai1 channels are gated exclusively by STIM1 oligomerization following ER store depletion. In the absence of concomitant STIM1 activation, excess Orai1 at the plasma membrane remains largely inactive and does not augment Ca²⁺ flux. Studies also reported that individual knockdown of STIM1 and Orai1 resulted in diminished intracellular calcium mobilization induced by IP₃R agonists or thapsigargin (phenotype similar to PIGBOS knockdown) [24, 25].

PIGBOS-CLCC1 interaction is crucial for Ca²⁺ signaling

Chu et al. previously demonstrated that PIGBOS interacts with CLCC1 via its C-terminal region [20]. To assess the functional significance of this interaction in terms of mediating intracellular Ca²⁺ homeostasis, we generated a C-terminal deletion mutant lacking residues 30–54 (ΔC-PIGBOS) (Fig. S5A), cloned into pICherryNeo backbone. Consistent with previous reports, ΔC-PIGBOS localized normally to mitochondria (Fig. S5B) but failed to enhance histamine-induced cytosolic or mitochondrial Ca²⁺ transients compared with full-length PIGBOS (Fig. 5A-B). We next tested whether ΔC-PIGBOS could rescue the Ca²⁺ signaling defects in PIGBOS KO cells. While full-length PIGBOS restored the histamine-induced [Ca²⁺]_cyto increase in PIGBOS KO cells, ΔC-PIGBOS did not (Fig. 5C), indicating that the C-terminal region of PIGBOS is essential for its effects on ER Ca²⁺ release.

To further probe the role of CLCC1 in cellular Ca²⁺ homeostasis, we reduced its expression by ~ 40% using shRNA (Fig. 6A). CLCC1 knockdown phenocopied PIGBOS depletion, resulting in reduced histamine-triggered [Ca²⁺]_ER release (Fig. 6B), lower basal [Ca²⁺]_ER levels (Fig. 6C), diminished ER depolarization (Fig. 6D), and decreased mitochondrial Ca²⁺ uptake (Fig. 6E). To demonstrate whether overexpression of PIGBOS could rescue the CLCC1 mediated Ca²⁺ defects, we measured the histamine-mediated ER Ca²⁺ release in CLCC1 knockdown cells and observed that PIGBOS was able to rescue the levels of ER Ca²⁺ release (Fig. S6A-B). Together, these results demonstrate that the PIGBOS-CLCC1 interaction is essential for coupling ER and mitochondrial Ca²⁺ signaling.

Fig. 6.

CLCC1 depletion alters histamine-induced ER Ca²⁺release, ER potential change and mitochondrial Ca²⁺ uptake. A Representative western blot obtained from whole-cell lysates showing reduced CLCC1 protein expression relative to vinculin following shRNA-mediated knockdown. The accompanying bar graph shows the densitometric quantification of CLCC1 protein levels (N = 4). B ER Ca²⁺ release following histamine stimulation. CLCC1 knockdown decreases histamine-induced ER Ca²⁺ release compared with control cells. The bar graph summarizes data from four independent biological replicates. C Basal ER Ca²⁺ levels imaged with GCEPIA1-SNAP_ER. Representative confocal images of scramble shRNA-transfected control and CLCC1-knockdown cells are shown. The bar plot summarizes quantified fluorescence intensity from five independent experiments, indicating a significant reduction in basal ER Ca²⁺ content in CLCC1-depleted cells. D ER membrane potential changes in response to histamine. CLCC1-deficient cells show a smaller histamine-induced ER voltage change than scramble controls (N = 3), as summarized in the bar graph. E Mitochondrial Ca²⁺ dynamics following histamine treatment. Histamine-induced mitochondrial Ca²⁺ increase is significantly lower in CLCC1-knockdown cells compared with control cells (N = 3). Data are presented as mean ± SD. *P < 0.05, ***P < 0.001 (A: Mann-Whitney U test; B-E: unpaired Student’s t-test)

