Calcium flow at ER-TGN contact sites facilitates secretory cargo export

Abstract

Ca²⁺ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca²⁺-ATPase SPCA1 and the luminal Ca²⁺ binding protein Cab45. Cab45 oligomerizes upon local Ca²⁺ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca²⁺ flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca²⁺ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), allowing protein-mediated exchange of molecular species such as lipids. Here, we show that the TGN export of secretory proteins requires the integrity of ER-TGN MCS and inositol 3 phosphate receptor (IP3R)-dependent Ca²⁺ fluxes in the MCS, suggesting Ca²⁺ transfer between these organelles. Using an MCS-targeted Ca²⁺ FRET sensor module, we measure the Ca²⁺ flow in these sites in real time. These data show that ER-TGN MCS facilitates the Ca²⁺ transfer required for Ca²⁺-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.

• Secreted proteins, including hormones, growth factors, and extracellular matrix proteins, undergo sorting in the trans-Golgi network (TGN). Although a Ca²⁺-dependent, cargo receptor-independent sorting mechanism in the TGN had been revealed, the source of Ca²⁺ remained unclear due to its nanomolar cytosolic concentrations.

• In this paper, we investigated endoplasmic reticulum-TGN membrane contact sites and demonstrated their role in transient Ca²⁺ transfer during cargo entry into the TGN.

• These findings reveal new insights into the mechanism of sorting at the TGN and unveil tools that will be helpful for researchers in the field.

INTRODUCTION

Protein secretion, a pivotal process crucial for maintaining the integrity and enabling cell−cell communication in multicellular organisms (Uhlen et al., 2015), involves synthesizing secreted proteins in the Endoplasmic Reticulum (ER). These proteins are transported to the Golgi apparatus via COPII-coated tubules and vesicles (Gillon et al., 2012; Barlowe and Miller, 2013; Zanetti et al., 2013; Raote and Malhotra, 2021; Raote et al., 2023). Subsequently, in their journey through the Golgi apparatus, they traverse from the cis- to the trans-Golgi cisterna, ultimately reaching the trans-Golgi Network (TGN) (De Matteis and Luini, 2008; Guo et al., 2014; Kienzle and von Blume, 2014; Di Martino et al., 2019). At the TGN, these proteins undergo sorting and packaging into various transport carriers destined for the cell surface, the endosomal system, or secretory granules in specialized cells (Mostov and Cardone, 1995; Tang, 2001; Stalder and Gershlick, 2020).

The sorting mechanism for soluble secretory proteins in the TGN presents a challenge due to the lack of direct membrane connections, and cargo receptors for these proteins remain elusive (Kienzle and von Blume, 2014; Pakdel and von Blume, 2018; Ramazanov et al., 2021). Research indicates that Calcium (Ca²⁺) is a significant regulator of cargo sorting at the TGN. The secretory pathway ATPase 1 (SPCA1) plays a pivotal role by pumping Ca²⁺ from the cytoplasm into the TGN lumen in an ATP-dependent manner (Missiaen et al., 2007; Sepulveda et al., 2008; Lissandron et al., 2010; Pizzo et al., 2010, 2011; von Blume et al., 2011; Wong et al., 2013b; Kienzle et al., 2014; Lebreton et al., 2021). In response to luminal Ca²⁺ influx, the Golgi resident protein Cab45 undergoes oligomerization, capturing cargo molecules before they are packed into vesicles budding from the TGN (Scherer et al., 1996; von Blume et al., 2012; Crevenna et al., 2016; Deng et al., 2018). Despite the critical role of Ca²⁺ in these processes, the source of Ca²⁺ pumped into the TGN lumen by SPCA1 remains unknown, given the low nanomolar range of cytosolic Ca²⁺ concentrations (Berridge et al., 2003).

The TGN forms membrane contact sites (MCS) with the ER, facilitating non-vesicular inter-organelle communication through lipid transfer (Masone et al., 2019; Venditti et al., 2019a, 2020). These ER-TGN MCSs contain tethering proteins such as vesicle-associated membrane proteins A and B (VAPA and VAPB) and lipid transfer proteins like Oxysterol-binding protein 1 (OSBP1) (Lehto and Olkkonen, 2003). OSBP1, with dual organelle targeting motifs, can bind ER proteins (VAPA and VAPB) and the TGN through its Pleckstrin homology (PH) domain. At the ER/TGN interface, OSBP1 counter-transports PI4P from the TGN and cholesterol from the ER, mediated by its oxysterol-binding domain (OBD) (Kawano et al., 2006; Mesmin et al., 2017; Kumagai and Hanada, 2019).

Recent studies emphasize the significance of lipid transfer in ER-TGN MCSs for regulating protein export from the TGN (Wakana et al., 2021). Beyond lipid transfer, MCSs enable Ca²⁺ transfer between the ER and other organelles, such as the mitochondrion (Kelly, 1985; Pfeffer and Rothman, 1987; Rizzuto et al., 1993, 1998). Hypothesizing that ER-TGN MCSs could provide Ca²⁺ for SPCA1 and Cab45-dependent cargo sorting at the TGN, our research demonstrates that the trafficking of secretory proteins relies on the IP3 receptor (IP3R) in ER membranes and the integrity of ER-TGN MCSs. Additionally, we developed an MCS-specific sensor to measure changes in Ca²⁺ levels within these sites, revealing the essential role of Ca²⁺ flow in cargo export from the TGN, dependent on the tethering between the ER and TGN. This finding addresses an unresolved question in cell biology.

RESULTS

The inhibition of IP3 receptors delays the TGN export of secretory proteins

Our previous work demonstrated that sorting of soluble secretory proteins requires a transient SPCA1-mediated Ca²⁺ influx into the TGN to facilitate the Cab45 oligomerization (von Blume et al., 2012; Crevenna et al., 2016; Deng et al., 2018). However, the cytosolic Ca²⁺ concentrations at steady state are in the low nanomolar range and thus may not provide the amount of Ca²⁺ ions required to promote the sorting of secretory proteins (Crevenna et al., 2016; Pizzo et al., 2011). With the ER being the largest Ca²⁺ store within the cell, we hypothesized that the MCS between the ER and the TGN is a likely candidate to provide Ca²⁺ for sorting at TGN, which could be mediated through an IP3 receptor (IP3R)-dependent mechanism.

