Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression

Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment.

In mammals, four different genes code for at least six distinct acid-sensing ion channel (ASIC) subunits: ASIC1a 1 and ASIC2a 2,3 and their splice variants ASIC1b 4,5 and ASIC2b 6 , ASIC3 7 and ASIC4 8,9 . Functional ASICs are homoor hetero-trimeric assemblies of individual subunits 10 . They are activated by a drop in extracellular pH and desensitize during sustained acidification 11 . ASICs are members of the degenerin/epithelial Na + channel (DEG/ENaC) superfamily and share about 25% sequence identity with ENaC subunits 12 .
In heterologous expression systems, ASIC1a, ASIC1b, ASIC2a, and ASIC3 form functional homomeric channels 1,[3][4][5]7 , while ASIC2b and ASIC4 do not 6,8,9 . Whereas ASIC2b contributes to functional heteromeric channels 6 , mammalian ASIC4 does also not contribute to functional heteromeric channels, because it apparently does not change the electrophysiological properties of other ASIC subunits, when co-expressed 8 . Thus, ASIC4 is not a bona fide ASIC. It has, however, been reported that, in heterologous expression systems, ASIC4 down regulates the expression of ASIC1a and ASIC3 13 .
There is compelling evidence that ASIC1a, ASIC2a, ASIC2b and ASIC3 contribute to functional ASICs in the plasma membrane of neurons [14][15][16][17][18][19][20][21][22] . ASIC1b-containing ASICs have not been unequivocally identified in neurons, but the presence of ASIC1b in the plasma membrane of a subpopulation of sensory neurons is likely 4,5 . In contrast to all other ASICs, function and location of the ASIC4 protein are unknown. ASIC4 has been cloned from neuronal tissue and its mRNA is faintly expressed all over the brain with highest abundance in pituitary gland 8 . Transgenic reporter mice confirmed strong expression of ASIC4 in pituitary gland and revealed restricted expression in other neurons, including a subpopulation of interneurons and cerebellar granule cells. It is possible that in some, but not all, of these cells ASIC4 is co-expressed with ASIC1a and modulates its expression 23 . It has been reported that ASIC4 is present in the plasma membrane of CHO cells, when heterologously expressed 13 . Thus, although current evidence suggests that ASIC4 is present at the plasma membrane, subcellular location and trafficking of ASIC4 are not well understood.
In this study, we investigated the subcellular location of ASIC4, heterologously expressed in COS-7 and HEK293 cells. We consistently found that ASIC4 mainly localizes to vacuoles related to early endosomes. We found that a conserved amino-terminal domain was important for accumulation in early endosome-related vacuoles. Moreover, we identified a carboxyl-terminal di-arginine motif that retained ASIC4 in early endosome-related vacuoles and prevented its passage to late endosomes. In contrast, we could not detect plasma membrane expression of ASIC4. Collectively, our results show that heterologously expressed ASIC4 mainly resides in an intracellular compartment related to early endosomes.