PIGBOS is associated with the Ca²⁺ signaling network

To assess whether PIGBOS is a functional component of the cellular Ca²⁺ signaling network, we performed a STRING-based protein-protein interaction analysis. PIGBOS was directly linked to CLCC1, which in turn was predicted to associate with key Ca²⁺ signaling components, including STIM1, SERCA, Orai1, and IP₃R (Fig. S7A). To test these network predictions, we quantified mRNA expression of representative Ca²⁺ signaling genes by RT-qPCR. PIGBOS overexpression increased CLCC1, IP₃R, and SERCA expression, whereas PIGBOS knockdown had opposite effects (Fig. S7B), indicating that PIGBOS influences the transcriptional landscape of the Ca²⁺ signaling pathway. To determine whether PIGBOS influences the expression of the mitochondrial Ca²⁺ uniporter (MCU), the principal channel mediating Ca²⁺ import across the inner mitochondrial membrane, we quantified MCU mRNA levels in both PIGBOS-overexpressing and knockdown cells. MCU transcript levels remained unchanged under either condition (Fig. S7B), suggesting that PIGBOS does not transcriptionally regulate the mitochondrial Ca²⁺ import machinery. PIGBOS KO also showed a similar trend to knockdown cells (Fig. S7C).

PIGBOS modulates mitochondrial metabolism and redox balance

Cytosolic and mitochondrial Ca²⁺ levels critically regulate mitochondrial metabolism and oxygen consumption rate (OCR), with reduced mitochondrial Ca²⁺ ([Ca²⁺]_mito) or [Ca²⁺]_cyto often associated with impaired respiration [26–29]. Given the role of PIGBOS in Ca²⁺ homeostasis, we assessed mitochondrial function using Seahorse metabolic flux analysis. PIGBOS overexpression enhanced basal respiration and ATP production without significantly altering proton leak or spare respiratory capacity (Fig. 7A–B). In contrast, PIGBOS knockdown or knockout decreased all respiratory parameters (Fig. 7C–D; Fig. S8A–B) and increased mitochondrial ROS levels, indicating elevated oxidative stress (Fig. 7E).

Fig. 7.

PIGBOS modulates mitochondrial metabolism and reactive oxygen species (ROS) levels. A, B Overexpression of PIGBOS in HEK293T cells increases basal respiration and ATP production compared to empty vector controls (N = 3–4). C, D Conversely, PIGBOS knockdown reduces basal respiration, ATP production and spare respiratory capacity relative to scramble controls (N = 3–4). E Mitochondrial ROS levels are elevated in PIGBOS knockdown cells compared with controls, whereas PIGBOS overexpression does not significantly alter ROS levels (N = 13). Data are presented as mean ± SD. n.s., not significant; *P < 0.05 (B-D: Mann-Whitney U test; E: Kruskal-Wallis and Dunn’s multiple comparison test)

Discussion

Recent studies have identified numerous functional microproteins, typically under 100 amino acids in length, encoded by small open reading frames (sORFs) [30]. These microproteins regulate essential cellular processes such as ion transport, oxidative phosphorylation, and stress responses [31, 32]. Several localize to the sarco/endoplasmic reticulum (SR/ER) and mitochondria, where they control key functions including Ca²⁺ homeostasis and ATP production [33–36]. In the SR, the microprotein DWORF activates SERCA [37], whereas Phospholamban [38], Sarcolipin [39, 40], Myoregulin [41], and Endoregulin [42] inhibit it. Together, these regulators fine-tune cytosolic Ca²⁺ clearance, [Ca²⁺]_ER levels, and downstream processes such as muscle contraction and apoptosis [43–45]. Mitochondria localized microproteins like ASAP (ATP synthase–associated peptide) [46], BRAWNIN [47, 48], Humanin [49, 50], Mitoregulin [51], and MOTS-c [52] regulate several core aspects of mitochondrial metabolism [53].