To investigate if the release of Ca²⁺ from the ER has an impact on the sorting and export of soluble secretory cargo molecules from the TGN, we analyzed trafficking and secretion of the well-established Cab45-clients: cartilage oligomeric protein (COMP) and Lysozyme C (LyzC) (von Blume 2011; von Blume 2012) in the presence and absence of the IP3R antagonist (2-APB) (Maruyama et al., 1997). We used the Retention Using Selective Hooks (RUSH) system to quantify the trafficking and packaging of COMP or LyzC, respectively, into secretory vesicles in HeLa cells in the presence of DMSO (control) or 2-APB (70 µM) (Boncompain et al., 2012). To this end, HeLa cell lines were transfected with the RUSH constructs containing COMP-EGFP or LyzC-EGFP to analyze intracellular trafficking of the EGFP-fused proteins at different time points after synchronous release from the ER by biotin addition (Figure 1A). We observed simultaneous export of COMP-EGFP from the ER in control and 2-APB treated cells (Figure 1B [0, 20 min]). However, the appearance of cytosolic vesicles (TGN carriers) was significantly delayed in cells treated with 2-APB compared with control cells at later time points (Figure 1, B and C [30, 40, 60 min]).

FIGURE 1:

Inhibition of IP3R activity delays TGN export of secretory proteins. (A) Schematic representation of the RUSH assay with fluorescent-tagged client molecules. (B) Representative immunofluorescence images of the RUSH experiments showing COMP-EGFP transport in HeLa lines treated with DMSO and 70 µM 2-APB (30 min pretreatment of cells with 2-APB and kept in the imaging medium throughout the experiments). HeLa cells were transfected with KDEL-IRES-SBP-COMP-EGFP and fixed at 0, 20, 40, and 60 min after adding 40 µM biotin. Z-stack images (d = 0.2 µm) were analyzed. The arrowheads indicate cytoplasmic vesicles. Scale bars, 10 µm. (C) The numbers of COMP budding vesicles from RUSH experiments with KDEL-IRES-SBP-COMP-EGFP in HeLa lines treated with DMSO and 2-APB were quantified. The cytoplasmic vesicles were counted at each time point by analyzing z-stack images (d = 0.2 µm). The scatter dot plot represents the means ± SD of at least three independent experiments (n > 30 cells per condition). Statistical test, Kruskal–Wallis. (D) Plot representing normalized average fluorescence intensity of COMP-EGFP in cells by FACS at 0, 60, and 120 min in biotin. (E) The plot represents the normalized fluorescence intensity of LyzC-EGFP within TGN (the GALNT1 area defined ROI) in cells treated with DMSO and 2-APB. (F) Western blot showing LyzC-GFP in Hela cells treated with DMSO and 2-APB in cell lysates (30 min pretreatment of cells with 2-APB and kept in the medium throughout the experiment), secreted medium (top), and β-actin as a loading control.

To confirm the observed phenotype on a cell population, we performed RUSH experiments using COMP-EGFP as cargo in HeLa cells treated with DMSO (control) or 2-APB with subsequent fluorescence-activated cell sorting (FACS) analysis. We used this assay to quantify the intracellular accumulation of EGFP-COMP in DMSO or 2-APB-treated cells. Cells were fixed at 0, 30, 60, and 120 min after biotin addition, and 10⁴ cells for each time-point were analyzed by FACS. We calculated the average arithmetical value for the fluorescent intensity of COMP-EGFP obtained from each sample’s green emission fluorescence channel. The arithmetical average values for fluorescence intensity of COMP-EGFP from 2-APB−treated cells were 1.8- and 2.1-fold higher than in control cells after 60 and 120 min after biotin addition, respectively, indicating a more extended residence of the EGFP-COMP inside cells in 2-APB−treated cells confirming our microscopy observations (Figure 1D; Supplemental Figure S1, A and C).

To correlate these results with the actual TGN exit of the cargo molecules, we applied live-cell imaging of the exiting EGFP-tagged cargo molecules in the presence of the GALNT1-BFP TGN marker. We measured time-dependent changes in fluorescence intensity of EGFP-tagged protein within the region of interest (ROI) of TGN defined by the GALNT1-BFP signal. These results showed that the reduction in the number of vesicles in 2-APB-treated cells in the RUSH experiments was consistent with a prolonged residence of cargo in the TGN compared with DMSO-treated cells (Figure 1E). We applied a non-linear regression function of the intensity values on the plot shown in Figure 1D (Supplemental Figure S1E) to quantify this phenotype. In addition, we calculated span values for each curve representing changes in LyzC-EGFP intensity within Golgi ROI. The span was defined as the difference between the fluorescence intensity of EGFP at the starting point and the predicted plateau for each curve, representing a change in fluorescence intensity during the experiment. The span value calculated for LyzC-EGFP expressing cells in the presence of 2-APB exhibited a two-fold decrease compared with control cells (span value for 2-APB and DMSO samples were 0.8 and 1.5, respectively), indicating a significant defect in TGN export of LyzC-EGFP in these cells.

Next, we performed a secretion assay to validate further that the inhibition of the IP3R reduces the secretion of the Cab45 client LyzC from the cells. We generated stable cell lines expressing EGFP-tagged LyzC under a constitutive promoter by lentiviral transduction of HeLa cells. Secretion assays were performed in a complete growth medium, and secreted LyzC-EGFP in the supernatant was immunoprecipitated using GFP trap agarose beads before analysis by Western blotting. The Western blot analysis revealed a 48% reduction in secreted LyzC-EGFP in the 2-APB−treated condition compared with DMSO-treated control cells (Figure 1F).

These data suggested that the activity of IP3 receptors impacts the TGN export and secretion of the secretory proteins COMP and LyzC. Previous work has shown that Golgi-localized IP3 receptors do not affect the SPCA1-dependent Ca²⁺ uptake (Wong et al., 2013b). Therefore, we speculated that Ca²⁺ flow between these organelles might be facilitated by ER-TGN MCS (Ramazanov et al., 2021).

