Abstract
A main requisite in the phagocytosis of ingested material is a coordinated series of maturation steps which lead to the degradation of ingested cargo. Photoreceptor outer segment (POS) renewal involves phagocytosis of the distal disk membranes by the retinal pigment epithelium (RPE). Previously, we identified melanoregulin (MREG) as an intracellular cargo-sorting protein required for the degradation of POS disks. Here, we provide evidence that MREG-dependent processing links both autophagic and phagocytic processes in LC3-associated phagocytosis (LAP). Ingested POS phagosomes are associated with endogenous LC3 and MREG. The LC3 association with POSs exhibited properties of LAP; it was independent of rapamycin pretreatment, but dependent on Atg5. Loss of MREG resulted in a decrease in the extent of LC3-POS association. Studies using DQ™-BSA suggest that loss of MREG does not compromise the association and fusion of LC3-positive phagosomes with lysosomes. Furthermore, the mechanism of MREG action is likely through a protein complex that includes LC3, as determined by colocalization and immunoprecipitation in both RPE cells and macrophages. We posit that MREG participates in coordinating the association of phagosomes with LC3 for content degradation with the loss of MREG leading to phagosome accumulation.
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Abbreviations
- POS:
-
Photoreceptor outer segment
- RPE:
-
Retinal pigment epithelium
- TR:
-
Texas red
- LC3:
-
Microtubule-associated protein 1 light chain 3
- IP:
-
Immunoprecipitation
- MREG:
-
Melanoregulin
- IEM:
-
Immuno-electron microscopy
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Acknowledgments
This work was supported by grants from PHS; R01EY010420, R01DE022465, and R21EY018705 to KBB, R01 EY013434 to CHM, Vision Research Core Grant EY001583 (KBB and CHM) and P30EY00331 and R01EY07042 to DSW. DSW is an RPB Jules and Doris Stein Professor. The authors would like to thank Dr. Anuradha Dhingra and Mr. Frank P. Stefano for their expert technical assistance as Managers of the PDM-Live Cell Imaging Core.
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Supplemental Figure 1
MREG and LC3 associate with Ingested OS phagosomes. ARPE19 (C2) cells challenged with TR-OS for 2h, were washed, external fluorescence quenched with trypan blue, fixed and stained for LC3 (Cell Signaling) and labeled with anti-rabbit Alexa Fluor 488. Cells were imaged and co-distribution analyzed using a binary submask Pearson’s coefficient 0.68. panel depicts representative co-localization images at t=1h depicting association of both MREG and LC3 with ingested TR-OS. Scale bar = 10 micron (PDF 41 kb)
Supplemental Figure 2
Atg5 knockdown and rapamycin treatment of ARPE19 cells. (A) Atg5 knock down RPE cells challenged with TR-OS for 2h, were washed, external fluorescence quenched with trypan blue, fixed and stained for LC3. Cells were imaged and co-distribution analyzed using a binary submask Pearson’s coefficient 0.68. Error bars represent ± SEM, (***p<0.001). Individual channels are indicated. (B) Atg5 knockdown in ARPE19 cells. siRNA-mediated silencing of Atg5 expression detected by western blot showed 71% knockdown compared to scrambled siRNA control. (C) Atg5 knockdown or scrRNA control cells were incubated with TR-OS at a density of 10 particles/cell for 2h at 37°C. Cells were washed and processed for immunofluorescence as described in methods. TR-OS were identified and quantitated. (D) Atg5 siRNA knockdown or scrRNA RPE cells were processed for immunofluorescence as described in methods. LC3 puncta were identified and quantitated. Error bars represent ±SEM, (* p<0.01). The data are an average of three independent Atg5 knock-down experiments. Error bars represent ±SEM. (E) TR-OS co-distribution with LC3 is unaffected by rapamycin. ARPE19 cells incubated with 100nM rapamycin for 4h prior to 2h TR-OS challenge and remained in the media for the duration of the study. Cells were imaged and co-distribution analyzed using a binary submask. Individual channels are indicated. (F) Rapamycin (100nM) inhibits S6 ribosomal protein phosphorylation in ARPE19 cells. ARPE19 cells were fed 20% FBS for 30 min, kept under regular growth conditions (10% FBS), or challenged with 100nM Rapamycin for 4 or 24h. Cells were washed and cleared lysates prepared for immuno-blotting. Western blots probed with anti-phospho S6 (Cell Signaling) and anti-actin as a loading control are shown. (PDF 281 kb)