In 2019, Chu et al. [20] discovered PIGBOS, a microprotein localized to the mitochondrial outer membrane that interacts with CLCC1 and modulates ER stress. However, the implications of PIGBOS-CLCC1 interaction in the context of ER-mitochondria signaling were not explored. It is possible that PIGBOS acts through its interacting partner, CLCC1. Since ER stress can result from dysregulated [Ca²⁺]_ER homeostasis and CLCC1 contributes to [Ca²⁺]_ER balance [22], we hypothesized that PIGBOS may play a key role in the cellular Ca²⁺ signaling network. Previously, we reported that PIGBOS overexpression in HEK293T cells enhanced histamine/ionomycin-induced intracellular Ca²⁺ rise and mitochondrial Ca²⁺ uptake [23]. In the present study, we provide an in-depth analysis showing that the PIGBOS-CLCC1 interaction governs intracellular Ca²⁺ dynamics, regulating Ca²⁺ transfer from the ER to mitochondria and thereby influencing metabolic coupling. The ER stress-regulatory role of PIGBOS possibly arises from its control over cellular Ca²⁺ signaling, particularly through maintaining ER Ca²⁺ homeostasis.

PIGBOS overexpression elevates basal [Ca²⁺]_ER and enhances histamine-induced Ca²⁺ release, whereas its knockdown or knockout reduces both parameters (Figs. 1 and 2). Mechanistically, PIGBOS sustains ER ionic balance by interacting with CLCC1, which provides counter-ion conductance required for optimum Ca²⁺ transport [22]. Deletion of the CLCC1-binding C-terminal region of PIGBOS abolishes its effect on Ca²⁺ signaling (Fig. 5A-B), and ΔC-PIGBOS fails to rescue the signaling defects in PIGBOS-null cells (Fig. 5C). Similarly, CLCC1 knockdown decreases basal [Ca²⁺]_ER, histamine-induced Ca²⁺ release, and mitochondrial Ca²⁺ uptake, mirroring the PIGBOS knockdown phenotype (Fig. 6B, C and E). These findings indicate that PIGBOS likely affects CLCC1 function and acts through it to modulate [Ca²⁺]_ER dynamics, though whether it does so directly or via intermediary proteins remains to be determined.

Although the precise mechanism of CLCC1-mediated Ca²⁺ regulation requires further investigation, previous studies showed that CLCC1 depletion lowers basal [Ca²⁺]_ER and Ca²⁺ release while increasing steady-state [Cl⁻]_ER and [K⁺]_ER [22]. During [Ca²⁺]_ER release, Cl⁻ efflux and compensatory K⁺ influx maintain charge balance and osmolarity; depletion of CLCC1 may disturb this equilibrium, increasing luminal osmolarity and causing ER swelling, which subsequently hampers proper Ca²⁺ signaling [22]. Consequently, PIGBOS knockdown may produce similar effects by reducing CLCC1 activity. Additionally, PIGBOS overexpression may enhance SERCA function, given that CLCC1 is predicted to interact directly with SERCA (Fig. S7A) and SERCA levels rise upon PIGBOS expression (Fig. S7B), thereby possibly contributing to elevated basal [Ca²⁺]_ER.

We found that PIGBOS enhances ER-to-mitochondria Ca²⁺ transfer without affecting Ca²⁺ uptake from extracellular sources. Deletion of the CLCC1-interacting domain of PIGBOS abolished the effect of Ca²⁺ uptake in the mitochondria or cytosol, confirming the necessity of this specific interaction. Further, overexpression of PIGBOS in CLCC1 knockdown cells rescued the histamine-induced [Ca²⁺]_ER calcium levels, thereby supporting our hypothesis that PIGBOS modulates CLCC1 activity and thereby may contribute to intracellular Ca²⁺ regulation. It is well established that MERCs play a critical role in facilitating IP₃R-mediated Ca²⁺ transfer from the ER to mitochondria. Interestingly, previous studies showed that PIGBOS is not an integral component of MAMs and that its deletion does not alter ER-mitochondria tethering sites [20], a change typically observed upon loss of canonical MAM-tethering proteins. Our findings are consistent with the interpretation that the PIGBOS-CLCC1 complex modulates both global ER Ca²⁺ availability and local Ca²⁺ microdomains at MERCs, collectively shaping IP₃R-mediated Ca²⁺ release and subsequent mitochondrial uptake.