Results
ASIC4 accumulates in early endosome-related vacuoles. Individual ASIC subunits show a topology with a large extracellular domain, relatively short intracellular amino-and carboxyl-termini and two transmembrane domains 24 . We fused ASIC4 and, for comparison, ASIC2a at their cytoplasmic amino-termini to GFP (GFP-ASIC4 and GFP-ASIC2a, respectively), transiently transfected them into COS-7 cells, and examined their subcellular distribution by confocal laser scanning microscopy. When ASIC1, ASIC2 and ASIC3 are over-expressed in heterologous cells they predominantly localize in the ER [25][26][27][28] . In agreement, GFP-ASIC2a showed a reticular distribution pattern associated with a slight membrane staining, suggesting a predominant location in the ER (Fig. 1a). In stark contrast, GFP-ASIC4 mainly accumulated in large vacuolar-like structures (Fig. 1a). In addition, it usually showed a perinuclear staining, suggesting that GFP-ASIC4 partially localized to the ER. Transfection of GFP-ASIC4 in HEK293 cells revealed a similar accumulation in vacuolar-like structures (see Supplementary Fig. S1 online). We examined cells after different times of transfection (12,24 and 48 h) to investigate whether GFP-ASIC4 might accumulate in the vacuolar structures after passage through a different cellular compartment. But already after 12 h, GFP-ASIC4 showed the typical vacuolar distribution pattern (see Supplementary Fig. S1 online). ASIC4 fused to GFP at its carboxyl-terminus (ASIC4-GFP) showed an identical distribution (see Supplementary Fig. S1 online), excluding that the position of GFP affected the distribution pattern of ASIC4. Moreover, transfection of COS-7 cells with untagged ASIC4 and staining cells with an anti-ASIC4 antibody revealed an identical accumulation of ASIC4 in large vesicles, excluding that the GFP induced the formation of these vesicles (Fig. 1b). Importantly, the antibody did not stain such vacuoles in untransfected control cells (Fig. 1b). Location in vacuolar structures suggests that over-expression of ASIC4 leads to fusion of vesicles and in accumulation in large endocytic structures. Resembling these findings, it has previously been reported that transient receptor potential mucolipin-1 (TRPML1), a resident protein of lysosomes 29 , when over-expressed in HeLa cells localizes in vacuolar structures containing lysosomal markers 30 , showing that fusion of vesicles is not an indication for mislocalization of an over-expressed channel.
To exclude that over-expression of mRFP-Rab5 resulted in formation of vesicular endosome-like structures or in mislocalization of ASIC4, we transfected GFP-ASIC4 or untagged ASIC4 in COS-7 cells and stained with an antibody against an endogenous marker protein of early endosomes, early endosomal antigen 1 (EEA1). In both cases this revealed a strong overlap of staining for ASIC4 and the marker for early endosomes (Fig. 3), confirming that over-expressed ASIC4 predominantly resides in large vacuolar-like structures that are related to early endosomes.
Over-expression of ASIC4 traps endocytosed proteins in large early endosome-related vesicles. We wondered whether the large vacuolar-like structures that formed upon over-expression of ASIC4 are bona fide early endosomes or some specialized organelles that served to deposit ASIC4. To address this question we investigated the passage of low-density lipoprotein (LDL) along the endocytic pathway in cells over-expressing ASIC4. We incubated HEK293 cells, which were transiently transfected with mRFP-Rab5 or Lamp1-RFP, with acetylated LDL (AcLDL) labelled with AlexaFluor488. In cells not over-expressing ASIC4, AcLDL appeared in early endosomes after 10 and 30 min as revealed by overlap of fluorescence with mRFP-Rab5 in these cells (Fig. 4a). After 2 and 3 h, AcLDL had reached lysosomes as revealed by overlap of fluorescence with Lamp1-RFP. In contrast, in cells over-expressing Cerulean-ASIC4, AcLDL accumulated in the large early-endosome related vesicles, where they co-localized with ASIC4 (Fig. 4b), and even after 3 h there was no overlap of fluorescence with Lamp1-RFP (Fig. 4b). This result identifies ASIC4-expressing endosomes as bona fide endosomes of the endocytic pathway and suggests that over-expression of ASIC4 and formation of vesicles trapped endocytosed LDL in these early-endosome-related structures.
The amino-terminus is important to direct ASIC4 into an early endosome-related organelle. Having identified early endosome-related vacuoles as the primary site of ASIC4 expression, we used the distinct distribution patterns of ASIC2a and ASIC4 to identify the parts of ASIC4 that are relevant for its location in these organelles. First, we constructed chimeras, in which we replaced either the intracellular amino-terminus of ASIC4 by the corresponding part of ASIC2a (chimera ASIC4-Nterm2a) or its intracellular carboxyl-terminus Scientific RepoRts | 5:18242 | DOI: 10.1038/srep18242 (chimera ASIC4-Cterm2a). The chimeras were fused to GFP. Similar to ASIC4, chimera ASIC4-Cterm2a localized in large vacuolar-like structures that co-stained with mRFP-Rab5 (PCC = 0.74 ± 0.04, n = 5; Fig. 5a,c and Supplementary Fig. S2 online). In contrast, chimera ASIC4-Nterm2a had a reticular distribution pattern and did not co-stain with mRFP-Rab5, similar to ASIC2a (PCC = 0.27 ± 0.08, n = 5; Fig. 5a,c and Supplementary Fig. S2 online), identifying the N-terminus of ASIC4 as necessary for its location in vacuolar structures.