MCS integrity is essential for the TGN export of secretory proteins

VAPA and VAPB proteins are essential in maintaining ER-TGN MCS integrity by tethering ER and TGN. Deleting these proteins from cells reduces contacts between the organelles (Lev, 2010; Phillips and Voeltz, 2016; Venditti et al., 2019b). To examine the role of ER-TGN MCSs in the trafficking of secretory proteins, we performed RUSH experiments. First, we analyzed the trafficking of COMP-EGFP in control and VAPA/B-KO HeLa cells. RUSH experiments were performed, and samples were fixed at different time points after biotin addition. Immunofluorescence images captured at 30, 40, and 60 min after biotin addition showed that compared with control cells, VAPA/B-KO cells exhibited a significant delay in the formation of COMP-EGFP containing post-Golgi vesicles (Figure 2A [30, 40, 60 min]). Consistent with this result, quantifying post-Golgi vesicles at different time points from a randomly selected population of control or VAPA/B-KO cells revealed a decrease in TGN-derived vesicles (Figure 2B).

FIGURE 2:

VAPA/B-KO cells exhibit an impaired TGN export of secretory proteins. (A) Representative immunofluorescence images of RUSH experiments showing COMP-EGFP transport in HeLa WT and VAPA/B-KO cell lines. Cells were transfected with KDEL-IRES-SBP-COMP- EGFP and fixed at 0, 20, 40, and 60 min after adding 40 µM biotin. Z-stack images (d = 0.2 µm) were analyzed. Scale bars, 10 µm. (B) The numbers of COMP budding vesicles were quantified. The cytoplasmic vesicles were counted at each time point by analyzing z-stack images (d = 0.2 µm). The scatter dot plot represents the means ± SD of at least three independent experiments (n > 30 cells per condition). Statistical test, Kruskal–Wallis. (C) The plot represents the normalized average fluorescence intensity of COMP-EGFP in cells by FACS at 0, 60, and 120 min in biotin in HeLa WT and VAPA/B-KO lines. (D) The plot represents the normalized fluorescence intensity of LyzC-EGFP within TGN (the GalNT1 area defined ROI) in cells treated with nontargeting (control) siRNA and siRNA targeting VAPA and VAPB. (E) VAPA and VAPB proteins were expressed in the HeLa cells transfected with control (nontargeting) siRNA, the HeLa cells transfected with siVAPA/siVAPB, and the HeLa VAPA/B-KO line. β-actin was used as a loading control.

We confirmed the delayed export of secretory proteins seen in VAPA/B-KO with RUSH LyzC or COMP, respectively, in control, VAPA/B-KO or VAPA/B siRNA treated cells (Dong et al., 2016) (see immunofluorescence in Supplemental Figures S2A and S2B [0, 20 min]), and quantification (Supplemental Figures S2C and S2D). In parallel, we measured the time-dependent intracellular decrease of COMP-EGFP by FACS in control versus VAPA/B-KO cells. The average intensity values in the GFP channel from VAPA/B-KO cells were 1.5-fold higher than in control cells at 60 and 120 min after biotin addition, indicating accumulation of COMP-EGFP in these cells (Figure 2C; Supplemental Figure S1A and S1C).

To demonstrate that the secretion defect is caused by impaired TGN export, we performed live-cell imaging of the exiting EGFP-tagged secretory proteins in the presence of the GALNT1-BFP TGN marker. We observed time-dependent changes in LyzC-EGFP intensity within the TGN ROI and a 1.6-fold difference in span values in control versus VAPA/B-KO cells. In addition, we observed a prolonged residence of LyzC in the TGN of HeLa cell lines transfected with siRNA to VAPA/B compared with control cells (Figure 2D; Supplemental Figure S1F). The efficiency of the VAPA/B-KO and siRNA knockdown was analyzed by Western blotting (Figure 2E).

To investigate whether this phenotype is specific for certain soluble secretory proteins and does not affect other proteins transported by bulk flow secretion, we measured the time-dependent intracellular decrease of COMP-EGFP and soluble form of equinatoxin (EQ-sol) by FACS in control versus VAPA/B-KO cells and 2-APB treated cells. These data indicated that the secretion of EQ-sol-GFP, a non-specific marker of bulk flow secretion (Deng et al., 2016), is unaffected by VAPA/B-KO (Supplemental Figure S1A–S1C).

These data showed that the TGN export of secreted COMP and LyzC from TGN requires intact MCSs between ER and TGN (Figure 2) and IP3R-dependent Ca2+ release from the ER (Figure 1). These data also suggest that ER-TGN MCS could serve as potential sites for Ca²⁺ transfer.

Targeting Twitch-based FRET sensors to ER-TGN MCS

Our next goal was to test potential Ca²⁺ flow at ER-TGN MCSs directly. These MCSs could serve as hotspots for Ca²⁺ transfer between the organelles. To evaluate possible Ca²⁺ flows in these sites, we used Förster resonance energy transfer (FRET)-based Ca²⁺ biosensors called Twitch. These sensors contain a minimal Ca²⁺ binding moiety derived from the C-terminal domain of troponin C incorporated between mCerulean3 and cpVenuscd (Thestrup et al., 2014). To target the sensors to ER-TGN MCS in living cells, we introduced an amino acid sequence coding the N-terminal region of OSBP1, including a PH domain and an FFAT motif (Figure 3A). As the N-terminal disordered domain of OSBP seems to be crucial for active lipid transport in the ER-TGN MCS (Jamecna et al., 2019), we generated sensors containing a disordered domain (N-PH-FFAT) as well as a sensor without that domain (PH-FFAT) (Supplemental Figure S3A and S3B). We constructed stable HeLa cell lines expressing the MCS-Twitch sensors under a doxycycline-inducible promoter to obtain stable and equal protein expression levels. To investigate the correct localization of the sensors at the TGN, cells were fixed and stained with antibodies recognizing GM130 or TGN46. Immunofluorescence microscopy data confirmed that the MCS-Twitch sensors in HeLa cells are correctly localized (Supplemental Figure S3B and S3C).

