Research ArticleCell Biology

Phagocytosed photoreceptor outer segments activate mTORC1 in the retinal pigment epithelium

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Science Signaling  29 May 2018:
Vol. 11, Issue 532, eaag3315
DOI: 10.1126/scisignal.aag3315
  • Fig. 1 Diurnal variation of mTORC1 activity in the RPE during morning burst of POS shedding.

    (A and C) Images of immunostained RPE/choroid whole mounts with antibodies against rhodopsin (A) or phosphorylated ribosome protein S6 (C) at the indicated times after lights on. (B and D) Quantification of the imaging data from three independent experiments (n = 3 mice per condition), presented as mean ± SEM. (E and F) Western blot analysis of phosphorylated S6 levels in RPE/choroid tissues. Data are means of five independent experiments (mean ± SEM). A.U., arbitrary units. **P < 0.01 relative to lights on [one-way analysis of variance (ANOVA) and Dunnett’s post hoc test]. Scale bar, 20 μm.

  • Fig. 2 mTORC1 activation by POS in cultured RPE cells.

    ARPE-19 cells were treated with purified POS at the indicated ratios for 1 hour (A) or at a 10:1 POS/RPE ratio for the indicated times (B). Phosphorylation status of S6, 4E-BP1, and ULK1 were examined by Western blot analyses. Data are means from five independent experiments (mean ± SEM). *P < 0.05, **P < 0.01 relative to cells without POS exposure (one-way ANOVA and Dunnett’s post hoc test). (C) mTORC1 activation in cells depleted of integrin β5 using siRNA or cells exposed to control scrambled (Scr) siRNA. POS were added at a 10:1 POS/RPE ratio for 1 hour. Blots are representative of three independent experiments. (D) mTORC1 activity in cells exposed to latex beads for the indicated times. Data are means from three independent experiments (mean ± SEM). No statistically significant differences were detected between control and bead-loaded RPE cells (one-way ANOVA).

  • Fig. 3 Localization of mTOR protein on POS-containing organelles.

    ARPE-19 cells were incubated with purified POS for up to 4 hours. Colocalization of POS with mTOR or endosome and lysosome marker proteins was monitored by costaining with antibodies against rhodopsin and mTOR, EEA1 (early endosome marker), Rab5 (early endosome marker), or LAMP2 (lysosomes) at the indicated times. Colocalization of each protein to rhodopsin-positive phagosomes was analyzed by determining Pearson’s correlation coefficients. Thirty rhodopsin-positive particles were analyzed for each protein from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 relative to cells exposed to POS for 15 min (one-way ANOVA and Dunnett’s post hoc test). Scale bars, 20 μm.

  • Fig. 4 Localization of mTORC1 pathway proteins on POS-containing organelles.

    (A to C) Colocalization of rhodopsin with p18 (A), RagA (B), or Rheb (C) in cells exposed or not exposed to POS for 30 min. Scale bar, 20 μm. Images are representative of three independent experiments. (D) Quantification of data in (A) to (C). Twenty fields with an area of 50 × 50 μm2 were analyzed for each protein. (E) Western blot analyses of POS-enriched organelles from ARPE-19 cells exposed to POS for 3 hours. Blots are representative of three independent experiments. WL, whole-cell lysate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Representative images of immunostaining of flat-mounted RPE/choroid tissues with antibodies against mTOR, RagA, p18, and rhodopsin. Small panels are enlarged images of white insets. Arrows indicate proteins associated with rhodopsin-positive phagosomes. Samples were prepared 1 hour after lights on. Images are representative of three independent experiments. Scale bars, 20 μm. (G) Quantification of data in (F). Six fields with an area of 100 × 100 μm2 were analyzed for each protein.

  • Fig. 5 POS-stimulated mTORC1 activation in cells with reduced expression of KLC1 or Myo6.

    (A) POS-induced mTORC1 activation in ARPE-19 cells transfected with either KLC1 siRNA or control scrambled siRNA. S6 phosphorylation and KLC1 knockdown were analyzed by Western blots. Quantification data are means from five independent experiments (mean ± SEM). *P < 0.05 (Kruskal-Wallis test and Dunn’s multiple comparisons test). n.s., not significant. (B and C) Coimmunostaining of rhodopsin and Myo6 in POS-treated ARPE-19 cells and RPE/choroid whole mounts, respectively. Images are representative of three independent experiments. Scale bars, 5 μm. (D) Quantitation of the data in (B) and (C). Twenty fields with an area of 50 × 50 μm2 were analyzed. (E) Depletion of Myo6 in ARPE-19 cells expressing Cas9 endonuclease and transfected with guide RNA (gRNA) targeting Myo6. Blots are representative of three independent experiments. (F) Degradation of POS in cells depleted of Myo6 by CRISPR/Cas9. Quantification data are means from six independent experiments (mean ± SEM). *P < 0.05 (Kruskal-Wallis test and Dunn’s multiple comparisons test). (G) POS-induced mTORC1 activation in cells depleted of Myo6 by CRISPR/Cas9. Quantification data are means from five independent experiments (mean ± SEM). ***P < 0.001 (one-way ANOVA and Tukey-Kramer multiple comparsions test). (H) Amino acid (AA)–induced mTORC1 activation in cells depleted of Myo6 by CRISPR/Cas9. Data are means of five independent experiments (mean ± SEM). ***P < 0.001 (one-way ANOVA and Tukey-Kramer multiple comparsions test).