We then did inverse chimeras. The chimera, in which we replaced the amino-terminus of ASIC2a by the amino-terminus of ASIC4 (chimera ASIC2a-Nterm4), localized to vesicular structures, which co-stained with mRFP-Rab5 (PCC = 0.57 ± 0.05, n = 5; Fig. 5b,c and Supplementary Fig. S2 online), indicating that the ASIC4 amino-terminus is sufficient to direct this chimera into early-endosome-related vesicles. The smaller size and number of these vesicles, however, indicates that this chimera did not fully reproduce the trafficking of ASIC4. In contrast, the chimera, in which we replaced the carboxyl-terminus of ASIC2a by the carboxyl-terminus of ASIC4 (ASIC2a-Cterm4), retained the reticular distribution of ASIC2a and showed no overlap of red and green fluorescence when co-expressed with mRFP-Rab5 (PCC = 0.18 ± 0.11, n = 5; Fig   The first eighteen amino acids of the N-terminus are important for the anterograde transport of ASIC4. To confirm the role of the cytoplasmic amino-terminus for trafficking of ASIC4, we successively truncated it. When we removed the first eighteen amino-terminal amino acids (GFP-ASIC4-M18), the channel had a reticular distribution pattern (Fig. 6a), similar to ASIC2a. Stainings with anti-PDI antibody revealed co-localization, suggesting that GFP-ASIC4-M18 indeed localized to the ER (Fig. 6a). Thus, the first eighteen amino acids are important for the anterograde transport of ASIC4 and their truncation leads to a retention of the channel in the ER. A truncation of the first thirty amino acids (GFP-ASIC4-M30) including a di-leucine motif at position 29/30 (see below) resulted in the same reticular distribution pattern as truncation of the first eighteen amino acids (Fig. 6b). Co-stainings with the anti-PDI antibody confirmed a localization of GFP-ASIC4-M30 in the ER (Fig. 6b). These results confirm an important role of the amino-terminus of ASIC4 for its location in early endosome-related organelles.
Mutation of a C-terminal di-arginine motif at position 478 changes the distribution pattern of ASIC4. ASIC4 contains two di-leucine motifs at its cytoplasmic termini, which often mediate endocytosis of membrane proteins 34 , one at its amino-terminus (LL29/30; Fig. 7a) and one at its carboxyl-terminus (LL519/520; Fig. 7b). To investigate whether the di-leucine motifs of ASIC4 are relevant for its vacuolar distribution pattern, we substituted them by alanines (GFP-A4LL29AA and GFP-A4LL519AA, respectively) and assessed the effect of these substitutions on ASIC4 localization in COS-7 cells (Fig. 7a,b). Examination with confocal laser scanning microscopy revealed that cells expressing GFP-A4LL29AA or GFP-A4LL519AA showed an accumulation in vacuolar structures, similar to ASIC4 wild-type (Fig. 7a,b). Co-expression of the mutants with mRFP-Rab5 confirmed their accumulation in early endosome-related vacuoles (PCC = 0.91 ± 0.02 and 0.78 ± 0.08, respectively, n = 5; Fig. 7a,b,d and Supplementary Fig. S3 online). In contrast, they did not co-localize with Lamp1-RFP, the marker Top, in COS-7 cells expressing GFP-ASIC4, staining with an antibody against the early endosomal antigene 1 (EEA1, red) identifies the same organelles as GFP fluorescence. Bottom, co-labelling of ASIC4 and EEA1 in cells expressing untagged ASIC4 (green). Strong overlap of staining for ASIC4 and EEA1 in both cases confirms that over-expressed ASIC4 predominantly localizes to vacuolar structures that are related to early endosomes. Scale bars: 20 μ m.

