Figure 7. ESCRT-mediated microaggrephagy... | Rockefeller University Press

Figure 7.

$ESCRT-mediated microaggrephagy targets tauFL (P301L) in SH-SY5Y cells. (A) Representative image of tauFL (P301L)-Venus aggregate–positive SH-SY5Y cells before and after seeding. Images were acquired by a confocal microscope. Scale bar: 10 µm. (B and C) TauFL (P301L)-Venus aggregate–positive SH-SY5Y cells were transfected with control or individual sgRNA, as indicated, and successfully transfected cells were selected by puromycin. TauFL (P301L)-Venus fluorescence was measured by flow cytometer (B), and whole-cell lysates were immunoblotted with the indicated antibodies (C) 1 wk after transfection. Data were normalized to the Venus fluorescence of sgControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05, ***P < 0.001. n.s., not significant. (D) TauFL (P301L)-Venus aggregate–positive SH-SY5Y cells were treated with 500 nM TAK-243 for 20 h, and tauFL (P301L)-Venus fluorescence was measured by flow cytometry. Data were normalized to the Venus fluorescence of DMSO-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with ***P < 0.001. (E) TauFL (P301L)-Venus aggregate–positive SH-SY5Y cells were transfected with control, TSG101, or PTPN23 sgRNA, and successfully transfected cells were selected by puromycin. Cells were lysed 1 wk after transfection, and Triton X-100–soluble and Triton X-100–insoluble fractions were immunoprecipitated with a GST-tagged anti-GFP nanobody. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of tauFL (P301L)-Venus or ubiquitin in lane 7. (F) The ESCRT-I complex binds to ubiquitylated tau aggregates via the UEV domain in TSG101. The CC domain in PTPN23 interacts with the PBR domain in the ESCRT-I subunit UBAP1. The ESCRT-III subunit CHMP4 is recruited to PTPN23 via its Bro1 domain. ESCRT-III then recruits the AAA ATPase VPS4 to complete the endosomal microautophagy of tau aggregates. Source data are available for this figure: SourceData F7.$

ESCRT-mediated microaggrephagy targets tauFL (P301L) in SH-SY5Y cells. (A) Representative image of tauFL (P301L)-Venus aggregate–positive SH-SY5Y cells before and after seeding. Images were acquired by a confocal microscope. Scale bar: 10 µm. (B and C) TauFL (P301L)-Venus aggregate–positive SH-SY5Y cells were transfected with control or individual sgRNA, as indicated, and successfully transfected cells were selected by puromycin. TauFL (P301L)-Venus fluorescence was measured by flow cytometer (B), and whole-cell lysates were immunoblotted with the indicated antibodies (C) 1 wk after transfection. Data were normalized to the Venus fluorescence of sgControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05, ***P < 0.001. n.s., not significant. (D) TauFL (P301L)-Venus aggregate–positive SH-SY5Y cells were treated with 500 nM TAK-243 for 20 h, and tauFL (P301L)-Venus fluorescence was measured by flow cytometry. Data were normalized to the Venus fluorescence of DMSO-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using two-tailed Student’s t test with ***P < 0.001. (E) TauFL (P301L)-Venus aggregate–positive SH-SY5Y cells were transfected with control, TSG101, or PTPN23 sgRNA, and successfully transfected cells were selected by puromycin. Cells were lysed 1 wk after transfection, and Triton X-100–soluble and Triton X-100–insoluble fractions were immunoprecipitated with a GST-tagged anti-GFP nanobody. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of tauFL (P301L)-Venus or ubiquitin in lane 7. (F) The ESCRT-I complex binds to ubiquitylated tau aggregates via the UEV domain in TSG101. The CC domain in PTPN23 interacts with the PBR domain in the ESCRT-I subunit UBAP1. The ESCRT-III subunit CHMP4 is recruited to PTPN23 via its Bro1 domain. ESCRT-III then recruits the AAA ATPase VPS4 to complete the endosomal microautophagy of tau aggregates. Source data are available for this figure: SourceData F7.

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