  • Fig. 6 POS-induced mTORC1 activation occurred independently of lysosomal functions.

    (A) Amino acid– or POS-induced mTORC1 activity in cells cultured in the absence or presence of 20 μM chloroquine (CQ). Blots are representative of three independent experiments. (B) Immunofluorescence microscopy of POS-exposed RPE cells labeled with antibodies against mTOR (green) and rhodopsin (red), with or without chloroquine treatment. Blue, nuclei (N). Scale bar, 10 μm. Colocalization of mTOR and rhodopsin-positive phagosomes was analyzed by determining Pearson’s correlation coefficients. Thirty rhodopsin-positive particles were included from three independent experiments for each condition. No statistically significant differences were detected between samples with or without chloroquine treatment (Student’s t test). (C) Western blot analysis of amino acid–induced mTORC1 activation. Cells were transfected with siRNAs against subunits of V-ATPase (V1A + V0D or V1A + V0C) or control scrambled siRNA and then stimulated with amino acids. Blots are representative of three independent experiments. (D) Western blot analysis of POS-induced mTORC1 activation. Quantification data are means from seven independent experiments (mean ± SEM). *P < 0.05; ***P < 0.001 (Kruskal-Wallis test and Dunn’s multiple comparisons test). dsRNA, double-stranded RNA.

  • Fig. 7 In vitro reconstitution of POS-mediated mTORC1 activation.

    (A) Schematic illustration of preparation of cell extracts depleted of lysosomes. C/M, cytosol/membrane fraction; C/M-Lyso, C/M fraction immunodepleted of lysosomes. (B) Fractions prepared according to scheme illustrated in (A) were separated on SDS–polyacrylamide gel electrophoresis (PAGE) and labeled with antibodies against lysosomal proteins LAMP1, LAMP2, and cathepsin B. EEA1, early endosome marker; HSC70, cytosolic marker. Blots are representative of three independent experiments. (C) mTORC1 activity in different fractions with or without incubation with purified POS. Blots are representative of four independent experiments. Quantification of phosphorylation of S6K (D) and S6 (E) in C/M and C/M-Lyso fractions (mean ± SEM). *P < 0.05 (Student’s t test). (F) mTORC1 activity in different fractions with or without incubation with liposomes. Blots are representative of three independent experiments. (G) Quantification of phosphorylation of S6K in the C/M-Lyso fraction in the presence or absence of liposomes (Lipo) (mean ± SEM). *P < 0.05 (Student’s t test).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/532/eaag3315/DC1

    Fig. S1. Rapamycin-sensitive mTORC1 activation by POS in cultured RPE cells.

    Fig. S2. POS-induced mTORC1 activation in cultured human fetal RPE cells.

    Fig. S3. Uptake of latex beads by cultured ARPE-19 cells.

    Fig. S4. Time course of POS phagocytosis in ARPE-19 cells.

    Fig. S5. Delayed degradation of ingested POS in RPE cells transfected with siRNAs against KLC1.

    Fig. S6. POS-stimulated mTORC1 activation in cells with reduced expression of Myo6.

    Fig. S7. Hypothetical working model of POS-stimulated mTORC1 activation.

    Table S1. Source of antibodies.

  • Supplementary Materials for:

    Phagocytosed photoreceptor outer segments activate mTORC1 in the retinal pigment epithelium

    Bo Yu, Anuoluwapo Egbejimi, Rachayata Dharmat, Pei Xu, Zhenyang Zhao, Bo Long, Hongyu Miao, Rui Chen, Theodore G. Wensel, Jiyang Cai, Yan Chen*

    *Corresponding author. Email: yachen1{at}utmb.edu

    This PDF file includes:

    • Fig. S1. Rapamycin-sensitive mTORC1 activation by POS in cultured RPE cells.
    • Fig. S2. POS-induced mTORC1 activation in cultured human fetal RPE cells.
    • Fig. S3. Uptake of latex beads by cultured ARPE-19 cells.
    • Fig. S4. Time course of POS phagocytosis in ARPE-19 cells.
    • Fig. S5. Delayed degradation of ingested POS in RPE cells transfected with siRNAs against KLC1.
    • Fig. S6. POS-stimulated mTORC1 activation in cells with reduced expression of Myo6.
    • Fig. S7. Hypothetical working model of POS-stimulated mTORC1 activation.
    • Table S1. Source of antibodies.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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