Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
Abnormal lysosome morphology and enlarged multivesicular body (MVB)-like structures in usp-50 mutants.
(A–C) Confocal fluorescence images of hypodermal cell 7 (hyp7) expressing the LAAT-1::GFP marker to highlight lysosome structures in L4 stage, young adult, and 3-day-old adult animals. Scale bar: 5 …
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Figure 1—source data 1
Excel file containing the quantified data of statistic analysis for Figure 1D.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig1-data1-v1.zip
Figure 1—figure supplement 1
ESCRT-0 components are stabilized by usp-50.
(A) Pearson’s correlation coefficient for co-localization of the lysosome membrane protein LAAT-1 (LAAT-1::GFP) and the lysosomal nuclease NUC-1 (NUC-1::mCherry) in wild-type and usp-50(xd413) …
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Figure 1—figure supplement 1—source data 1
Excel file containing the quantified data of statistic analysis for Figure 1—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig1-figsupp1-data1-v1.zip
Figure 2 with 2 supplements
Enlarged early endosomes (EEs) in usp-50/usp8 mutant cells.
(A) Confocal fluorescence images of hypodermis expressing YFP::2xFYVE to detect EEs in L4 stage animals. Compared to wild-type, EEs are enlarged in usp-50(gk632973) and usp-50(xd413) mutants. Scale …
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Figure 2—source data 1
Excel file containing the quantified data of statistic analysis for Figure 2B, K, and M.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig2-data1-v1.zip
Figure 2—figure supplement 1
usp-50 functions cell-autonomously.
(A) Pearson’s correlation coefficient for co-localization of early endosomes (EEs) (labeled with YFP::2xFYVE) and lysosomes (labeled with LAAT-1::mCherry) in wild-type and usp-50(xd413) animals. ns, …
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Figure 2—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 2—figure supplement 1C (anti-USP8 and anti-GAPDH).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF containing original scans of the relevant western blot analysis (anti-USP8 and anti-GAPDH) with highlighted bands and sample labels for Figure 2—figure supplement 1C.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig2-figsupp1-data2-v1.pdf
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Figure 2—figure supplement 1—source data 3
Excel file containing the quantified data of statistic analysis for Figure 2—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig2-figsupp1-data3-v1.zip
Figure 2—figure supplement 2
Mutation of usp-50 does not affect other organelles.
(A–C) In usp-50 mutant animals, no obvious alterations are detected in Golgi apparatus (MANS::GFP) (A), recycling endosomes (GFP::RME-1) (B), or retromers (VPS-29::GFP) (C). Confocal fluorescence …
Figure 3 with 2 supplements
USP8 is recruited to Rab5-positive vesicles.
(A–C) SUM159 cells genome-edited for USP8-mEGFP+/+ were transiently transfected with the indicated mScarlet-I-tagged proteins and then imaged by spinning-disk confocal microscopy. The single-frame …
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Figure 3—source data 1
Excel file containing the quantified data of statistic analysis for Figure 3H.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
The early endosome (EE) localization of USP-50 and USP8.
(A) USP-50::GFP is co-localized with mCherry::RAB-5 in hypodermal cell 7 (hyp7) of wild-type animals at L4 stage. (B) USP-50::GFP is not co-localized with mCherry::RAB-7 in hypodermal cell 7 (hyp7) …
Figure 3—figure supplement 2
The generation of endogenous tagged USP8.
(A) CRISPR/Cas9 genome-editing strategy used to incorporate mEGFP at the C-terminus of USP8 in SUM159 cells. The target sequence at the genomic USP8 locus recognized by the single-guide RNA is …
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Figure 3—figure supplement 2—source data 1
Original file for the genomic PCR analysis in Figure 3—figure supplement 2B.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
PDF containing original file for the genomic PCR analysis with highlighted bands and sample labels for Figure 3—figure supplement 2B.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig3-figsupp2-data2-v1.pdf
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Figure 3—figure supplement 2—source data 3
Original file for the western blot analysis in Figure 3—figure supplement 2C (anti-USP8 and anti-GAPDH).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig3-figsupp2-data3-v1.zip
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Figure 3—figure supplement 2—source data 4
PDF containing original scans of the relevant western blot analysis (anti-USP8 and anti-GAPDH) with highlighted bands and sample labels for Figure 3—figure supplement 2C.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig3-figsupp2-data4-v1.pdf
Figure 4
USP-50 interacts with RABX-5.