PIGBOS plays a broader role than regulating intracellular ER-mitochondria Ca²⁺ signaling; it also influences SOCE, a key pathway for Ca²⁺ influx in most cells. Cells overexpressing PIGBOS show higher SOCE, whereas its depletion produces the opposite effect. Mechanistically, PIGBOS overexpression increases the protein levels of STIM1 and Orai1, the principal components of the SOCE machinery, and enhances their physical interaction - the two molecular determinants that directly set SOCE amplitude. Conversely, PIGBOS knockdown and knockout reduce STIM1 and Orai1 levels and disrupt their coupling, confirming that PIGBOS is a positive upstream regulator of the STIM1-Orai1 axis. This bidirectional, loss- and gain-of-function symmetry indicates that PIGBOS regulation of SOCE is mediated primarily through its control of STIM1-Orai1 expression and interaction, rather than being a passive consequence of altered ER Ca²⁺ store size. In parallel, PIGBOS appears to suppress constitutive ER Ca²⁺ leak, thereby potentially elevating steady-state luminal [Ca²⁺]_ER - an effect that, if confirmed, would be independent of its transcriptional coordination of the STIM1-Orai1 axis. The precise molecular mechanism by which PIGBOS may suppress ER Ca²⁺ leak channels and coordinate STIM1-Orai1 upregulation remains an important direction for future investigation.

At the systems level, PIGBOS overexpression and depletion altered the expression of CLCC1 and several genes associated with the core Ca²⁺ signaling pathway, indicating that PIGBOS may link local ionic changes to global transcriptional adaptation, possibly through Ca²⁺-responsive transcription factors. Further, MCU levels remain unaffected upon PIGBOS overexpression/knockdown, suggesting that PIGBOS does not directly modulate the mitochondrial Ca²⁺ import machinery. Functional analysis of mitochondria showed that PIGBOS overexpression increases respiration and ATP production, whereas its depletion reduces oxidative phosphorylation and elevates ROS levels. These observations suggest that PIGBOS may function as a microprotein that connects Ca²⁺ signaling to mitochondrial energy metabolism and redox balance through its interaction with CLCC1.

Although CLCC1 has been implicated in several diseases, such as ALS [22] and autosomal recessive retinitis pigmentosa [54], PIGBOS has not yet been associated with any disease, likely due to its recent discovery. Considering its critical role in Ca²⁺ signaling, ER stress, and mitochondrial function, it is reasonable to predict that future studies will reveal its involvement in diseases such as Parkinson’s disease, Alzheimer’s disease, or cancer, where Ca²⁺ signaling, the unfolded protein response (UPR), and mitochondrial functions are disrupted. Therefore, PIGBOS may emerge as a potential drug target for multiple diseases in the near future. In summary (Fig. 8), we demonstrate that PIGBOS coordinates plasma membrane-ER-mitochondria Ca²⁺ communication, coupling ionic balance to bioenergetic output. This work expands the paradigm that small mitochondrial proteins can exert broad regulatory influence on inter-organelle signaling and cellular stress adaptation.

Fig. 8.

Schematic representation of how PIGBOS affects cellular Ca²⁺ homeostasis involving ER-mitochondria-plasma membrane. Model summarizing how PIGBOS coordinates ER-mitochondria-plasma membrane Ca²⁺ signaling. PIGBOS engages CLCC1 via its C terminus to augment IP₃R-dependent agonist-evoked ER Ca²⁺ release, and STIM1-Orai1 coupling to potentiate SOCE, thereby amplifying cytosolic and mitochondrial Ca²⁺ transients and supporting bioenergetic and redox homeostasis. Conversely, PIGBOS loss attenuates IP₃R, STIM1, and Orai1 function, blunts ER-to-mitochondria Ca²⁺ transfer, and promotes ROS accumulation, ATP depletion, and mitochondrial injury (figure created with BioRender)

