The Calcium Channel Mucolipin-3 is a Novel Regulator of Trafficking Along the Endosomal Pathway

Keywords: autophagy, EGFR, endosomes, mucolipin, TRP

MCOLN3 localizes to the endosomal pathway in human epithelial cells

We addressed the distribution of MCOLN3 in the human retinal pigmented epithelial cell line ARPE19 (35), which has been extensively used as a model system in multiple studies (36,37). ARPE19 cells are polarized, possessing a highly specialized endosomal system, and express endogenous MCOLN3 as revealed by quantitative Reverse Transcription-Polymerase Chain Reaction (Q-RT-PCR) analysis (Figure 7).

The complete open reading frame of MCOLN3 was isolated from a human kidney complementary DNA (cDNA) library and cloned into the pMonomeric-GFP-C1 vector to produce a chimera that expresses green fluorescent protein (GFP) at the N-terminus of MCOLN3 (GFP-MCOLN3). We established that the addition of the GFP tag did not affect the channel activity or specificity of MCOLN3 (data not shown). Transient expression of GFP-MCOLN3 in ARPE19 cells revealed that the protein mainly localizes to the plasma membrane and intracellular vesicles (Figure 1A).

To determine the subcellular localization of GFP-MCOLN3 in more detail, cells were immunostained with antibodies against specific compartments of the endosomal pathway (Figure 1A and B). We found that 60% of the GFP-MCOLN3 positive vesicles also contained Hrs (59.84% SD ± 7.3, n = 707). Colocalization with other early endosomal markers such as EEA1 (10.6% SD ± 1.5, n = 365) and sorting nexin-2 (SNX2) (7.98% SD ± 3.14, n = 653) was much lower (Figure 1B). These data indicate that GFP-MCOLN3 localizes to a subfraction of early endosomes that are starting to mature into late endosomes.

We also found a high incidence of colocalization of GFP-MCOLN3 with late endosomal/lysosomal markers such as lysobiphosphatidic acid (LBPA) (52.91% SD ± 12.21, n = 1203), CD63 (61.43% SD ± 9.6, n = 623) and Lamp-1 (50.34% SD ± 9.5, n = 1913) (Figure 1A and B). In contrast, no colocalization was found with the Golgi marker GM130 (data not shown).

To corroborate these data we also made a functional fusion of the red fluorescent protein mCherry to the amino-terminus of MCOLN3 (Cherry-MCOLN3). We found that Cherry-MCOLN3 extensively colocalizes with GFP-Hrs in vesicular structures (Figure 1C). These data show that GFP-MCOLN3 is mainly located to early and late endosomes, although we cannot rule out that a fraction of the protein is present in lysosomes.

It was previously described that transiently expressed MCOLN3 remains trapped in the endoplasmic reticulum (ER) unless coexpressed with MCOLN1 or MCOLN2 (5); however, we did not observe retention of MCOLN3 in the ER in ARPE19 cells. To solve this discrepancy, we compared the distribution of transiently expressed MCOLN3 between control and MLIV fibroblasts. As seen in Figure S1, MCOLN3 localized to plasma membrane and intracellular vesicles (many of which were positive for CD63) in both cell types, indicating that MCOLN1 is not necessary for transport of MCOLN3 to the endosomal system. Although we cannot discard the possibility that endogenous MCOLN2 is sufficient to promote exit of MCOLN3 from the ER in MLIV cells, this seems improbable as the levels of recombinant MCOLN3 were over 10 000-fold higher than the levels of endogenous MCOLN2 as measured by quantitative RT-PCR (data not shown).

Overexpression of MCOLN3 causes enlargement of Hrs-positive endosomes

Next we sought to determine the effect of MCOLN3 overexpression on the endosomal pathway. In order to achieve high levels of protein expression, we generated recombinant adenoviruses expressing either GFP-MCOLN3 (Ad. GFP-MCOLN3) or GFP alone (Ad. GFP). As seen in Figure 2, infection of ARPE19 cells with the adenovirus expressing GFP did not cause measurable changes in the distribution or morphology of Hrs-positive vesicles, which showed a similar pattern to the one found in uninfected cells. In contrast, infection with Ad. GFP-MCOLN3 caused a dramatic enlargement of Hrs-labeled structures that seemed to accumulate close to the perinuclear region of the cell (Figure 2). Both Hrs and GFP-MCOLN3 clearly colocalize to the same enlarged early endosomes induced by MCOLN3 overexpression (see inset in Figure 2). Therefore, we observed remarkable alterations in the morphology of the endosomal pathway in cells overexpressing GFP-MCOLN3.