FIGURE 3:

The MCS-targeted FRET sensor responds to changes in Ca²⁺ levels at ER/TGN MCS. (A) Schematic representation of OSBP and the Twitch2b Calcium Sensor targeting ER/TGN MCS. The PH domain of OSBP binds to PI4P on the outer leaflet of the TGN membrane, and the FFAT domain interacts with VAPA on the ER membrane. (B) The calibration curve for the Twitch2b sensor used a buffer with increasing concentration of free Ca²⁺ ions, demonstrating a correlation between FRET indices and free Ca²⁺ (see Materials and Methods for a detailed description). (C) Pseudocolor map of FRET index and dot plot (D) representing FRET indices for PH-FFAT-Twitch2b and PH-FFAT-Twitch9x within the TGN ROI of live cells and at steady state (***p < 0.05). (E) Pseudocolor map of FRET index and dot plot (F) representing FRET indices for PH-FFAT-Twitch2b at steady state and treated with 25 µM BAPTA-AM for 20 min (***p < 0.05). (G) Pseudocolor map of FRET index and dot plot (H) representing FRET indices for PH-FFAT-Twitch2b at steady state and treated with 1 µM ionomycin for 20 min (***p<0.05). (I) Pseudocolor map of FRET index and dot plot (J) representing FRET indices for PH-FFAT-Twitch2b at steady state and treated with 1 µM Histamine for 2 min (***p < 0.05). The pseudocolor bar FRET index value is in the range of 0–0.4.

The Twitch2b-MCS FRET sensor detects Ca ²⁺ fluxes

To determine the range of Ca²⁺ signals detectable at the MCS at steady-state (nontreated cells incubated at 37°C), we constructed four MCS-Twitch sensors with different Ca²⁺ affinities (depicted in Supplementary Tables S1 and S2; and Supplemental Figure S3D). For quantitative FRET measurements of Ca²⁺, we calculated the FRET index value in the cell line expressing the respective sensor as an approximation of the FRET/molecule (Grashoff et al., 2010; Kumar et al., 2016). The normalized FRET index values were acquired by measuring FRET intensity, subtracting the background noise and the bleed-through for the two fluorophores, and normalizing them to FRET acceptor intensity. Comparing FRET indices from the MCS-Twitch2b and Twitch9 showed that Twitch 2b elicits a significantly higher FRET index than Twitch9x (Figure 2, C and D). We obtained the same result when we compared the FRET of MCS-Twitch 2b to the other three sensors, MCS-Twitch9x, Twitch7x, and Twitch8x (Supplemental Figure S3D).

Next, we performed a series of control experiments to test if the Twitch2b sensor was responsive to Ca²⁺ perturbations in the cells. First, we determined the FRET values in HeLa cells expressing the MCS-Twitch2b sensor by live-cell fluorescence microscopy in cells treated with DMSO (control) or the Ca²⁺ chelating agent BAPTA-AM. As expected, chelating intracellular Ca²⁺ ions with BAPTA-AM significantly decreased the FRET index of MCS-Twitch2b (Figure 3, E and F). The FRET indices of MCS-Twitch2b after BAPTA treatment were similar to the baseline levels of the low Ca²⁺ affinity MCS-Twitch7x, 8x, and 9x sensors (Figure 3, E and F; Supplemental Figure S3D). Therefore, Twitch2b was selected for all further experiments.

The N-terminal domain of OSBP controls its orientation in the MCS (Jamecna et al., 2019). To test if the N-terminal domain impacts the functionality of MCS-Twitch2b, we compared the Ca²⁺ response of PHFAAT-Twitch2b and NPHFAAT-Twitch2b. We observed that both sensors exhibited similar FRET indices at steady state (Supplemental Figure S3E) and upon ionomycin (Figure 3H, PHFAAT; Supplemental Figure S3F, NPHFAAT) or histamine treatment (Figure 3J, PHFAAT; Supplemental Figure S3G). Therefore, we used PHFAAT-Twitch2b in further experiments.

To quantify the range of free Ca²⁺ concentrations, we calibrated the Twitch2b sensor. To calibrate Twitch2b in live cells, we used HeLa cells that stably expressed CYTO-Twitch2b. We applied a reciprocal dilution of buffers containing increasing ratios of Ca-EGTA/K2-EGTA concentrations, and the free Ca²⁺ ion concentrations were calculated as described in Materials and Methods (Supplemental Figure S4A and S4B). The FRET indexes obtained from Twitch2b at different ratios of Ca-EGTA/K2-EGTA in the calibration experiment allowed us to build a calibration curve demonstrating a strong correlation between the FRET index value and the concentration of free Ca²⁺ (calibration plot shown in Figure 3B). Thus, we developed a powerful tool to measure Ca²⁺ levels in the ER-TGN-MCS. Furthermore, we developed a tool that accurately measures Ca²⁺ levels in the ER-TGN-MCS.

To measure the effects of increased intracellular Ca²⁺ concentrations on the FRET signals from MCS-Twitch2b sensors, we utilized active and passive means of increasing cytosolic Ca²⁺ levels. Treatment of cells with ionomycin, which raises the intracellular Ca²⁺ level, significantly increased the FRET index (Figure 3, G and H). Several signaling pathways, including cell surface receptors, are known to utilize Ca²⁺ ions as second messengers for the downstream signaling (Carafoli, 2002; Dickenson and Hill, 1994; Thillaiappan et al., 2017). In addition, the activation of histamine receptors (H1-receptors) at the plasma membrane causes the activation of PLC, elevating intracellular Ca²⁺ through an IP3-dependent mechanism. To test if the MCS sensor detects these signals, we performed the FRET measurements in HeLa cells expressing PH-FFAT-Twitch2b incubated with either DMSO (control) or after treatment with histamine. (Figure 3, I and J). The data showed an increase in the FRET index – revealing a physiological link between MCS Ca²⁺ levels and the signaling receptor IP3.

The data indicated that the release of Ca²⁺ caused by IP3R stimulation leads to increased Ca²⁺ levels in MCS and that our sensor sensitively detects these changes.

TGN protein abundance and Ca ²⁺ flux at MCS are coupled

Our previous work showed that TGN Ca²⁺ influx is necessary for the TGN export of secretory proteins (von Blume et al., 2011; Kienzle et al., 2014; Crevenna et al., 2016; Deng et al., 2018). Therefore, we hypothesized that there must be a correlation between cargo influx into the TGN and Ca²⁺ flow in the MCS. To test if Ca²⁺ in ER-TGN MCSs is influenced by protein abundance, we treated HeLa cells expressing MCS-Twitch 2b with cycloheximide (CHX) to inhibit de novo protein synthesis. We incubated cells for 1, 2, and 4 h and analyzed FRET signals. We observed a time-dependent decrease in the FRET index of cells treated with CHX. To further demonstrate that this is correlated with cargo abundance in the TGN, we incubated HeLa cells expressing MCS-Twitch 2b at 20°C to arrest secretory proteins in the TGN (Figure 4A) (Matlin and Simons, 1983; Ladinsky et al., 2002). Notably, FRET values were significantly decreased in cells incubated at 20°C (Figure 4B). However, the 20°C block duration for more than 1 h did not affect the average FRET index values (Figure 4C). More importantly, incubation of cells at 37°C to release the 20°C block resulted in a complete recovery of the FRET index (Figure 4, B and C). To validate that the changes in the FRET indices were not due to other factors, cells were incubated at 37° and 20°C in the presence of ionomycin to promote maximal Ca²⁺ influx (Figure 4E). Independent of the incubation temperature, ionomycin treatment led to a recovery of FRET indices, demonstrating that the effects are specific to Ca²⁺.

