Research ArticleLIFE SCIENCES

A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy

See allHide authors and affiliations

Science Advances  02 Sep 2016:
Vol. 2, no. 9, e1601167
DOI: 10.1126/sciadv.1601167
  • Fig. 1 Isolation of C9ORF72-associated proteins.

    (A) Silver staining analysis of Flag affinity–purified fractions from cytoplasmic extracts of Flag-GFP and Flag-C9ORF72 HEK293 cell lines. Asterisks indicate common contaminants of Flag purification (SKB1, α-tubulin, and MEP50). Flag-C9ORF72–associated proteins as identified by mass spectrometry are indicated. MW, molecular weight; IP, immunoprecipitation. (B) Superose 6 gel filtration fractions from C9ORF72 cytoplasmic Flag affinity purification. Fractions were resolved by 4 to 12% SDS-PAGE and analyzed by silver staining (top) and Western blot with corresponding antibodies (bottom). The gel filtration purification scheme and fraction numbers are indicated on the top. (C and D) Purification of the WDR41-associated proteins from a HEK293 stable cell line expressing Flag-WDR41. Gel filtration fractions and Flag eluates were resolved on a 4 to 12% SDS-PAGE gel, followed with silver staining (left) and Western blot with corresponding antibodies (right). The gel filtration purification scheme and fraction numbers are indicated on the top. WDR41-associated proteins and molecular markers are indicated.

  • Fig. 2 The C9ORF72 complex displays a GTPase activity and acts as a GEF for RAB39B.

    (A) GTPase assays were performed using WDR41 gel filtration fractions with [α-32P]GTP. The guanine nucleotides were separated by thin-layer chromatography plate. The positions of [α-32P]GTP and [α-32P]GDP are indicated on the right. (B) GTPase assay with Flag affinity–purified eluates from different Flag-tagged proteins in the C9ORF72 complex. Free [α-32P]GTP and Flag-GFP elutes were treated as controls. [α-32P]GTP and [α-32P]GDP are indicated on the right. (C and D) GFP-RAB39B, Flag-C9ORF72, or DENN domain–depleted C9ORF72ΔDENN was transfected into N2A cells. C9ORF72 proteins or RAB39B proteins were immunoprecipitated with M2 beads (anti-Flag) (C) or anti-GFP beads (D) followed by Western blot analyses using antibodies as listed. IB, immunoblot. (E) GEF assay of C9ORF72 protein complex and RAB39B. Purified His-tagged RAB39B proteins were preloaded with fluorescence-labeled BODIPY-GDP followed by addition of control or C9ORF72 protein complex. Fractions without C9ORF72 complex serve as the negative control. C9ORF72 complex promotes the release of GDP from RAB39B, suggesting its GEF activity against RAB39B.

  • Fig. 3 C9ORF72 interacts with SMCR8 in a DENN domain–dependent manner.

    (A and B) Flag-tagged SMCR8 or WDR41 was transfected into N2A cells. SMCR8 or WDR41 proteins were immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analysis using antibodies against endogenous C9ORF72. (C) Flag-tagged full-length or DENN domain–deleted SMCR8 was transfected into HEK293 cells. SMCR8 proteins were immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analysis using antibodies against C9ORF72 or WDR41. (D) Flag-tagged full-length or DENN domain–deleted C9ORF72 constructs were transfected into HEK293 cells. C9ORF72 proteins were immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analysis using antibodies against SMCR8 or WDR41. (E) GFP-tagged C9ORF72 was cotransfected with red fluorescent protein (RFP)–tagged SMCR8 or WDR41 into N2A cells. Confocal micrographs of N2A cells stained with antibodies against GFP (C9ORF72; green) and RFP (SMCR8 or WDR41; red). Hoechst stains the nuclei (blue). Scale bars, 10 μm. (F) GFP-tagged DENN domain–depleted C9ORF72 was cotransfected with RFP-tagged SMCR8 into N2A cells (upper panels). GFP-tagged C9ORF72 was cotransfected with RFP-tagged DENN domain–depleted SMCR8 into N2A cells (lower panels). Confocal micrographs of N2A cells stained with antibodies against GFP (C9ORF72ΔDENN or C9ORF72; green) and RFP (SMCR8 or SMCR8ΔDENN; red). Hoechst stains the nuclei (blue). Scale bars, 10 μm.

  • Fig. 4 Autophagy induction is compromised in Smcr8-deficient cells.