Overexpression of MCOLN3 affects EGFR trafficking

To analyze whether the accumulation of Hrs-positive endosomes induced upon GFP-MCOLN3 overexpression correlates with defects in trafficking of proteins along the endosomal pathway, we monitored delivery of EGF and EGFR from the plasma membrane to lysosomes. EGFR is a prototypical member of the receptor tyrosine kinase family and its activation and trafficking have been extensively characterized. Ligand binding results in dimerization (38), activation and autophosphorylation of the receptor followed by the subsequent binding and phosphorylation of downstream signaling proteins (39). Termination of EGFR signaling occurs via endocytosis and delivery of the receptor to lysosomes for degradation.

We first examined the fate of the internalized EGF over time. ARPE19 cells were infected with adenovirus expressing GFP or GFP-MCOLN3. Forty hours after infection, cells were incubated with Alexa-EGF for 10 min and chased for 1, 2 or 3 h. In control cells the disappearance of internalized EGF was essentially complete after 3 h (Figure 3A). In cells overexpressing GFP-MCOLN3, however, a substantial amount of internalized EGF could still be detected at this time (Figure 3A). EGF remained trapped in enlarged GFP-MCOLN3-positive structures at 3 h after internalization (Figure 3B). Furthermore, ≅70% (67.7% SD ± 5.5, n = 836) of the vesicles positive for EGF and MCOLN3 also contained Hrs, suggesting that they correspond to early endosomes (Figure S2). These experiments indicate that increased levels of MCOLN3 impair EGF degradation and cause accumulation of internalized EGF into aberrant early endosomes.

To corroborate our data we also monitored degradation of EGFR after ligand stimulation. We stimulated cells with EGF for different periods of time and determined the amount of remaining EGFR by immunoblot analysis. Interestingly, we observed that EGFR degradation was considerably delayed in GFP-MCOLN3 expressing cells when compared to cells expressing GFP alone (Figure 4A). Quantitative analysis of three independent experiments revealed that GFP-MCOLN3 overexpression resulted in a 40% increase in the fraction of EGFR that remained undegraded after 4 h of EGF induction compared with control cells (Figure 4B). Therefore, our results suggest that MCOLN3 has a role in sorting and/or degradation of activated EGF receptors.

Cells overexpressing MCOLN3 also showed evident accumulation of ubiquitin at enlarged Hrs-positive endosomes (Figure 5). These data indicate that MCOLN3 overexpression not only delays EGFR degradation but may also have a broader effect in the trafficking of ubiquitinated proteins along the endosomal pathway.

Increased levels of MCOLN3 do not affect EGFR internalization

To determine whether the effect of overexpressing GFP-MCOLN3 on the downregulation of EGFR was due to a change in the internalization of EGFR, ARPE19 cells overexpressing GFP-MCOLN3 or GFP alone were tested for their ability to internalize Alexa-EGF. We did not find any noticeable difference in the amount of internalized Alexa-EGF in cells overexpressing GFP-MCOLN3 or GFP as measured by confocal immunofluorescence microscopy (Figure 3A), indicating that overexpression of MCOLN3 has little effect on ligand-induced endocytosis of EGFR.

To confirm these data, we also monitored EGFR internalization by using 125I-labeled EGF as ligand (Figure 6). Mock- and adenovirus-infected cells expressing GFP-MCOLN3 were serum-starved, and stimulated with 125-IEGF for different periods of time. Endocytosis of EGFR was very fast in ARP19 cells and by 5 min over 50% of the label was intracellular. Quantitative analysis of three independent experiments revealed no statistically significant differences in the amount of 125I-EGF internalized after ligand stimulation in MCOLN3 expressing cells compared with control cells (Figure 6). Taken together, our data suggest that MCOLN3 inhibits downregulation of EGFR by affecting delivery of the receptor to lysosomes for degradation.