(A) The affinity-purified RABX-5::GFP from worm lysates is immunoprecipitated by USP-50::4xFLAG purified from HEK293T cells using anti-FLAG beads. Only the area of the blot containing the …
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Figure 4—source data 1
Original file for the western blot analysis in Figure 4A (anti-GFP and anti-FLAG).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig4-data1-v1.zip
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Figure 4—source data 2
PDF containing original scans of the relevant western blot analysis (anti-GFP and anti-FLAG) with highlighted bands and sample labels for Figure 4A.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig4-data2-v1.pdf
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Figure 4—source data 3
Original file for the western blot analysis in Figure 4B (anti-GFP and anti-FLAG).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig4-data3-v1.zip
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Figure 4—source data 4
PDF containing original scans of the relevant western blot analysis (anti-GFP and anti-FLAG) with highlighted bands and sample labels for Figure 4B.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig4-data4-v1.pdf
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Figure 4—source data 5
Original file for the western blot analysis in Figure 4D (anti-MYC and anti-FLAG).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig4-data5-v1.zip
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Figure 4—source data 6
PDF containing original scans of the relevant western blot analysis (anti-MYC and anti-FLAG) with highlighted bands and sample labels for Figure 4D.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig4-data6-v1.pdf
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Figure 4—source data 7
Excel file containing the quantified data of statistic analysis for Figure 4G and I.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig4-data7-v1.zip
Figure 5 with 3 supplements
USP-50 dissociates RABX-5 from endosomes.
(A–D) The punctate distribution of RABX-5::GFP KI (knock-in) in wild-type (A) and usp-50(xd413) (B). The increased RABX-5::GFP KI signal in usp-50(xd413) is rescued by expressing wild-type (C) but …
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Figure 5—source data 1
Excel file containing the quantified data of statistic analysis for Figure 5I, L, and M.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-data1-v1.zip
Figure 5—figure supplement 1
USP8 inhibits the early endosome (EE) localization of Rabex5.
(A) USP8-KO leads to enlarged EEs and enhanced Rabex5 (Rabex5-mEGFP+/+) and Rab5 (mScarlet-I-Rab5c) signals. Rabex5-mEGFP+/+ SUM159 cells with or without USP8 expression were transiently transfected …
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Figure 5—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 5—figure supplement 1E (anti-USP8 and anti-GAPDH).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
PDF containing original scans of the relevant western blot analysis (anti-USP8 and anti-GAPDH) with highlighted bands and sample labels for Figure 5—figure supplement 1E.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp1-data2-v1.pdf
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Figure 5—figure supplement 1—source data 3
Excel file containing the quantified data of statistic analysis for Figure 5—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp1-data3-v1.zip
Figure 5—figure supplement 2
Loss of usp-50/USP8 leads to more activated RAB-5.
(A, B) The GFP::RAB-5 KI signal in wild-type (A) and usp-50(xd413) (B). Scale bar: 5 μm. (C) Quantification of the volume of individual RAB-5::GFP KI vesicles in L4 animals (10 animals for wild-type …
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Figure 5—figure supplement 2—source data 1
Original file for the Coomassie blue staining of purified GST-EEA1-NT protein in Figure 5—figure supplement 2E.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp2-data1-v1.zip
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Figure 5—figure supplement 2—source data 2
PDF containing the Coomassie blue staining of purified GST-EEA1-NT protein with highlighted bands and sample labels in Figure 5—figure supplement 2E.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp2-data2-v1.pdf
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Figure 5—figure supplement 2—source data 3
Original file for the western blot analysis in Figure 5—figure supplement 2F (anti-GFP and anti-GST).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp2-data3-v1.zip
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Figure 5—figure supplement 2—source data 4
PDF containing original scans of the relevant western blot analysis (anti-GFP and anti-GST) with highlighted bands and sample labels for Figure 5—figure supplement 2F.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp2-data4-v1.pdf
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Figure 5—figure supplement 2—source data 5
Original file for the western blot analysis in Figure 5—figure supplement 2G (anti-GFP and anti-Tub).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp2-data5-v1.zip
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Figure 5—figure supplement 2—source data 6
PDF containing original scans of the relevant western blot analysis (anti-GFP and anti-Tub) with highlighted bands and sample labels for Figure 5—figure supplement 2G.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp2-data6-v1.pdf
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Figure 5—figure supplement 2—source data 7
Excel file containing the quantified data of statistic analysis for Figure 5—figure supplement 2C, D, H, and J.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig5-figsupp2-data7-v1.zip
Figure 5—figure supplement 3
The ubiquitin modification sites on RABX-5.