Limitations

Although we presented a detailed study demonstrating the role of PIGBOS in inter-organelle Ca²⁺ signaling and SOCE, our work has several limitations. All experiments were performed in HEK293T cells, which raises concerns about the generalizability of our findings to other cell types. Since our primary goal was to delineate the molecular mechanism of PIGBOS in intracellular Ca²⁺ flux rather than to characterize cell-type-specific physiological outputs, we believe that our mechanistic insights reflect core features of Ca²⁺ signaling networks. The mechanism we propose, in which the PIGBOS-CLCC1 interaction possibly regulates [Ca²⁺]_ER homeostasis and facilitates IP₃R-mediated Ca²⁺ transfer to mitochondria, is the best fit with our data and existing literature. However, because the precise role of CLCC1 in [Ca²⁺]_ER regulation is not fully understood, further studies are needed to validate this model using the CLCC1 mutant that lacks PIGBOS binding capacity. We identified PIGBOS as a positive modulator of CLCC1, but this requires confirmation by examining how PIGBOS influences CLCC1 channel activity in lipid bilayer. Finally, in vivo validation is essential to establish the translational relevance of our findings. Future studies using PIGBOS KO animal models will be crucial to assess the physiological consequences of impaired PIGBOS-mediated Ca²⁺ homeostasis.

Materials and methods

Cell culture and transfection

HEK293T cells were procured from the National Centre for Cell Science (NCCS), India. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1x antibiotic–antimycotic solution (Gibco #154240-062). Cells were cultured under standard conditions at 37 °C in a cell culture incubator maintaining 5% CO₂ levels and passaged when approximately 80% confluency was reached. For imaging experiments, cells were seeded at equal densities on 35 mm dishes containing glass coverslips.

For transfection, cells were grown to 50–60% confluency and transfected using JetPrime Transfection Reagent (Polyplus #10100051) according to the manufacturer’s instructions. A total of 2–3 µg of plasmid DNA was used per transfection, with equal amounts of each construct when multiple plasmids were co-transfected. For western blotting and RNA isolation, cells were transfected under identical conditions after plating at equal densities in 60 mm dishes. Culture medium was replaced 5 h post-transfection.

In overexpression experiments, control cells were transfected with the empty pcDNA3.1 vector to equalize total DNA content with groups overexpressing PIGBOS-FLAG. For knockdown assays, a scrambled siRNA served as the negative control, and a PIGBOS-specific siRNA (Dharmacon #N-192024-01-0002) was used for targeted silencing. The scrambled siRNA (MISSION siRNA Universal Negative Control #1, SIC001) was purchased from Sigma-Aldrich.

Generation of PIGBOS knockout HEK293T cells

CRISPR–Cas9 constructs were generated by cloning a single-guide RNA (sgRNA) sequence reported by Chu et al. [20], which targets exon 2 of the PIGBOS1 locus, into the PX458 vector backbone (Addgene #48138). HEK293T cells were transfected with the resulting CRISPR-Cas9 plasmids, and GFP-positive cells were isolated 48 h post-transfection by fluorescence-activated cell sorting (FACS) into 96-well plates. Single-cell-derived PIGBOS KO clones were expanded and screened by western blot analysis to confirm loss of PIGBOS expression.

Plasmids

pcDNA3.1(+) was used as the empty vector control for overexpression experiments. The PIGBOS-FLAG plasmid was kindly provided by Dr. A. Saghatelian (Salk Institute). Additional constructs included pCMV G-CEPIA1_ER (Addgene #58215) and pCMV CEPIA-2mt (Addgene #58218) from Dr. M. Iino; GCEPIA1–SNAP_ER (Addgene #172659) from Dr. P. Xu; pGP-CMV-jGCaMP7s (Addgene #104463) from Dr. D. Kim and the GENIE Project; and pCAG-ASAP3_ER (Addgene #195356) from Dr. B. Bloodgood. Orai-YFP and STIM1-mCherry plasmids were kindly provided by Dr. Shmuel Muallem (National Institute of Dental and Craniofacial Research, USA). Full-length PIGBOS and a C-terminal deletion (ΔC) mutant were cloned into pICherryNeo (Addgene #52119). For knockdown experiments, CLCC1 shRNA (Sigma-Aldrich #TRCN0000257146) and a scrambled shRNA control (Addgene #1864) were used.