Depletion of endogenous MCOLN3 accelerates EGFR degradation

The decreased EGFR degradation caused by overexpression of MCOLN3 prompted us to investigate whether depletion of endogenous MCOLN3 might also have an effect on the delivery of EGFR to lysosomes. To deplete endogenous MCOLN3, we transfected ARPE19 cells with specific siRNAs. Because antibodies that recognize endogenous MCOLN3 are not commercially available, the efficiency of MCOLN3 siRNA was assessed by Q-RT-PCR. As seen in Figure 7A, we achieved close to 90% reduction in the levels of MCOLN3 messenger RNA (mRNA) after 72 h transfection with MCOLN3 siRNA when compared with cells transfected with control nontarget siRNA.

The kinetics of EGFR degradation was examined at 72 h after transfection with nontarget or MCOLN3 siRNAs. Interestingly, we observed that the rate of EGFR degradation was enhanced at all time points after ligand stimulation in cells treated with MCOLN3 siRNA compared with cells treated with control siRNA (Figure 7B). Quantitative analyses of three independent experiments revealed over 40% reduction in the amount of remaining EGFR at 90 and 120 min after ligand stimulation in MCOLN3-depleted cells when compared with control cells (Figure 7C). Our results confirm a role of MCOLN3 in the trafficking of EGFR to lysosomes.

Abnormalities at the endosomal pathway induced by overexpression of MCOLN3 cause defective autophagic degradation

Recent evidence has shown that defects in the endosomal pathway affect autophagic degradation (19,40–42). Autophagy is a crucial clearance mechanism that prevents accumulation of protein aggregates and abnormal organelles (43–45). The last step of autophagy requires fusion of autophagosomes with the endosomal system to guarantee the degradation of the autophagosome's content. Autophagosomes can fuse with lysosomes to produce autolysosomes, but they also undergo fusion with earlier parts of the endocytic pathway (46–49). The pre-autolysosomal compartments containing both autophagic and endocytic material are known as amphisomes (50).

To determine whether the alterations of the endosomal pathway caused by MCOLN3 overexpression also affected autophagic degradation, we analyzed the distribution of LC3 in cells transiently expressing GFP-MCOLN3. LC3 is considered to be one of the most specific markers for autophagosomes. Under normal conditions the majority of LC3 is cytosolic; however, after autophagy induction LC3 is lipidated and recruited to the autophagosome's membrane. As expected, LC3 was mostly cytosolic in nontransfected ARPE19 cells, although occasional small LC3-labeled vesicles could be observed (Figure 8A). In contrast, cells expressing GFP-MCOLN3 showed a dramatic accumulation of LC3-positive structures (see arrow in Figure 8A). Colocalization experiments determined that over 75% (76.47% SD ± 10.3, n = 764) of the LC3-positive vesicles also contained GFP-MCOLN3 while 55% (54.62% SD ± 10.36, n = 557) were positive for LC3, GFP-MCOLN3 and CD63 suggesting that they correspond to amphisomes (Figure 8B). In addition, 15% (14.9% SD ± 9.3, n = 164) of the LC3-positive vesicles contained CD63 but not GFP-MCOLN3; these likely correspond to autolysosomes (Figure 8B). Therefore, the majority of LC3 accumulates at the endosomal pathway suggesting that fusion of autophagosomes with endosomes/lysosomes is not blocked by MCOLN3 overexpression but degradation is impaired.

Autophagy can also be monitored by immunoblot, as the lipidated form of LC3 (also known as LC3II) migrates faster than the nonlipidated LC3 (LC3I) during SDS-PAGE. We prepared lysates from mock-infected cells and from cells infected with adenovirus expressing GFP or GFP-MCOLN3. As seen in Figure 8C, a robust increase in the level of LC3II was observed in cells expressing GFP-MCOLN3, confirming that MCOLN3 causes alterations in the autophagic process. Quantification of four independent experiments showed a 20-fold increase in the LC3II/LC3I ratio in MCOLN3-overexpressing cells (Figure 8D). We observed considerable variation in the LC3II/LC3I ratio among different experiments (varying from a minimum of 11-fold increase to a maximum of 38-fold increase). This is probably due to variability in the level of MCOLN3 overexpression; however, it was clear that MCOLN3 caused a dramatic increase in the levels of LC3II. We also observed a significant accumulation of LC3-positive vesicles in cells infected with Ad. GFP-MCOLN3 when compared with cells infected with Ad. GFP as revealed by confocal microscopy (data not shown).