FIGURE 4:

Ca²⁺ at ER-TGN MCS is coupled to protein trafficking. (A) Schematic representation of experiment and effect on protein trafficking in live cells during 20°C block and cycloheximide (CHX) treatment. (B) Pseudocolor maps of FRET index for PH-FFAT-Twitch2b within Golgi ROI of live cells at described above conditions. The pseudocolor bar FRET index value is in the range of 0–0.4. (C) Dot plot representing FRET index for PH-FFAT-Twitch2b at steady state, after 20°C block for 1 and 2 h and after 10 min recovery at 37°C (***p < 0.05). (D) The dot plot represents the FRET index for PH-FFAT-Twitch2b at steady state, treated with 50 µg/ml CHX for 1, 2, 4 h, and after 1 h 20°C block (***p < 0.05). (E) The dot plot represents the normalized FRET index for PH-FFAT-Twitch2b at steady state after 1 h 20°C incubation and the effect of ionomycin at 37° and 20°C conditions (***p < 0.05). (F) Cartoons depicting PH-FAAT-Twitch2b and the cytosolic control sensor CYTO-Twitch2b. (G) IF images of HeLa lines stably expressing MCS Twitch2b and CYTO-Twitch. (H) The expression of CYTO-Twitch2b was induced by adding 1 µg/ml doxycycline to HeLa-CYTO-Twitch2b cells. FRET measurements were performed in control, 4 h 50 µg/ml CHX treated, or in 1 h incubated cells at 20°C. Images show a pseudocolor FRET map of FRET indices within the cell. The range of FRET indices is between 0 and 0.3. (I) The graph represents the quantification of an average FRET value within a cell of each condition. At least 40 cells were quantified in each condition. While there is no change in average FRET values upon CHX treatment, there is a 10% reduction upon incubation of cells at 20°C. ***p < 0.05. Size bars: 10 µm.

These data supported the idea that newly synthesized protein stimulates the Ca²⁺ flux. Furthermore, we showed that this is specific to the cargo abundance in the TGN. In the current study, we quantify the abundance of Ca²⁺ flux in the ER-TGN MCS, which has remained unknown. The data also suggest that cargo entering the TGN elicits a Ca²⁺ release in the MCS required for sorting secretory proteins into a TGN-derived carrier (Figure 5).

FIGURE 5:

The model depicts the role of ER/TGN MCS and IP3R-dependent release of calcium ions for SPCA1-dependent sorting at TGN. Previously published work showed that SPCA1, Cab45, and SMS1 deficiencies significantly impact the TGN export of secretory proteins. Sphingomyelin synthase 1 (SMS1) controls SPCA1 activity and provides the sphingomyelin for the TGN export carrier. The current study demonstrates that the TGN export of COMP and LyzC requires the integrity of the ER-TGN MCS and IP3R-dependent Ca²⁺ fluxes in these sites. These data suggest that the Ca²⁺ transferred through the MCS is the primary source for SPCA1.

DISCUSSION

In the current study, we identified the source of Ca²⁺ required for secretory cargo export from the TGN. Our work elucidates that Ca²⁺ transfer is facilitated by ER/TGN MCS and IP3-Rs and regulated by protein abundance in the TGN. Our conclusions are based on the following results: TGN export of secretory proteins requires 1) ER-localized IP3 receptors (Figure 1); 2) VAPA and VAPB (Figure 2); 3) we measure Ca²⁺ flow through ER/TGN MCS in real-time (Figure 3); 4) Ca²⁺ signals through ER/TGN MCS can be modulated by the abundance of newly synthesized proteins (Figure 4, B and D); or by 5) arresting proteins in the TGN by incubating cells 20°C and releasing cargo by incubation at 37°C (Figure 4, B and C). With this work, we provide new insights into cellular Ca²⁺ signaling that supports cargo export from the TGN.

Our prior research demonstrated the essential role of SPCA1, the exclusive Ca²⁺ ATPase in the trans-Golgi network (TGN), in sorting secretory cargo within the TGN (von Blume et al., 2011; Kienzle et al., 2014; Deng et al., 2018). Contrarily, Golgi-localized IP3 receptors (IP3Rs) and SERCA in the cis and medial Golgi cisterna did not impact the TGN Ca²⁺ homeostasis (Lissandron et al., 2010; Pizzo et al., 2011; Wong et al., 2013a). Intriguingly, our investigations revealed that Cab45, a luminal Ca²⁺ binding protein, undergoes oligomerization and serves as a scaffold for secreted proteins (von Blume et al., 2012; Crevenna et al., 2016). The oligomerization of Cab45 necessitates millimolar Ca²⁺ concentrations for capturing secreted proteins (Crevenna et al., 2016). However, a significant gap in understanding persisted regarding how SPCA1 facilitates millimolar Ca²⁺ influx from the cytosol into the TGN lumen, given that the “bulk” cytosolic Ca²⁺ concentration hovers around 200 nM (Berridge et al., 2003). The endoplasmic reticulum (ER) serves as the primary Ca²⁺ store within cells, maintaining steady-state Ca²⁺ levels of 1 mM through the action of SERCA, which pumps Ca²⁺ into the ER lumen. IP3 receptors can swiftly release this Ca²⁺ (Camello et al., 2002).

Notably, ER-mitochondria MCS exist, with mitochondrial Ca²⁺ influx levels exceeding cytosolic ranges by more than tenfold (Rizzuto et al., 1998; Csordas et al., 2010). This local increase in Ca²⁺ results from IP3 receptors accumulating in this microdomain, transferring Ca²⁺ from the ER lumen to mitochondria via interaction with the mitochondrial voltage-dependent anion channel 1 (VDAC1) on the mitochondrial membrane and the ER chaperone glucose-related regulated protein 75 (Grp75) facilitating their interaction (Szabadkai et al., 2006).