    (A) HEK293 cells infected with lentiviruses expressing control shRNA, SMCR8 shRNA1, or SMCR8 shRNA2 were cultured 72 hours before protein lysate collection. The cell lysates were subjected to Western blot analyses using antibodies as indicated. (B) Quantification of LC3-II/actin ratio. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). (C) Confocal imaging of GFP-LC3 expression in SMCR8 knockdown HEK293 cells using antibodies against GFP. Hoechst stains the nuclei (blue). Scale bars, 10 μm. (D) Quantification of GFP-LC3–positive puncta per cell in (C). Error bars represent SEM of three independent experiments; ~100 GFP-positive cells were randomly selected for each experiment. *P < 0.05 (Student’s t test). (E) Confocal microscope images of wild-type and Smcr8 mutant MEFs stained with antibodies against LC3 (green) under normal or starvation conditions. Hoechst stains the nuclei (blue). Scale bars, 10 μm. (F) Quantification of LC3-positive puncta per cell in (E). Error bars represent SEM of three independent experiments; ~100 cells were randomly selected for each experiment. *P < 0.05 (Student’s t test). Two-way analysis of variance (ANOVA) detects a significant decrease in the magnitude of up-regulation of LC3-positive puncta after starvation in Smcr8 mutant MEFs compared to that in wild-type MEFs (P < 0.05). (G) Western blot analysis of LC3 expression. Wild-type or Smcr8 mutant MEFs were treated with dimethyl sulfoxide (DMSO) or rapamycin (0.1 μM) for 3.5 hours before protein lysate collection. β-Actin serves as the loading control. (H) Quantification of LC3-II/actin ratio from (G). Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). n.s. represents no significant difference in LC3-II expression between DMSO and rapamycin treatment in Smcr8 mutant MEFs. Two-way ANOVA detects a significant decrease in the magnitude of LC3-II up-regulation after rapamycin treatment in the Smcr8 mutant MEFs compared to wild-type MEFs (P < 0.05).

  • Fig. 5 C9orf72 knockdown disrupts autophagy induction.

    (A) Western blot analysis of LC3 expression. MEF cells infected with lentiviruses expressing control or C9orf72 shRNA were treated with DMSO or rapamycin (0.1 μM) for 3.5 hours before protein lysate collection. β-Actin serves as the loading control. (B) Quantification of LC3-II/actin ratio. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). n.s. represents no significant difference in LC3-II expression between DMSO and rapamycin treatment in C9orf72 knockdown MEFs. Two-way ANOVA detects a significant decrease in the magnitude of LC3-II up-regulation after rapamycin treatment in the C9orf72 knockdown MEFs compared to that in wild-type MEFs (P < 0.05). (C) Confocal imaging of MEFs stained with antibodies against LC3 (green). MEF cells were infected with lentiviruses expressing control or C9orf72 shRNA followed by starvation. Hoechst stains the nuclei (blue). Scale bars, 10 μm. (D) Quantification of LC3-positive puncta per cell in (C). Error bars represent SEM of three independent experiments; ~100 cells were randomly selected for each experiment. *P < 0.05 (Student’s t test). (E) Western blot analysis of LC3 expression. Wild-type or Smcr8 mutant MEF cells were infected with lentiviruses expressing control or C9orf72 shRNA as indicated followed by DMSO or rapamycin (0.1 μM) treatment for 3.5 hours before protein lysate collection. β-Actin serves as the loading control. (F) Quantification of LC3-II/actin ratio. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). n.s. represents no significant difference in LC3-II expression between DMSO and rapamycin treatment. Two-way ANOVA detects a significant decrease in the magnitude of LC3-II up-regulation after rapamycin treatment in the C9orf72 knockdown, Smcr8 mutant, and C9orf72/Smcr8 double depletion MEFs compared that in to wild-type MEFs (P < 0.05).

  • Fig. 6 The C9ORF72/SMCR8 complex regulates ULK1.