To confirm that increased levels of LC3II after MCOLN3 overexpression were due to impaired autophagosome degradation, we starved cells expressing Ad. GFP or Ad. GFP-MCOLN3 to further induce accumulation of autophagosomes, then allowed cells to recover for 20 min in nutrient-rich medium to permit autophagosome degradation. Quantification of the total number of autophagosomes per cell revealed that after the 20-min recovery period only 22% (n = 93) of the cells expressing GFP showed more than 20 autophagosomes. In contrast, 59% (n = 71) of the cells expressing GFP-MCOLN3 contained more than 20 autophagosomes after the same recovery period (Figure 8E). Therefore, these results suggest that MCOLN3 overexpression inhibits autophagic degradation. Although we cannot discard that MCOLN3 also enhances autophagosome formation, we did not observed increased levels of beclin-1 in MCOLN3-expressing cells (data not shown).

Altogether, our data reveal that MCOLN3 localizes to the endosomal pathway and is required for efficient sorting of cargo proteins and autophagosomes to lysosomes for degradation.

Overexpression of MCOLN3 alters endosomal pH

To gain additional insight on the function of MCOLN3 at the endosomal pathway we addressed whether MCOLN3 overexpression might alter endosomal pH. The reason for this is that MCOLN3 is a Ca2+ channel and release of Ca2+ has been linked to endosomal acidification (51) (see discussion).

The kinetics of endosomal acidification was measured in control and MCOLN3-expressing cells by fluorescence ratio imaging (52). Cells were simultaneously loaded with fluorescein- and Alexa555-conjugated dextrans. Because fluorescein fluorescence decreases at acidic pH, while Alexa555 fluorescence is pH independent, the ratio of green (fluorescein) to red (Alexa555) fluorescence is indicative of the pH in endocytic vesicles. As shown in Figure 9A endocytic vesicles appear mostly red after 1 h dextran internalization in noninfected cells or cells infected with a control adenovirus, suggesting that at this time point the dextrans have reached an acidic compartment and much of the green fluorescence has been lost. In contrast, most of the green signal is still present in MCOLN3-expressing cells indicating that overexpression of MCOLN3 inhibits proper endosomal acidification.

In a control experiment we determined that MCOLN3 overexpression does not affect trafficking of dextran along the endosomal pathway, as the degree of colocalization of dextran with early endosomal (EEA1) and late endosomal/lysosomal (Lamp-1) markers at different time points was the same in control and MCOLN3-expressing cells (Figure S3).

To convert data to absolute values of pH we measured the fluorescein/Alexa555 ratios and fit them to a curve constructed by calculating ratios in permeabilized cells equilibrated with calibration solutions (Figure 9B). Quantification of many different random fields of cells confirmed that endosomal pH is clearly higher in cells overexpressing MCOLN3. Thus, after 25 min dextran internalization, average endosomal pH was close to 5.5 in noninfected cells (5.38) or in cells infected with a control adenovirus (5.30), whereas the average pH was over 6 (6.20) in MCOLN3-expressing cells (Figure 9C). Therefore, our data suggest that MCOLN3 function is required for efficient acidification of endosomal vesicles. In addition, endosomal acidification is known to be crucial for endosomal maturation and vesicle fusion thus explaining the defective sorting of proteins along the endosomal pathway caused by MCOLN3 overexpression.

