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
). 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 Depletion of MCOLN3 accelerates degradation of EGFR. A) Q-RT-PCR analysis of MCOLN3 mRNA levels from ARPE19 cells treated with either siRNA nontarget or siRNA to MCOLN3. Results are mean ± SD (n = 3). B) ARPE19 cells treated with siRNA duplexes (more ...)
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 1 MCOLN3 distribution in ARPE19 cells. A) ARPE19 cells transiently transfected with GFP-MCOLN3 were fixed, permeabilized, immunostained with the indicated antibodies, and analyzed by confocal fluorescence microscopy. Insets show a fourfold magnification (more ...)
To determine the subcellular localization of GFP-MCOLN3 in more detail, cells were immunostained with antibodies against specific compartments of the endosomal pathway (). 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 (). 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) (). 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 (). 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 , 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 (). Both Hrs and GFP-MCOLN3 clearly colocalize to the same enlarged early endosomes induced by MCOLN3 overexpression (see inset in ). Therefore, we observed remarkable alterations in the morphology of the endosomal pathway in cells overexpressing GFP-MCOLN3.
Figure 2 MCOLN3 overexpression induces accumulation of enlarged endosomes. ARPE19 cells infected with adenovirus expressing either GFP (upper panel) or GFP-MCOLN3 (lower panel) were fixed, permeabilized, and immunostained with antibodies to Hrs. Insets show a (more ...)
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 (). In cells overexpressing GFP-MCOLN3, however, a substantial amount of internalized EGF could still be detected at this time (). EGF remained trapped in enlarged GFP-MCOLN3-positive structures at 3 h after internalization (). 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.
Figure 3 Overexpression of GFP-MCOLN3 delays degradation of Alexa555-labeled EGF. A) ARPE19 cells were infected with adenovirus expressing either GFP (upper panel) or GFP-MCOLN3 (lower panel). Infected cells were incubated with Alexa555-labeled EGF for 10 min (more ...)
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 (). 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 (). Therefore, our results suggest that MCOLN3 has a role in sorting and/or degradation of activated EGF receptors.
Figure 4 Overexpression of GFP-MCOLN3 affects normal trafficking of EGFR along the endo/lysosomal pathway. A) ARPE19 cells infected with adenovirus expressing either GFP or GFP-MCOLN3 were starved for 4 h in medium containing 0.1% BSA and incubated with or without (more ...)
Cells overexpressing MCOLN3 also showed evident accumulation of ubiquitin at enlarged Hrs-positive endosomes (). 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.
Figure 5 MCOLN3 overexpression induces accumulation of ubiquitinated proteins in endosomes. ARPE19 cells were transiently transfected with GFP-MCOLN3, fixed, permeabilized, immunostained with the indicated antibodies, and analyzed by confocal fluorescence microscopy. (more ...)
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 (), 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 (). 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 (). Taken together, our data suggest that MCOLN3 inhibits downregulation of EGFR by affecting delivery of the receptor to lysosomes for degradation.
Figure 6 Overexpression of GFP-MCOLN3 does not affect internalization of EGFR. ARPE19 cells mock-infected (●) or infected with adenovirus expressing GFP-MCOLN3 (▲) were starved for 4 h in medium containing 0.1% BSA. Cells were allowed to bind 125I-EGF (more ...)
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 , 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 (). 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 (). 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
). Autophagy is a crucial clearance mechanism that prevents accumulation of protein aggregates and abnormal organelles (43
). 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
). 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 (). In contrast, cells expressing GFP-MCOLN3 showed a dramatic accumulation of LC3-positive structures (see arrow in ). 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 (). 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 (). 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.
Figure 8 Overexpression of GFP-MCOLN3 inhibits autophagosome degradation. A) ARPE19 cells transiently transfected with GFP-MCOLN3 were fixed and immunostained to detect LC3. The arrow points to a GFP-MCOLN3 expressing cell. Dotted lines delineate untransfected (more ...)
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 , 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 (). 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 (). 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 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.
Figure 9 MCOLN3 overexpression decreases acidification of the endosomal pathway. A) ARPE19 cells were mock-infected, infected with a control adenovirus encoding only the cytomegalovirus (CMV) promoter (Ad. Control), or infected with an adenovirus expressing untagged (more ...)
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 (). 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 (). 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.