    (A) The interaction between C9ORF72/SMCR8 complex with ULK1 is enhanced under starvation conditions. Flag-C9ORF72 and RFP-SMCR8 were transfected into HEK293 cells with or without amino acid starvation for 1 hour before protein lysate collection. C9ORF72 protein was immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analyses using antibodies as listed. (B) Western blot analysis of Ulk1 expression in wild-type or Smcr8 mutant MEFs. β-Actin serves as the loading control. (C) Quantification of Ulk1 expression relative to actin. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). (D) Ulk1 protein in wild-type or Smcr8 mutant MEFs was immunoprecipitated with Ulk1 antibodies followed by Western blot analyses. IgG, immunoglobulin G. (E) Quantification of relative Ulk1 and Atg13 protein levels. Error bars represent SEM of three measurements from three independent experiments; *P < 0.01 (Student’s t test). (F) Western blot analysis of phospho-Ulk1 (p-Ser757) expression. Wild-type or Smcr8 mutant MEFs were treated with DMSO or rapamycin (0.1 μM) for 3.5 hours before protein lysate collection. (G) Quantification of phospo-Ulk1 (p-Ulk1)/actin ratio. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). (H) Western blot analysis of phospho-Ulk1 (p-Ser757) expression. Wild-type or Smcr8 mutant MEFs were infected with lentiviruses expressing control or C9orf72 shRNA constructs. β-Actin serves as the loading control. (I) Quantification of relative levels of phospho-Ulk1. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test).

  • Fig. 7 Autophagic flux is defective in Smcr8-deficient cells.

    (A) Confocal imaging of HEK293 cells infected by lentiviruses expressing control or SMCR8 shRNA. Double-tagged LC3 (mCherry-GFP-LC3) protein was used to indicate autophagic flux. The percentage of yellow dots, which maintains both GFP and mCherry signal, out of total red dots is listed in the merged figures. Values represent SEM of three independent experiments; ~50 cells were randomly selected for each statistical analysis. *P < 0.05 (Student’s t test). (B) Western blot analysis of p62 expression. β-Actin serves as the loading control. (C) Quantification of p62 protein levels relative to actin. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). (D and F) Western blot analyses of LC3 (D) or p62 (F) expression in MEFs. MEFs were cultured in normal conditions or treated with lysosome inhibitors LP (100 μM each) for 3.5 hours before protein lysate collection. β-Actin serves as the loading control. (E and G) Quantification of LC3-II levels relative to actin (E) and p62 levels relative to actin (G). Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). n.s. represents no significant difference of LC3-II or p62 expression. Two-way ANOVA detects a significant decrease in the magnitude of LC3-II or p62 up-regulation after LP treatment in Smcr8 mutant MEFs compared to that in wild-type MEFs (P < 0.05). (H and J) Western blot analysis of cathepsin D (H) or cathepsin L (J) expression. β-Actin serves as the loading control. CtsDp, procathepsin D; CtsDm, mature cathepsin D; CtsLp, procathepsin L; CtsLm, mature cathepsin L. (I and K) Quantification of cathepsins D or L expression. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test); n.s. represents no significant difference detected.

  • Fig. 8 C9orf72 knockdown results in an increase in autophagic flux.

    (A) Confocal imaging of GFP-mCherry expression in N2A cells infected with lentiviruses expressing control or C9orf72 shRNA. (B) Quantification of the percentage of yellow dots, which maintains both GFP and mCherry signal, out of total red dots. Values represent SEM of three independent experiments; ~50 cells were randomly selected for each statistical analysis. *P < 0.05 (Student’s t test). (C) Western blot analyses of p62 expression. Wild-type or Smcr8 mutant MEFs were infected with lentiviruses expressing control or C9orf72 shRNA followed by 72 hours of culture before protein lysate collection. β-Actin serves as the loading control. (D) Quantification of p62 levels relative to actin. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). n.s. represents no significant difference detected. (E and G) Western blot analysis of cathepsin D (E) or cathepsin L (G) expression in the control or C9orf72 knockdown MEFs. β-Actin serves as the loading control. (F and H) Quantification of cathepsins D or L expression. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05, ***P < 0.001 (Student’s t test); n.s. represents no significant difference detected.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1601167/DC1

    fig. S1. Isolation of SMCR8-associated proteins.

    fig. S2. Identifying C9orf72- or SMCR8-associated Rab GTPases.

    fig. S3. Characterization of SMCR8 and C9orf72 shRNA constructs.

    table S1. Polypeptide composition through Flag-C9ORF72 immunopurification by mass spectrometry.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Isolation of SMCR8-associated proteins.
    • fig. S2. Identifying C9orf72- or SMCR8-associated Rab GTPases.
    • fig. S3. Characterization of SMCR8 and C9orf72 shRNA constructs.
    • table S1. Polypeptide composition through Flag-C9ORF72 immunopurification by mass spectrometry.

    Download PDF

    Files in this Data Supplement:

Navigate This Article