Recombinant DNA constructs and adenovirus preparation

The full-length cDNA of human Mucolipin-3 (MCOLN3; GenBankTM/EMBL/ DDBJ accession number NM_018298) was obtained by PCR amplification from a human kidney cDNA library (Clontech Laboratories, Inc.), followed by in-frame cloning into HindIII-EcoRI sites of pmGFP-C1 and pmCherry-C1 vectors, or HindIII-BamHI sites of pCDNA3.1/Hygro(+) vector. Constructs were confirmed by DNA sequencing. GFP-Hrs construct was a kind gift of Silvie Urbe (University of Liverpool, Liverpool, United Kingdom). Control adenovirus and adenovirus expressing GFP, GFP-MCOLN3 or untagged-MCOLN3 were prepared, amplified, and purified by Welgen, Inc.

Cell culture, transfection, siRNA knockdown and adenoviral infection

ARPE19 cells (American Type Culture Collection) were grown at 37^C in a 1:1 mixture of DMEM and Ham's F12 media supplemented with 10% fetal bovine serum (Invitrogen Co.), 2 mm GlutamaxTM, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified 5% CO2 atmosphere. Cells were transiently transfected using FuGENE-6 reagent (Roche Applied Science) following manufacturer's recommendations. Transfected cells were analyzed 24–36 h post-transfection. For siRNA knockdown, cells were transfected with either ON-TARGETplus smart pool siRNA duplexes against MCOLN3 or ON-TARGETplus nontargeting pool siRNA duplexes using DharmaFECT transfection reagent (Dharmacon-ThermoScientific). Treated cells were analyzed 72–80 h after transfection. For infection experiments, cells were infected with adenoviruses according to the manufacturer's recommendations. Analyses were performed 36–40 h post-infection.

Immunofluorescence microscopy and internalization assays

For immunofluoresence, cells grown on coverslips were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature or with methanol/acetone (1:1, v/v) for 10 min at −20^C. Cells then were incubated with the indicated primary antibodies in PBS containing 10% Fetal Bovine Serum and 0.1% (wt/v) saponin for 1 h at room temperature, followed by incubation with the corresponding secondary antibodies conjugated to Alexa Fluor568 or Alexa Fluor647. After staining, the coverslips were mounted onto glass slides with Fluoromount-G (SouthernBiotech). For Alexa555-EGF internalization, cells were incubated with Alexa555-EGF (500 ng/mL) in medium containing 0.1% (wt/v) bovine serum albumin for 10 min at 37^C. Cells were then washed with PBS and chased with medium containing BSA for the indicated times at 37^C. Finally, cells were washed two times with PBS and fixed with methanol/acetone as indicated above. Samples were examined and images were acquired on a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc).

For colocalization analysis, the percentage of colocalized vesicles was calculated as the number of GFP-MCOLN3-positive vesicles that colocalized with the indicated organelle markers divided by the total number of GFP-MCOLN3-positive vesicles in a given cell and multiplied by 100. The same calculation was applied for colocalizations with LC3-positive vesicles.

For starvation and recovery experiments, cells were treated as described previously (19).

Electrophoresis and Immunoblotting

Cells were washed with ice-cold PBS, resuspended in lysis buffer (25 mm Hepes-KOH, pH 7.4, 250 mm NaCl, 1% Triton X-100 (wt/v) supplemented with protease inhibitors cocktail) and lysed by passing the samples 10 times through a 25 gauge needle. Cell lysates were centrifuged at 16 000 × g for 15 min at 4^C, and the soluble fractions were collected. Samples were analyzed by SDS-PAGE (4–20% gradient gels) under reducing conditions and transferred to nitrocellulose. Membranes were immunoblotted using the indicated antibodies. Horseradish peroxidase-chemiluminiscence was developed by using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).

Degradation of EGFR

Twenty four hours after infection with adenovirus expressing GFP or GFP-MCOLN3, ARPE19 cells were serum-starved for an additional 4 h and then stimulated with EGF (200 ng/mL) in presence of 100 µg/mL cycloheximide for the indicated times at 37^C. Cells were washed with ice-cold PBS, harvested and lysed in Laemmli buffer. Cell extracts were subjected to SDS-PAGE and immunoblotting as described above. The amount of EGFR was quantified by using the public domain software ImageJ version 1.42 (NIMH, NIH), normalized with the clathrin content in the same sample and presented as the fraction of remaining EGFR relative to unstimulated control cells.