Discussion
Our study shows that ASIC4 has a subcellular location, which is unique among heterologously expressed ASICs: it mainly resides in endosome-related vacuoles. Location of ASIC4 in large vacuolar organelles was striking but consistently observed, in two different cell systems, and with and without a GFP-tag. It is likely that vacuoles formed because of the over-expression of ASIC4. We considered that location in an early endosome-related organelle is a consequence of aggregation of over-expressed ASIC4. In this case, however, we would have expected that the vacuolar-like structures were related to the ER and not to early endosomes. Our assay testing endocytosis of LDL also identified ASIC4-expressing vacuoles being within the endo-lysosomal pathway. Furthermore, truncation of only 18 amino-terminal amino acids resulted in a reticular distribution pattern as for ASIC2a and mutation of the carboxyl-terminal di-arginine motif resulted in the appearance of ASIC4 in late endosomes. Both results are unexpected if ASIC4 formed aggregates in the ER. They rather suggest that ASIC4 specifically accumulates within  To the left of the scheme, a representative image shows that the mutant (GFP-A4RR478AA) accumulated in smaller vesicles than ASIC4 wild type. Below the scheme, co-expression with mRFP-Rab5 (early endosomes) or with mRFP-Rab7 (late endosomes) showed clear overlap, while there was only slight overlap with Lamp1-RFP (lysosomes). Scale bars: 20 μ m. (d) Colocalization of ASIC4 with mRFP-Rab5, mRFP-Rab7 and Lamp1-RFP was quantified by Pearson´s correlation coefficient. n = 5 representative images; one image of each condition is shown in (a-c) and the other four images in Supplementary Fig. S3 online. PCC values of the different marker proteins were compared with Rab5 by Student's t-test and revealed a significantly higher co-localization with mRFP-Rab5 than Lamp1 for all three mutants. For the mutant of the di-arginine motif (A4-RR478AA), colocalization with mRFP-Rab7 was not significantly different from co-localization with Rab5; ***p < 0.001. early endosomes. In summary, we conclude that endosomal location of ASIC4 in COS-7 and HEK293 cells was not a simple artefact due to aggregation or over-expression.
The amino-terminus of ASIC4 had a pivotal role for forward-trafficking of ASIC4 from the ER into early-endosomes. It was not only necessary for location of ASIC4 in early-endosome related vacuoles (Figs 5 and 6) but was also sufficient to direct ASIC2a to early-endosomes (Fig. 5). In addition, it appears to be responsible for the formation of large vacuolar-like structures.
Cytoplasmic amino-termini of ASIC1, ASIC2a and ASIC3 are approximately 40 amino acids long; the amino-terminus of ASIC4, however, is 25 amino acids longer 8 and this amino-terminal extension is highly conserved in orthologs from other species 36 . It is also well conserved in ASIC1b 5 , where deletion of the domain increases current amplitudes 5-to 10-fold in Xenopus oocytes 5,37 , suggesting that it impairs surface expression. In zebrafish, ASIC4 has two paralogs, zASIC4.1 and zASIC4.2. zASIC4.1 is a proton-gated ion channel, whereas zASIC4.2 is insensitive to protons 36 . Similar to ASIC1b, deletion of the amino-terminal domain of zASIC4.1 increases current amplitudes and surface expression about 10-fold 38 . The amino-terminal domain of ASIC4 contains several lysine residues and it has been proposed that ASIC4 gets polyubiquitinated at these lysines 13 , which may lead to enhanced degradation and reduced surface expression. Lysines are not present in the amino-terminal domain of ASIC1b, however, indicating that the presence of lysine residues and polyubiquitination cannot fully explain the role of this domain in surface expression of ASICs. Moreover, in our study we found no evidence for location of ASIC4 in lysosomes. Our results rather suggest that in ASIC4 the amino-terminal domain is important for forward trafficking into an early-endosome-related compartment. Although the exact role of this domain may be different in different ASICs, the previous observations that deletion of the domain dramatically increases surface expression of ASICs is consistent with an important role in trafficking.