We propose a similar mechanism at ER/TGN MCS, where an ER microdomain rich in IP3R interfaces with an SPCA1-rich domain in the TGN membrane. Consequently, IP3 receptors release elevated Ca²⁺, which is pumped into the TGN lumen by SPCA1. This hypothesis gains further support from observing that SPCA1 localizes in TGN microdomains overlapping with Cab45 and cargo proteins (Crevenna et al., 2016).

We show that the Ca²⁺ signal in the MCS is triggered by protein abundance in the TGN while it is reduced in its absence. How can the presence of cargo trigger the Ca²⁺ response? Phospholipase β3 (PLCβ3) is predominantly recruited to membranes upon Gβγ subunit activation (Fogg et al., 2001). Research in previous years has shown that activated PLCβ3 localizes to TGN membranes and facilitates cargo export (Diaz Anel and Malhotra, 2005; Anitei et al., 2017). PLCβ3 catalytically hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), generating diacylglycerol (DAG) in the cytoplasmic leaflet of the TGN - Golgi-localized phosphatidylinositol 5 kinase 1 α  (PIP5K1α) sustains PIP2 pools in the TGN (Anitei et al., 2017). Accumulated DAG in the TGN recruits Protein Kinase D for vesicular fission and export (Yeaman et al., 2004; Malhotra and Campelo, 2011; Campelo and Malhotra, 2012). The DAG produced by PLCβ3 also generates IP3, and in our model, soluble IP3 triggers Ca²⁺ release by binding to ER-localized IP3Rs. The released Ca²⁺ from IP3Rs is subsequently pumped into the TGN lumen by SPCA1. How does this cytosolic signaling cascade relate to luminal cargo proteins?

Recent studies have discovered a G-protein coupled receptor (GPCR) within TGN membranes. GPRC5A engages with cargo proteins, activating PLCβ3 and PKD (Di Martino et al., 2019). This signaling cascade potentially governs cargo movement in and out of the TGN. Subsequent research will investigate whether GPRC5A plays a role in the Ca²⁺-dependent sorting and export of cargo. The local arrangement of these components at ER/TGN MCS is expected to spatially organize the involved elements, facilitating precisely timed signaling responses that facilitate TGN export.

MATERIALS AND METHODS

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DNA techniques and plasmid construction

Restriction enzymes for molecular biology were obtained from New England Biolabs. PCRs were performed with a Phusion Polymerase (Thermo Fisher Scientific) and a Mastercycler Nexus (Eppendorf). All plasmids used in this study bear ampicillin resistance for selection in Escherichia coli and are listed in Table 1, where the transgenes and inserts are described. The DNA sequences encoding PH-FFAT/N-PH-FFAT domains of OSBP1 fused with Twitch sensors were integrated into the donor plasmid of the transposon-based piggyBac system for stable transgene-expressing cell lines generation (Li et al., 2013). The piggyBac backbone vector (PB-T-PAF) and PB-RN and PBase were gifts from James Rini, University of Toronto, Ontario, Canada (Li et al., 2013). In brief, to generate PB-T-PAF-PHFFAT-Twitch constructs, the PB-T-PAF vector was linearized with NheI and NotI-HF restriction enzymes (NEB). PH-FFAT/N-PH-FFAT sequences (5′-NheI/3′-AscI) were amplified by PCR from pLJM1-FLAG-GFP-OSBP plasmid (Addgene#134659). The sequences encoding Twitch (Twitch2b/Twitch7x/Twitch8x/Twitch9x) Calcium sensors were obtained from plasmids generously provided by Oliver Griesbeck Lab and amplified by adding corresponding restriction sites (5′-AscI/3′-NotI). All fragments were ligated in the PB-T-PAF backbone. All cloning experiments were conducted using Phusion High-Fidelity Polymerase and T4 ligase (Thermo Fisher Scientific) according to the manufacturer’s instructions. Similar strategies generated VAPA-Twitch2b and CYTO-Twitch2b constructs. The sequences encoding Twitch2b for these constructs were amplified by adding corresponding restriction sites (5′-NheI/3′-AscI). The VAPA fragment was amplified from plasmid coding full-length VAPA protein and was gifted from Pietro De Camilli with the addition of AscI and NotI restriction sites. CYTO-Twitch2b construct was generated from VAPA-Twitch2b by replacing the VAPA sequence with a short stop-codon containing fragment, annealed using the following sequences (5′CGC­GCCAGAGGAGTTTTAAGC3′ and 5′GGCCGCTTAAAACTCCT­CTGG3′). pLenti-LyzC-EGFP for secretion assay lines was generated using gateway cloning reaction by amplifying LyzC-EGFP from plpcx-LyzC-EGFP and cloning into pDONR221 using BP cloning reaction and then subsequently using LR cloning reaction into the destination vector to generate the desired construct. The correct sequence of all constructs was confirmed by DNA sequencing using the SmartSeq Kit from Eurofins Genomics or KECK sequencing (Yale University).

Cell culture and generation of stable cell lines expressing Calcium sensor constructs.

Cell lines were maintained in DMEM (Life Technologies) containing 10% FBS (Sigma 12306C-500ML) at 37°C and 5% CO₂. For transfection, cells were plated in antibiotic-free media 24 h before the procedure. DNA transfections were performed using Lipofectamine 2000 reagent per the manufacturer’s protocol. After 8 h, the media was replaced. Transgene expression was estimated at 48 h after transfection. HeLa lines were used to generate cell lines stably expressing the mentioned transgenes. In brief, HeLa cells at 70% confluency were transfected with PB-T-PAF (with the corresponding transgene), PB-RN, and PBase (total DNA 1.5 µg; at ratio 8:1:1) using Lipofectamine 2000 in OptiMEM-I media. In addition, cells were selected for 48 h with 2 µg/ml puromycin dihydrochloride (Sigma-Aldrich) and for 7 d with 400 µg/ml G418 disulfate salt (A1720-5G, Sigma-Aldrich). Finally, cells were incubated with doxycycline (J63805 Alfa Aesar; 1 µg/ml) for 24 h to induce transgene expression. The generated lines were sorted on BD FACS Aria to exclude resistant cells without transgene expression.