EGF internalization

To study the rate of 125I-EGF internalization, ARPE19 cells were cultured in 6-well plates and mock-infected or infected with adenovirus expressing GFP-MCOLN3 for 40 h. Cells were serum-starved for 4 h in internalization medium (IM; 1:1 mixture of DMEM and Ham's F12 media and 20 mm Hepes containing 0.1% BSA), placed on ice, briefly washed one time with ice-cold IM, and incubated for 1 h on a rocker at 4^C in 0.6 mL IM containing 125I-EGF at 300 nCi/mL (1.5 ng/mL). The monolayers were then washed three times with ice-cold IM. To determinate the amount of surface-bound 125I-EGF for the zero time point, one plate of each condition was incubated for 5 min with 0.8 mL of acid wash (0.2 m acetic acid, pH 2.8, containing 0.5 m NaCl) on ice followed by a second short wash with the same acid solution. The remaining plates were incubated with 1 mL of prewarmed I m at 37^C for various times. To stop the internalization process, cells were placed on ice, the incubation medium was collected, and the cell surface-bound 125I-EGF was collected by acid wash as described above. Finally, cells were lysed with 0.8 mL of 1 m NaOH. Radioactivity in the medium, acid wash and NaOH extract was quantified using a gamma counter (PerkinElmer). Non-specific binding was measured for each time point in the presence of 250-fold molar excess of unlabeled EGF and was not more that 1% of the total counts.

RNA isolation

RNA was isolated from cells by using PureLink Total RNA Purification System (Invitrogen Co.) following manufacturer recommendations. RNA yield was quantified by measuring the optical density at 260 and 280 nm using an Eppendorf BioPhotometer.

Quantitative reverse transcription-polymerase chain reaction

RNA (2–4 µg) was reverse transcribed in a 20 µL reaction using oligo(dT)20 and SuperScript III First-Strand Synthesis System (Invitrogen Co.) following manufacturer recommendations. PCR was performed using 5 µL SYBR GreenER qPCR SuperMix (Invitrogen Co.), 2 µL cDNA, 1 µL gene specific primer mix (QuantiTect primer Assays, QIAGEN, Valencia, CA) and 2 µL water for a total reaction volume of 10 µL. Quantification of gene expression was performed using 7900HT Fast Real-Time PCR System (Applied Biosystems). The thermal profile of the reaction was: 50^C for 2 min, 95^C for 10 min and 40 cycles of 95^C for 15 seconds followed by 60^C for 1 min. All samples were run in triplicates. Amplification of the sequence of interest was compared with a reference probe (β-2-microglobulin) and normalized against a standard curve of cell line mRNA. The 7900HT Fast Real-Time PCR System Software was used for data analyses (Applied Biosystems).

Measurement of endosomal pH

ARPE19 cells were grown at 37^C and 5% CO2 in Nunc Lab-Tek chambered cover glasses (Thermo Fisher Scientific Inc.) with DMEM/F12 medium. Cells were incubated for 15 min at 37^C with both 500 µm dextran conjugated to fluorescein (pH sensitive) and 20 µm dextran conjugated to Alexa Fluor 555 (10 000 MW anionic, Invitrogen) in complete growth media. After dextran internalization cells were washed three to five times with complete media and incubate with a physiological buffer (140 mm NaCl, 4.7 mm KCl, 2 mm CaCl2, 1.1 mm MgCl2, 50 mm Hepes, 10 mm glucose, pH 7.4) to be processed for confocal microscopy directly after the rinsing or 1 h after the beginning of the dextran internalization. Confocal microscopy was performed using the 63 × 1.4 NA objective of the Zeiss LSM 510 confocal system equipped with a imaging chamber thermostated at 37^C and maintained at 5% CO2 (Carl Zeiss Inc.). After confocal acquisition, ImageJ version 1.42 software (NIMH, NIH, Bethesda MD) was used to measure the Green/Red fluorescence ratio of the endosomal compartment. Correlation between fluorescence ratio and pH was done by a calibration procedure in a high K+ buffer (44.7 mm NaCl, 100 mm KCl, 2 mm CaCl2, 1.1 mm MgCl2, 50 mm Hepes, 10 mm glucose) supplemented with 5 µm Nigericin (Sigma Aldrich) and a pH set up between 4 and 8.

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