(A) Peptide sequences identified from the ubiquitination proteomics analysis. (B) The ubiquitinated sites of RABX-5 detected by ubiquitination proteomics analysis.
Figure 6 with 1 supplement
Loss of usp-50/usp8 disrupts SAND-1/Mon1 localization.
(A–C) The reduction of punctate GFP::SAND-1 signals in rabx-5(null) and usp-50(xd413) mutant animals. (D) Line scan analyses for (A–C). (E–G) The reduced lysosomes (labeled by LAAT-1::GFP) in rabx-5(…
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Figure 6—source data 1
Original file for the western blot analysis in Figure 6P (anti-MYC and anti-FLAG).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig6-data1-v1.zip
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Figure 6—source data 2
PDF containing original scans of the relevant western blot analysis (anti-MYC and anti-FLAG) with highlighted bands and sample labels for Figure 6P.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig6-data2-v1.pdf
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Figure 6—source data 3
Excel file containing the quantified data of statistic analysis for Figure 6D, T, H, I, and X.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig6-data3-v1.zip
Figure 6—figure supplement 1
The distribution of endogenous Mon1a and Mon1b proteins.
(A) Quantification of the individual volume of LAAT-1::GFP vesicles in hyp7 of 3-day-old adults. Data are presented as mean ± SEM. ****p<0.0001; one-way ANOVA with Tukey’s test. (B) Western blotting …
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Figure 6—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 6—figure supplement 1B and E (anti-mEGFP and anti-GAPDH).
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
PDF containing original scans of the relevant western blot analysis (anti-mEGFP and anti-GAPDH) with highlighted bands and sample labels for Figure 6—figure supplement 1B and E.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig6-figsupp1-data2-v1.pdf
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Figure 6—figure supplement 1—source data 3
Excel file containing the quantified data of statistic analysis for Figure 6—figure supplement 1A, H, and I.
- https://cdn.elifesciences.org/articles/96353/elife-96353-fig6-figsupp1-data3-v1.zip
Author response image 1
(A) Confocal fluorescence images of hypodermis expressing YFP::2xFYVE to detect EEs in L4 stage animals in wild type and stam-1(ok406) mutants. Scale bar: 5 μm. (B) Confocal fluorescence images of …
Author response image 2
ESCRT-0 is adjacent to both early endosomes and late endosomes.
(A) Confocal fluorescence images of wild-type and usp-50(xd413) hypodermis at L4 stage co-expressing HGRS-1::GFP (hgrs-1 promoter) and endogenous wrmScarlet::RAB-5. (B) HGRS-1 and RAB-5 puncta were …
Author response image 3
The endogenous RABX-5 protein level is increased in usp-50 mutants.
(A) The RABX-5::GFP KI protein level is increased in usp-50(xd413). (B) Quantification of endogenous RABX-5::GFP protein level in wild type and usp-50(xd413) mutant animals.
Author response image 4
(A-C) Over-expression wild type RABX-5 causes enlarged EEs (labeled by YFP::2xFYVE) while RABX-5(K323R) mutant form does not. (D) Quantification of the volume of individual YFP::2xFYVE vesicles. …
Author response image 5
(A) RABX-5::GFP protein was purified from worm lysates using anti-GFP antibody. FLAG-tagged USP-50 was purified from HEK293T cells using anti-FLAG antibody. Purified RABX-5::GFP was incubated with …
