Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity
- State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, China
- Huffington Center on Aging, Baylor College of Medicine, United States
- Developmental Biology Graduate Program, Baylor College of Medicine, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Molecular and Cellular Biology Graduate Program, Baylor College of Medicine, United States
- Institute of Organic Chemistry and Biochemistry, Czech Republic
- Institute for Chemistry, Engineering and Medicine for Human Health (ChEM-H), Stanford University, United States
- Department of Chemical Engineering and Genetics, Stanford University, United States
- Department of Molecular and Cellular Biology, Baylor College of Medicine, United States
Figures
Figure 1 with 1 supplement
Rapid lysosome isolation coupled with proteomic profiling.
(A) Schematic of the workflow for immunoprecipitation-based lysosome purification (Lyso-IP) and mass spectrometry-based proteomic profiling to identify lysosome-enriched proteomes in C. elegans.(B) …
-
Figure 1—source data 1
Western blots shown in Figure 1.
- https://cdn.elifesciences.org/articles/85214/elife-85214-fig1-data1-v2.zip
Figure 1—figure supplement 1
Analysis of LysoTg lines and Lyso-IP profiling in wild-type (WT) worms.
(A) Developmental timing of WT and transgenic strains expressing LMP-1 and CTNS-1 Lyso-Tag (LMP-1 and CTNS-1 LysoTg). n.s. p > 0.05 by Chi-squared test. (B) Lifespan of WT, LMP-1 LysoTg, and CTNS-1 L…
Figure 2 with 1 supplement
Systematic view of lysosome-enriched proteome.
(A) Scatter plots showing candidate selection from four independent biological replicates in proteomics analyses. Proteins with at least 10-fold higher levels in Lyso-IP samples than in flow-through …
Figure 2—figure supplement 1
Pie chart showing the proportion of LMP-1 Lyso-IP candidates from wild-type (WT) worms with mammalian homologs.
Figure 3 with 1 supplement
Lysosomal proteome heterogeneity across tissues.
(A) Example images of transgenic strains carrying Lyso-Tag (LMP-1::RFP-3×HA) driven by four different tissue-specific promoters. Scale bar = 20 μm. Scatter plot showing the relative enrichment ratio …
Figure 3—figure supplement 1
Tissue-specific Lyso-IPs and candidate imaging.
(A–D) Pearson correlation matrices of tissue-specific lyso-IP (IP) samples and flow-through (FT) samples show the correlation among three different replicates. (A) Hypodermis, (B) muscle, (C) …
Figure 4 with 1 supplement
Lysosomal proteome in different pro-longevity models.
(A) Scheme showing four different longevity regulatory mechanisms used in this study. Loss-of-function mutants (lf) of isp-1, daf-2, and glp-1 reduce mitochondrial electron transport chain (ETC) …
Figure 4—figure supplement 1
Lyso-IP analyses from different long-lived strains.
Correlation analysis of three independent biological replicates of Lyso-IP (IP) and Flow-through (FT) from proteomics analyses of the long-lived lipl-4 transgenic strain lipl-4 Tg (A), the daf-2 …
Figure 5
Increased enrichment of lysosomal proteins upon lysosomal lipolysis.
(A) Normalized protein levels (z-score across samples) of autophagy-related components, mTORC1 signaling factors, lysosomal v-ATPase V0, V1, and transporting accessory (TA) subunits, lysosomal …
Figure 6 with 1 supplement
Enhanced lysosome–nucleus proximity contributing to longevity.
(A) The percentage of proteins with different subcellular localization is compared between lysosome-enriched proteomes from wild-type (WT) and lipl-4 Tg worms. *p = 0.019 by two-sample test for …
Figure 6—figure supplement 1
Lysosomal positioning in longevity regulation.
(A) Summary of the method flow for quantifying the lysosomal distribution in intestinal cells of C. elegans. Scale bar = 10 μm. Curve graph showing the normalized accumulated intensity of lysosomal …
Figure 7 with 2 supplements
Lysosome-enriched proteome identified with Cystinosin.
(A) Example images of transgenic strains carrying CTNS-1 Lyso-Tag (CTNS-1::RFP-3×HA) with LysoTracker staining to mark lysosomes in vivo. Scale bar = 5 µm. (B) Venn diagram showing the overlap …
Figure 7—figure supplement 1
The colocalization between LMP-1::mNeonGreen and CTNS-1::wrmScarlet in different tissues.
Representative images of knock-in lines with both LMP-1::mNeonGreen and CTNS-1::wrmScarlet show partial colocalization between LMP-1 and CTNS-1 signals in different tissues. Scale bar = 20 μm.
Figure 7—figure supplement 2
CTNS-1 Lyso-IPs and LMTR-3 imaging analyses.
(A) Correlation analysis of three independent biological replicates of CTNS-1 Lyso-IP (IP) and Flow-through (FT). (B) Principal components analysis (PCA) of three independent biological replicates …
Figure 8 with 1 supplement
Lysosome-enriched proteins regulating lysosomal functions.
Confocal fluorescence microscopy images of intestinal cells in worms stained with LysoSensor DND-189 and treated with empty vector (A), slc36.2 RNAi (B), R144.6 RNAi (C), vha-5 RNAi (D), and unc-32 …
Figure 8—figure supplement 1
LysoSensor intensity quantification in five candidates.
The LysoSensor signals are visualized by confocal fluorescence microscopy in empty vector (A), slc36.2 RNAi (B), R144.6 RNAi (C), vha-5 RNAi (D), and unc-32 RNAi (E) conditions. Scale bar = 50 μm. …
Additional files
-
Supplementary file 1
Lysosome-enriched proteins identified from LMP-1 Lyso-IP using wild-type (WT) worms.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp1-v2.xlsx
-
Supplementary file 2
Lysosome-enriched proteome exhibits tissue specificity.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp2-v2.xlsx
-
Supplementary file 3
Lysosome-enriched proteins identified from LMP-1 Lyso-IP using lipl-4 Tg worms.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp3-v2.xlsx
-
Supplementary file 4
Lysosome-enriched proteins identified from LMP-1 Lyso-IP using daf-2(lf) mutant.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp4-v2.xlsx
-
Supplementary file 5
Lysosome-enriched proteins identified from LMP-1 Lyso-IP using isp-1(lf) mutant.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp5-v2.xlsx
-
Supplementary file 6
Lysosome-enriched proteins identified from LMP-1 Lyso-IP using glp-1(lf) mutant in 25°C.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp6-v2.xlsx
-
Supplementary file 7
Lysosome-enriched proteins identified from LMP-1 Lyso-IP using wild-type (WT) worms in 25°C.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp7-v2.xlsx
-
Supplementary file 8
Summary of lifespan analyses.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp8-v2.xlsx
-
Supplementary file 9
Lysosome-enriched proteins identified from CTNS-1 Lyso-IP using wild-type (WT) worms.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp9-v2.xlsx
-
Supplementary file 10
LysoSensor screening of lysosome-enriched proteins shared between LMP-1 and the CTNS-1 Lyso-Ips.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp10-v2.xlsx
-
Supplementary file 11
File list of mass spectrum samples.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp11-v2.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/85214/elife-85214-mdarchecklist1-v2.docx
-
Source code 1
Matlab code for lysosome distribution quantification.
- https://cdn.elifesciences.org/articles/85214/elife-85214-code1-v2.zip