HeLa cell lines stably expressing LyzC-EGFP were generated using lentiviral transduction containing pLenti-LyzC-EGFP construct with followed Blasticidin 8 µg/ml (InvivoGen) selection for 48 h. siRNA transfections were performed using Lipofectamine RNAiMAX according to standard protocol. VAPA/B-KO lines were generated by Pietro De Camilli Lab and published previously (Dong et al., 2016).

RUSH cargo sorting assay and fluorescent microscopy

RUSH assays were performed as described previously (Boncompain et al., 2012; Deng et al., 2018). Studied cell lines were cultured in 6-well plates (Corning, catalog no. 353046,) on glass slides (catalog no. 72290-04, EMS) and transfected with RUSH-COMP-EGFP and RUSH-LyzC-EGFP constructs using Lipofectamine 2000 according to standard protocol. At 24 h posttransfection, cells were incubated with 40 µM d-Biotin (Sigma) ingrown media for different time points (20, 30, 40, 60, and 90 min or without d-Biotin (control). For IF slides, cells were washed once with PBS, fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 10 min, and mounted on 12-mm coverslips (Electron Microscopy Sciences) using ProLong Gold (Thermo Fisher Scientific). Nuclear chromatin was stained by short incubation in 2.5 µM DAPI (Biolegend) solution. EGFP was acquired using a Delta Vision system by imaging z-stacks with a step size of 0.2 µm.

We empirically measured the sizes of objects between 4 and 20 pixels for quantification of vesicles using the Analyze Particles function in ImageJ, which detects vesicular carriers but omits larger objects such as the Golgi. At the same time, small fragmented and isolated Golgi structures could be seen in error. Furthermore, only vesicles of cells expressing the RUSH construct were counted. The Fiji macro count_fixed_vesicles_V1.3, including the Particle Analyzer plug-in by Fiji, was used to determine the number of vesicles (Deng et al., 2018). Kruskal–Wallis one-way analysis of variance was used for comparisons in RUSH experiments.

Cells were cultured in six wells on glass slides for immunostaining and fixed for 10 min with 4% paraformaldehyde. After washing with PBS, cells were permeabilized for 5 min in 0.2% Triton-X 100 and 0.5% SDS in 4% BSA. After washing with PBS, cells permeabilized with Triton-X 100 were blocked with 4% BSA for 1 h. Next, cells were incubated with primary followed by the corresponding secondary antibody for 1 h at room temperature in a blocking buffer in the dark. Slides were washed three times with PBS after incubation with antibody. Glass slides were mounted with ProLong Gold (Thermo Fisher Scientific). Antibodies to TGN46 (AHP500G, Bio-Rad) and GM130 (610822, BD Biosciences) were used at dilution 1:200.

For FACS analysis, cells were fixed with 4% PFA for 10 min. After fixation, cells were washed with ice-cold DPBS and dissociated using a trypsin-EDTA solution. Next, cells were washed with DPRS thrice by centrifugation at 200 × g for 5 min each. FACS analysis was performed using the BD LSRII machine. 10,000 cells were analyzed for each sample. FACS data were analyzed using FlowJo 10.5.3 for Windows.

RUSH protein trafficking analysis

HeLa cells were seeded on Mattek dishes (p35gc-1.5-14-c, Mattek) and cultured at 37°C with 5% CO₂. The next day or the day before imaging, cells were transfected by Lipofectamine 2000 (Invitrogen) with two plasmids expressing GalT-BFP and RUSH COMP or RUSH Lysozyme C (see above for details of the plasmids). After five to eight hours, cells were washed and incubated with DMEM with 10% FBS overnight. Before imaging, cells were washed and briefly incubated with 37°C-warmed DMEM supplemented with 10% FBS and HEPES buffer (Life Technologies, 21063029). Cells were then staged on the microscope as described below. For 2-APB treatments, HeLa cells expressing RUSH cargos were preincubated for 30 min with 70 µM 2-APB, which was kept throughout the experiment.

Live-cell imaging was performed using a spinning disk confocal microscope CSUXfw-06p-01 (Yokogawa) on a Nikon eclipse Ti2 (LWD NA = 0.52) microscope stand with a motorized stage with stage top Piezo. sCMOS camera Photometrics Prim 95B and CFI Plan Apo Lambda 60x oil objective were used. Also, the Oko Lab temperature control system was set to 37°C, and the fluorescence (405 nm and 488 nm) was induced using an Agilent laser combiner. Images were acquired using Nikon Elements. Fluorescence images were taken every three minutes after adding biotin (see above for the methods for RUSH experiments). At each point in time, the Nikon Elements stitched 6 × 5 fields to generate relatively large field images that could accommodate at least 10 distinguished secretion events through 90 or 120 min.

The time course images generated above were imported to Fiji (ImageJ). Images containing individual cells with distinguishable Golgi and RUSH signals were cropped and stored separately. The Golgi masks were generated using GalT-BFP signals (405 nm excitation) and ImageJ Auto-threshold and imported as ROIs. The Golgi ROIs were used to measure mean RUSH intensity in the Golgi at each point in time. First, the mean RUSH intensities were subtracted from the background signals. Then, the maximum mean value of RUSH during the time course of the single-cell images was used to divide the RUSH intensities, thus normalizing the maximal RUSH intensities in the Golgi marker set to 1. The time course images for every cell were analyzed separately and combined to generate the mean RUSH intensity in each time point and its SD, as described in the Figures. Three independently cultured cells were analyzed, and their images were combined to generate the final data.

Secretion assay of LyzC-EGFP

5 × 10⁵ cells stably expressing LyzC-EGFP were plated in a 6-well plate for the secretion assay. After 24 h, cells were pretreated for 30 min with 70 µM 2-APB and DMSO. After that, cells were incubated in a growth medium containing 70 µM 2-APB and DMSO for 1 h. Cells and media were collected separately. Cells were lysed using RIPA buffer. The collected growth media was incubated overnight with GFP-trap beads at 4°C. The following day, the beads were washed 4 times with DPBS, and then protein was eluted from the beads by boiling it in 2X Laemmli SDS sample buffer (Bio-Rad). Cell lysates and IP fractions were analyzed by Western blotting.