In addition to the amino-terminal domain, we found evidence that a C-terminal di-arginine motif has a role in retaining ASIC4 in early-endosomes and preventing further trafficking into late endosomes/lysosomes (Fig. 7). The presence of chimeras ASIC4-Cterm2a and ASIC2a-Nterm4, which contain the ASIC2 C-terminus lacking the C-terminal di-arginine motif, in Rab5-positive vesicles suggests that this motif has a more subtle role in localizing ASIC4 to early endosome-related vesicles than the amino-terminal domain. Di-arginine motifs are known to retrieve some membrane proteins to the ER 35 . Retrieval is mediated by interaction with the coat protein complex I (COPI). Somewhat reminiscent of this situation, the di-arginine motif at position 478/479 of ASIC4 seems to retrieve the channel to early endosome-related vacuoles. Whether the di-arginine motif of ASIC4 interacts with COPI has to await further studies. Recently, a di-arginine motif in the proximal carboxyl-terminus of ASIC1a has been shown to interact with the adaptor protein complex and to mediate constitutive endocytosis of ASIC1a 39 .
Another feature of ASIC4 that fits with its location within the endo-lysosomal pathway is its high glycosylation, which is characteristic for proteins of acidic organelles like lysosomes. Most ASICs contain two to four N-glycans, whereas ASIC4 has eight consensus sequences for N-glycosylation most if not all of which are used 40 . Together, these results suggest that ASIC4 might routinely passage through or even reside in intracellular organelles that are related to early endosomes. Based on the expression of its RNA, ASIC4 has highest expression in pituitary gland 8,23 . At present we cannot exclude that ASIC4 localizes in its native cells in an unknown organelle not present in COS-7 or HEK293 cells and that early endosome-related vacuoles in heterologous cells are a surrogate of this organelle. In the future, it will therefore be important to identify the subcellular location of ASIC4 in cells that express it endogenously.
It should be emphasized that we cannot rule out that a small fraction of the total ASIC4 pool localizes to the plasma membrane. While mutation of two di-leucine motifs, which enhance endocytosis of other membrane proteins 34 , had no effect on the distribution of ASIC4, this is no strong evidence against plasma membrane expression of ASIC4.
An intracellular location would explain why the function of ASIC4 remains unknown. Whereas all other ASICs assemble into proton-activated channels and physiological functions are emerging for them 41 , activating stimuli and physiological functions of ASIC4 are unknown 8,9 . Previously, we also found no evidence that ASIC4 assembles into functional heteromeric ASICs 8 . Others, however, have reported that ASIC4 down regulates the expression of other ASICs 13 . Retention by ASIC4 of other ASIC subunits in an intracellular organelle could reconcile these findings. In the future it will therefore be important to investigate whether co-expression of ASIC4 with other ASICs changes their subcellular location.
In Xenopus oocytes, zASIC4 reaches the plasma membrane 38 . Since oocytes are a permissive expression system, we speculate that zASIC4 reaches the oocyte plasma membrane because their control systems are less stringent than that of HEK293 or COS-7 cells, used in the present study. In zASIC4-expressing oocytes, removal of extracellular divalent cations and low pH elicit a small sustained current, which is unselective for cations and insensitive to amiloride 38 . Thus, ASIC4 seems to mediate a current that is atypical for an ASIC. If ASIC4 had an intracellular of fluorescence overlap. (b) To the left of the scheme, a representative image illustrates that truncation of the carboxyl-terminus of GFP-ASIC4 at position 477 (GFP-ASIC4-C477), including the di-arginine motif at position 478,479, resulted in predominant localization in small vesicles. Below the scheme, GFP-ASIC4-C477 overlapped with fluorescence from Lamp1-RFP (lysosomes) but not from mRFP-Rab5 (early endosomes) or mRFP-Rab7 (late endosomes). Scale bars: 20 μ m. (c) Colocalization with mRFP-Rab5, mRFP-Rab7 and Lamp1-RFP was quantified by Pearson´s correlation coefficient. n = 5 representative images; one image of each condition is shown in (a,b) and the other four images in Supplementary Fig. S4 online. PCC values of the different marker proteins were compared with Rab5 by Student´s t-test and revealed a significantly higher colocalization with mRFP-Rab5 than Lamp1 for the truncation GFP-ASIC4-C518 but the opposite for truncation GFP-A4-C477; ** p < 0.01, *** p < 0.001. location, current evidence, thus, suggests that it could carry a monovalent cation current. Cations possibly involved include Na + , K + and protons. At present, we can only speculate on the function of this conductance, but studies with isolated ASIC4-expressing endosomes or studies investigating pH of such endosomes might give first hints.