Mitochondria isolation and western blotting

Mitochondria were isolated as described [55] with minor modifications. Cells were lysed in isolation buffer (75 mM sucrose, 225 mM mannitol, 5 mM HEPES pH 7.4, 1 mM EGTA) with PMSF and protease inhibitors (Sigma #P8340), vortexed 30 min, and centrifuged (1,300× g, 5 min × 3) to remove cellular debris. The supernatant was centrifuged (13,000 × g, 25 min) to pellet mitochondria, which were then lysed in radioimmunoprecipitation assay buffer (RIPA) buffer. Protein concentration was determined by the Lowry method [56]. Samples were resolved on 12% Tricine gels, transferred to PVDF membranes, blocked in 5% bovine serum albumin (BSA), and probed with antibodies against PIGBOS (Abgenex #ABP121, 1:500), CLCC1 (Thermo Fisher #A305-668 A-T, 1:1,000), STIM1 (CST #D88E10, 1:1,000), Orai1 (Novus #NBP1-85463, 1:1,000), ATF6 (CST #65880, 1:1,000), VDAC (CST #4866, 1:1,000), and vinculin (CST #4650, 1:5,000), followed by HRP-conjugated secondary antibodies: anti-rabbit (CST #7074, 1:5,000) and anti-mouse (CST #7076, 1:5,000) and Clarity ECL detection (Bio-Rad #1705060).

Confocal microscopy

Cells were seeded on 18-mm square glass coverslips (Bluestar), transfected the following day, and, at 48 h post-transfection, labeled with 20 nM MitoTracker Red CMXRos (Thermo Fisher #M7512) for 30 min at 37 °C, 5% CO₂. Cells were fixed in 4% paraformaldehyde (Himedia #TCL119), permeabilized with 0.1% Triton X-100, blocked in 5% BSA dissolved in phosphate-buffered saline (PBS) for 1 h, and stained with anti-FLAG primary antibody (CST #14793S, 1:100, overnight at 4 °C) followed by Alexa Fluor secondary antibody (Invitrogen #A11034, 1 h at room temperature); nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and coverslips were mounted in DPX (SRL #88417). Images were acquired using a 60 x oil-immersion objective on an Olympus FluoView 3000 confocal microscope.

For STIM1–Orai1 puncta analysis, cells seeded on 35-mm glass-bottom dishes were co-transfected with STIM1-mCherry and Orai1-YFP, 24 h later were treated with 2 µM thapsigargin (Sigma #T9033) for 5 min or left untreated. They were then imaged pre- and post-treatment using a 60 x oil-immersion objective (excitation: YFP 514 nm; mCherry 561 nm) on an Olympus FluoView 3000 confocal microscope.

Ca²⁺ imaging

HEK293T cells were cultured on 12-mm glass coverslips (Bluestar) and transfected with organelle-specific Ca²⁺ sensor plasmids, the indicated PIGBOS constructs, and siRNAs according to the respective experimental conditions. 48 h post-transfection, cells were washed with Hank’s balanced salt solution (HBSS; 140 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO₃, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 10 mM HEPES, pH 7.4) and transferred to a perfusion chamber (Warner Instruments) continuously supplied with HBSS. Fluorescence imaging was performed using an Olympus IX83 inverted microscope equipped with an sCMOS camera (ORCA-Flash4.0, Hamamatsu Photonics), appropriate dichroic mirrors and filter sets (Chroma Technology Corp.), and an LED light source (CoolLED pE-340fura). Exposure time and light intensity were maintained constant across all recordings. Time-lapse images were acquired at 200–250 ms intervals following agonist or test compound addition.