Supplemental Figure 3
(A) MREG expression in C2, M5 and M5 cells transfected with MREG, these cells are designated (R). Western blots probed with anti-MREG and anti-actin as a loading control shown. (B) POS uptake in C2, M5 and M5+MREG (R) cells. Cells were incubated with TR-OS at a density of 10 particles/cell for up to 2h at 37°C. Cells were washed and processed for immunofluorescence as described in methods. TR-OS were identified and quantitated. The data are an average of three independent experiments. Error bars represent ±SEM (C) Lysosomal pH remained acidic upon MREG knockdown. Lysosomal pH was measured as described. Error bars represent ±SEM (n=9). (PDF 141 kb)
Supplemental Figure 4
Opsin is degraded in a time-dependent manner in hfRPE cells. (A) Time course of opsin in RPE lysates from POS pulse/chase study. hfRPE cells were pulsed with POS for 20 min and phagocytosis was allowed to continue for the time points indicated, t=0h, no POS addition, t=5minute chase, t=0.5h chase and t=4h chase. RPE lysates from apical and basal chamber were collected and pooled at each time points. Proteins were separated by SDS-Page and immunoblotted with anti-opsin mAb 4D2. A representative western is shown in the inset. Western blots were quantified and opsin levels show progressive decrease over time of chase. Results are average of 3 independent experiments each analyzed in duplicate. Error bars represent ±SEM. (B) Primed hfRPE cells show normal Cathepsin D processing. Cleared cell lysates from polarized hfRPE cells were probed with anti-Cathepsin D Ab to follow Cat-D processing. (PDF 38 kb)
Supplemental Figure 5
MREG associates with LC3. (A). Immunoprecipitation (IP) studies were carried out with lysates prepared from Mreg +/+ and Mreg dsu/dsu RPE cells (6 month old animals, sacrificed 3h after light onset). Lysates(inout) for botth e antiLC3 and the MOPC1 a none specific IgG control are indicated Proteins were immunoprecipitated with a polyclonal anti-LC3 antibody and then immunoblotted (IB) anti-LC3. (B) Immunoprecipitation (IP) studies were carried out with lysates prepared from C2 and M5 RPE cells isolated either at t=0 (no OS addition, designated, −OS) or after 2h outer segment feeding (designated, +OS). The blots shown indicated the Bound (elute) and Unbound fractions (designated FT) for both anti-LC3 as well as MOPC1 control. Proteins were immunoprecipitated with a polyclonal anti-LC3 antibody and then immunoblotted (IB) using an anti-MREG mAb or anti-LC3. (C) MREG-GST pulls down LC3 from mouse RPE lysates. The lysates inputs from Mreg +/+ and Mreg dsu/dsu RPE cells (6 month old animals, sacrificed 3h after light onset) are indicated. (D) MREG does not immunoprecipitates with anti-LC3 in macrophages upon POS challenge. J774A.1 cells challenged with TR-OS for 2h at 37°C as described in methods for C2 cells. Cells were washed, lysates prepared and LC3 containing complexes were immunoprecipitated with anti-LC3 and IB probed with anti-MREG or anti-LC3. El indicates (bound fraction) and Fl indicates flow through for both anti-LC3 IP as well as MOPC1 control. (PDF 200 kb)
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Frost, L.S., Lopes, V.S., Bragin, A. et al. The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium. Mol Neurobiol 52, 1135–1151 (2015). https://doi.org/10.1007/s12035-014-8920-5
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DOI: https://doi.org/10.1007/s12035-014-8920-5