Live-cell imaging and FRET analysis

For live-cell imaging, DMEM without phenol red containing 4.5 mg/ml – glucose, 25 mM HEPES, and 2 mM glutamine (Life Technologies) supplemented with 10% FBS. Cells were seeded on glass bottom dishes (D35-14-1.5-N, Cellvis) at a density of 5 × 10⁴ per dish. To induce transgene expression the next day, doxycycline was added at a final 1 µg/ml concentration. After 24 h, doxycycline was removed, and cells were incubated in imaging media for an additional 24 h. ImageJ (National Institutes of Health) was used for basic image processing. All analyses were done using custom-written software (MATLAB R2014a; MathWorks). To manipulate intracellular Ca²⁺ levels as well as induce Ca²⁺ flux following drugs at corresponding concentrations were used: calcium ionomycin (I3909-1ML, Sigma) at 1 µM; 2-APB (100065-100MG, Millipore) at 70 µM, Histamine (H7125-1G, Sigma) 1 µM solution, BAPTA-AM (126150-97-8, Millipore) at 25 µM final concentration. Finally, cells were treated with cycloheximide to inhibit protein synthesis at a concentration of 10 µg/ml (DSC81040-5, Dot Scientific Inc).

FRET sensor calibration experiment.

Free Ca²⁺ calibration solutions were made using Calcium Calibration Buffer Kit #1 (Cat. No. C3008MP, Biotium) according to standard protocol. The HeLa cell line stably expressing CYTO-Twitch2b was used to perform the FRET calibration experiment. 1 × 10⁵ cells were plated into glass bottom dishes (D35-14-1.5-N, Cellvis), and doxycycline was added to the media at a final 1 µg/ml concentration. After 24 h, each plate was washed twice with 10 mM EGTA, 100 mM KCl, and 10 mM MOPS (pH 7.2). To chelate residual Ca²⁺, cells were incubated in 10 mM EGTA, 100 mM KCl, and 10 mM MOPS (pH 7.2) with 1 µM ionomycin for 20 min at RT. 2 ml of stock solutions with free Ca²⁺ concentrations ranging from 0 µM to 39 μM were added to cells, and FRET indexes were measured for each condition.

FRET imaging and analysis

These analyses were performed as previously described (Kumar et al., 2016). High-resolution live FRET imaging was performed on a Nikon Eclipse Ti widefield microscope equipped with a cooled charged-coupled device Cool SNAP HQ2 camera, using a × 63, 1.49 NA oil objective at 37°C. Images were acquired using Micromanager software. Three sequential images with 500 ms exposure time were obtained with the following filter combinations: donor (Teal) channel with 460/20 (excitation filter-ex), T455lp (dichroic mirror-di), and 500/22 (emission filter-em); FRET channel with 460/20 (ex), T455lp (di) and 535/30 (em); and acceptor (Venus) channel with 492/18 (ex), T515lp (di) and 535/30 (em) filter combinations. All filters and dichroic were purchased from Chroma Technology. For data analysis, donor leakage was determined from HeLa cells transiently transfected with Vinculin-Teal, whereas acceptor cross excitation was obtained from Vinculin-Venus transfected cells. For all the calculations, respective background subtraction, illumination gradient, and pixel shift correction were performed, followed by three-point smoothening. The pixel-wise donor or acceptor channel intensity slope versus FRET channel intensity gives leakage (x) or cross-excitation (y) fractions, respectively. FRET map and pixel-wise FRET index for the sensors were determined from FRETindex = [FRETchannel−x(Donorchannel)− y(Acceptorchannel)]/[Acceptorchannel].

ImageJ was used for primary image thresholding. First, the mean FRET index per cell was calculated for each region within the mask. Then, a Student’s t test was performed between the two groups to calculate statistical significance and p-value. At least p < 0.05 was considered significant. All analyses used custom-written software (MATLABR2020b; MathWorks) (Kumar et al., 2016).

siRNA delivery and Western blotting

Knockdown of VAPA and VAPB proteins was performed using siRNA (VAPA: 5′-AACTAATGGAAGAGTGTAAAA-3′; VAPB: 5′-AAGAAGG­TTATGGAAGAATGT-3′) (Wakana et al., 2021). Nontargeting siRNA was purchased from Qiagen (Cat no. 1027281) and used as a negative reference control. The knockdown of VAPA and VAPB proteins was achieved by combined transfection (siVAPA and siVAPB) using LipofectRNAiMAX according to standard protocol. Knockdown efficiency was confirmed by Western blotting of cell lysates in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4 [American Bioanalytical], 150 mM NaCl [American Bioanalytical], 1% Triton X100 [American Bioanalytical], 1% sodium deoxycholate [Sigma-Aldrich], and 0.1% SDS [American Bioanalytical] in Milli-Q water); protease and phosphatase inhibitor (Thermo Fisher Scientific) was added just before extraction. The cell lysate was resolved using SDS–PAGE and transferred to the nitrocellulose membrane (Bio-Rad Laboratories). The membrane was blocked using 5% skimmed milk in TBS with 0.1% Tween 20 (TBST) for 1 h and incubated with the following primary antibodies diluted in 5% milk with TBST overnight at 4°C: Anti-β-actin (1:5000, A5441-.2ML Sigma); Anti-VAPA (1:1000, SAB1402460-100G, Sigma); Anti-hVAPB (1:2000, MAB58551 R&D Systems); anti-GFP (1:1000, 11814460001, Roche). The membrane was washed three times with TBS-T and incubated with HRP-conjugated secondary antibodies (1:5000, 32230/32260; Invitrogen). Data were visualized using chemiluminescence detection on ChemiDoc Touch (Bio-Rad Laboratories).

Graphical data and image design

Graphs were plotted in GraphPad Prism version 9.2.0 for Mac, GraphPad Software, San Diego, California, USA. Images were compiled using Adobe Illustrator 2022 (Adobe Inc. (2022). Adobe Illustrator. Retrieved from https://adobe.com/products/illustrator). Schemes were designed using Biorender software (biorender.com).

Abbreviations used:

Cab45

calcium-binding protein 45

COMP

cartilage oligomeric protein

ER

endoplasmic reticulum

LyzC

lysozyme C

IP3

inositol 3 phosphate

IP3-R

IP3 receptor

MCS

membrane contact sites

OSBP1

oxysterol binding protein 1

ROI

region of interest

RUSH

retention using selective hooks

SPCA1

secretory pathway Ca ²⁺ ATPase 1

TGN

trans-Golgi network

VAPA/B

vesicle associated proteins A and B