Cytosolic Ca²⁺ dynamics were monitored using the genetically encoded sensor jGCaMP7s [57]. Ca²⁺ release from the ER was triggered by histamine (100 µM)/ ionomycin (5 µM). For steady-state [Ca²⁺]_ER measurements, HEK293T cells expressing GCEPIA1-SNAP_ER [58] were plated on 35-mm glass-bottom dishes, washed once with HBSS, and incubated with SNAP-Cell 647-SiR (New England Biolabs, #S9106) for 45 min at 37 °C. Following two washes, cells were imaged in HBSS using a 60 x oil immersion objective on an Olympus FluoView 3000 confocal microscope (excitation: 640 nm, emission: 661 nm). CEPIA1 fluorescence intensity was normalized to SNAP fluorescence in each cell to account for expression variability, as described previously [58]. For checking the effect of ER stress on [Ca²⁺]_ER dynamics, PIGBOS KO cells were treated with ER stress inhibitor 4-PBA (Sigma #P21005, 3mM for 12 h), followed by [Ca²⁺]_ER measurement after stimulation with histamine .

Mitochondrial Ca²⁺ levels were measured using the mitochondria-targeted reporter GCEPIA2-mt. For mitochondrial Ca²⁺ uptake assays in permeabilized cells, HEK293T cells co-transfected with GCEPIA2-mt [59], were washed with HBSS and transferred to a perfusion chamber. Images were recorded continuously for 5 min. During the first minute, cells were perfused with HBSS, followed by a Ca²⁺-free, high-K⁺ permeabilization buffer (125 mM KCl, 5 mM NaCl, 2 mM K₂HPO₄, 1 mM MgCl₂, 20 mM HEPES, pH 7.2, supplemented with 5 mM glutamate, 100 µM digitonin, 5 mM EGTA, and 1 µM thapsigargin). Thapsigargin depleted ER Ca²⁺ stores, and EGTA chelated residual Ca²⁺ in the digitonin-permeabilized cells. After 2 min, HBSS containing 1.8 mM Ca²⁺ was reintroduced, eliciting a rapid increase in GCEPIA2-mt fluorescence indicative of mitochondrial Ca²⁺ uptake from the extracellular medium.

Store-operated Ca²⁺ entry (SOCE) was assessed by first depleting ER Ca²⁺ stores using a Ca²⁺-free buffer (140 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO₃, 1 mM MgCl₂, 5 mM EGTA, 5.6 mM glucose, 10 mM HEPES, pH 7.4) containing 0.5 µM thapsigargin. After ~ 3.5 min of ER depletion and passive Ca²⁺ leak, HBSS supplemented with 1.8 mM Ca²⁺ was reintroduced to induce SOCE.

Measurement of ER voltage

ER membrane potential dynamics were monitored using the ER-targeted voltage sensor ASAP3_ER [60]. Upon histamine stimulation, a decrease in fluorescence intensity indicated ER membrane depolarization. Images were captured every 250 ms, and fluorescence traces were analyzed offline using ImageJ and custom MATLAB scripts.

Mitochondrial membrane potential and ROS measurement

Mitochondrial membrane potential was assessed using TMRE. Cells on coverslips were incubated in HBSS containing 200 nM TMRE for 5 min at room temperature in the dark, followed by two washes with HBSS. Time-lapse imaging was performed (excitation: 562 nm; emission: 641 nm), and 1 µM FCCP was added after 20 s to induce mitochondrial depolarization.

Mitochondrial ROS was measured using MitoSOX™ Red (Thermo Fisher #M36008). Cells were incubated with 2.5 µM MitoSOX in HBSS for 30 min at 37 °C with 5% CO₂, washed twice with HBSS, and imaged on an Olympus IX83 microscope using an mCherry filter set.

Image analysis and quantification

For Ca²⁺ imaging experiments, individual cells were manually delineated as regions of interest (ROIs) in ImageJ following background subtraction. Fluorescence intensity values across time were extracted using the Time Series Analyser plugin and processed in MATLAB (R2020b, MathWorks). The baseline fluorescence immediately preceding agonist application was defined as F₀. Ca²⁺ transients were identified using the cusum algorithm, and fluorescence changes were normalized as F/F₀. The half-time (t_1/2) was computed as the time to reach 50% of maximum or minimum fold change relative to F₀.