Collectively, our results show that in heterologous expression systems ASIC4 localizes to endosome-related vacuoles. While many questions regarding its function and location in its native environment remain open, it should be considered that ASIC4 is a resident ion channel of an intracellular compartment.
Heterologous cells were transiently transfected using X-treme gene 9 transfection reagent (Roche, Mannheim, Germany) or FuGene6 (Promega, Mannheim, Germany) according to manufacturers´ protocols. For antibody staining, transfection of single constructs and co-localization studies with organelle markers, cells were grown on 13 mm glass coverslips and transfected with 1 μ g of each construct. For endocytosis assay, HEK293 cells were transfected with 2 μ g of each construct. Cells were incubated for 24 h after transfection before staining, fixation with 4% paraformaldehyde, or endocytosis assays with Alexa Fluor ® 488 AcLDL.
Immunofluorescence. 24 h after transfection, coverslips were washed three times with PBS and fixed with PBS containing 4% paraformaldehyde for at least 10 min at room temperature. Cells were either directly mounted in Mowiol or Fluoromount-G (Southern Biotech, Biozol Diagnostica, Eching, Germany) or further used for antibody staining. For this purpose, cells were washed twice with PBS and permeabilized by incubation with PBS containing 0.3% Triton-X-100 for 20 min at room temperature. Cells were washed three times with PBS and incubated with blocking solution containing 10% FBS for 1 h. Coverslips were transferred to a humidified chamber and incubated with the first antibody (1:200 dilution) in blocking solution overnight at 4 °C. Cells were washed three times with PBS and incubated with Alexa Fluor ® 633-conjugated anti-rabbit or Alexa Fluor ® 635-conjugated anti-mouse secondary antibody (1:500 dilution) for 1 h at 37 °C. After two washing steps, coverslips were dried and mounted in Mowiol.
For antibody stainings using anti-EEA1 or anti-ASIC4, cells were washed three times with PBS and permeabilized with PBS containing 2% normal goat serum (NGS; Pan Biotech, Aidenbach, Germany) and 0.1% Triton-X-100 for 10 min at room temperature (RT). Cells were incubated with the first antibody (1:100 dilution) in PBS containing 2% NGS for 1 h at RT. Afterwards, cells were washed three times with PBS, blocked in PBS containing 2% NGS for 10 min at RT and incubated with either Alexa Fluor ® 488-conjugated anti-rabbit, Alexa Fluor ® 633-conjugated anti-rabbit, or Alexa Fluor ® 635-conjugated anti-mouse secondary antibody (1:500 dilution) for 1 h at RT. To stain the nuclei, cells were incubated with PBS containing DAPI (1:5000 dilution) for 5 min at RT prior to washing them three times with PBS and mounting them in Fluoromount-G.
Fluorescence imaging was performed with a LSM 700 confocal microscope (Carl Zeiss, Jena, Germany) with 40× oil-immersion objective, 4× line averaging, 200 mHz scanning speed and pinhole set to 1 Airy unit.
Quantification of co-localization. Co-localization was quantified using Pearson´s correlation coefficient (PCC), which measures the pixel-by-pixel covariance in the signal levels of two images 42 . PCC values range from 1 to − 1. Two images whose fluorescence intensities are perfectly, linearly related have a value of 1 and two images whose intensities are perfectly, but inversely, related have a value of − 1. Probes that are uncorrelated with one another are expected to have a PCC value close to zero 42 .
PCC was determined with the Zen Blue Software 2012 (Zeiss). Background in the GFP-and RFP-channels were chosen manually for each image by using the pipette tool and PCC was then calculated automatically by the software. At least 5 different, representative images from 2-3 independent transfections were analysed. Bar diagrams in Figs 5, 7 and 8 represent results from five representative images for each condition; the corresponding images are shown in the figures and in the supplementary Figs 2-4. Data are expressed as means ± SEM. Student′s t-test was used to compare experimental groups; p values < 0.05 were considered as statistically significant. Images, for which PCC was not determined, are representative for images of at least three independent transfections.