STIM1–Orai1 puncta were quantified after background subtraction using the Analyze Particles function in ImageJ (3–100 pixels, circularity 0.1–1.0). Data were expressed as puncta per µm². For plasma membrane fluorescence analysis, ROIs were manually defined, and fluorescence intensities from YFP and mCherry channels were summed to assess STIM1-Orai1 colocalization before and after thapsigargin treatment. Figures were generated in MATLAB and finalized in Adobe Illustrator (Adobe Inc.).

mRNA expression analysis

Cells were washed with ice-cold PBS, and total RNA was extracted using TRIzol reagent (Takara #9108). The aqueous RNA phase was isolated following chloroform extraction, precipitated with isopropanol, and washed with 70% ethanol. cDNA was synthesized using the cDNA Conversion Kit (Bio-Rad #170–8891). Quantitative real-time PCR was performed using 200 nM of each primer (listed in Supplementary Table 1). Gene expression was normalized to β-actin, which served as an internal reference.

Mitochondrial respiration analysis

Mitochondrial function in HEK293T cells was measured using the Seahorse XF96 Analyzer (Agilent Technologies) according to the manufacturer’s protocol. Approximately 5 × 10⁴ cells per well were seeded onto Seahorse XF96 cell culture plates and equilibrated in assay medium (DMEM supplemented with 2 mM glutamine, 1 mM pyruvate, and 10 mM glucose). The Seahorse XF Cell Mito Stress Test was performed using sequential injections of oligomycin (1.5 µM), FCCP (1 µM), and a mixture of rotenone and antimycin A (0.5 µM each). Oxygen consumption rate (OCR) was recorded and normalized to protein content for intersample comparison.

Statistical analysis

All experiments were repeated at least 3 times in three different passage numbers of cells, unless otherwise stated. In case of calcium imaging experiments, ROIs analyzed from independent biological replicates (at least ~ 10–15 cells per replicate) were averaged and assessed for their normality of distribution and variance. The Jarque-Bera test was used to determine the normality of the data. The equality of variance of the data was tested using the F-test. For datasets showing normal distribution and equal variance, unpaired Student’s t-test was performed to compare between two groups while one-way ANOVA followed by Tukey’s test was used to compare more than two groups. To analyze the non-parametric data, Mann-Whitney U test was used to compare between two conditions while Kruskal-Wallis test followed by Dunn’s multiple comparison test was performed to compare more than two conditions. All the statistical tests were performed using MATLAB (R2020b, MathWorks) software. Data are presented as mean ± SD (Standard Deviation). Statistical significance is denoted as P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Abbreviations

ATF6

Activating Transcription Factor 6

ATP

Adenosine Triphosphate

Ca²⁺

Calcium

[Ca²⁺]_cyto

Cytoplasmic Calcium; [Ca²⁺]_ER-ER Calcium; [Ca²⁺]_mito-Mitochondrial Calcium

CEPIA

Calcium-Measuring Organelle-Entrapped Protein Indicators

ER

Endoplasmic Reticulum

FCCP

Carbonyl Cyanide-4-(trifluoromethoxy)Phenylhydrazone

GRP78

Glucose-Regulated Protein 78

HEK293T

Human Embryonic Kidney 293T cells

IP₃R

Inositol 1,4,5-Trisphosphate Receptor

KO

Knockout

MAMs

Mitochondria-Associated Membranes

MCU

Mitochondrial Calcium Uniporter

OCR

Oxygen Consumption Rate

Orai1

Calcium Release-Activated Calcium Channel Protein 1

PIGBOS

PIGB Opposite Strand

ROS

Reactive Oxygen Species

SERCA

Sarco/ Endoplasmic Reticulum Ca²⁺ ATPase

SOCE

Store-Operated Calcium Entry

STIM1

Stromal Interaction Molecule 1

TG

Thapsigargin

UPR

Unfolded Protein Response

VDAC

Voltage-Dependent Anion Channel

Authors’ contributions

SA and AKB conceptualized the study and designed the research; SA performed all experiments and analyzed the data; SA and AKB wrote the paper; AKB secured funding.

Funding

The work was funded by the intramural fund provided by the Department of Biotechnology, IIT Madras.

Data availability

The datasets obtained and analyzed for this study will be made available from the corresponding author upon a reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have agreed to publish this manuscript.

Competing interests

The authors declare no competing interests.