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) Example images of transgenic strains carrying LMP-1 Lyso-Tag (LMP-1::RFP-3×HA) with LysoTracker staining to mark lysosomes in vivo. Scale bar = 5 µm. (C) Example images of beads carrying purified lysosomes from Lyso-IP with LysoTracker staining to mark intact lysosomes in vitro. Scale bar = 5 µm.(D) Western blot for protein markers of different subcellular compartments using purified lysosomes (Lyso-IP), paired non-lysosomal fractions (Flow-through) or Pellet. (E) Principal components analysis (PCA) of four independent biological replicates of Lyso-IP and Flow-through samples.
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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 LysoTg worms. The lifespan data are also in Supplementary file 8. (C) Correlation analysis of four independent biological replicates of Lyso-IP (IP) and Flow-through (FT) samples from proteomics analyses.
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 (FT) controls are highlighted with different colors based on repeated times in four replicates. (B) Scatter plot showing candidate selection with normalization to non-tagged controls using wild-type worms. 216 proteins with over twofold higher levels in Lyso-IP samples than in non-tagged controls are highlighted in red. (C) Pie chart showing molecular function categories of lysosome-enriched proteins. (D) The lysosomal enrichment ratio (Lyso-IP vs. FT) for each subunit of lysosomal vacuolar ATPase (v-ATPase) in four independent replicates is shown. Inserted scheme showing lysosomal V-ATPase assembly. (E) Pie chart showing subcellular location categories of lysosome-enriched proteins.
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 for each of 216 lysosome-enriched proteins identified from whole-body LMP-1 Lyso-IP in comparison with tissue-specific LMP-1 Lyso-IPs, hypodermis (B), muscle (C), intestine (D), and neuron (E). X-axis, enrichment ratio tissue-specific vs. whole-body; Y-axis, normalized protein abundance over LMP-1; each dot represents the average of three replicates. (F) Heatmap showing the relative enrichment of 216 lysosome-enriched proteins identified from whole-body LMP-1 Lyso-IP in comparison with tissue-specific LMP-1 Lyso-IPs. Group I, comparable ratios between whole-body and tissue-specific Lyso-IPs; Group II, increase in tissue-specific Lyso-IPs (p < 0.05 by Student’s t-test); Group III, decrease in tissue-specific Lyso-IPs (p < 0.05 by Student’s t-test); Group IV, absent in tissue-specific IPs.
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) intestine, and (D) neuron. (E) Representative images showing colocalization of Y58A7A.1::mNeonGreen and LysoTracker Red in the hypodermis. Scale bar = 20 μm.
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) complex III, insulin/IGF-1 signaling, and germline stem cell proliferation, respectively, leading to lifespan extension; while increasing lysosomal lipolysis by lipl-4 transgenic overexpression (lipl-4 Tg) promotes longevity. (B) Venn diagram showing the overlap between the lysosome-enriched proteomes from wild-type (WT) and lipl-4 Tg worms. (C) Upset graph showing the distribution and overlap of lysosome-enriched proteins across the four pro-longevity models. Inserted Venn diagram showing the overlaps between the lysosome-enriched proteomes of WT worms and the long-lived daf-2(lf) and isp-1(lf) mutants.
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 loss-of-function mutant (daf-2(lf)) (B), the isp-1 loss-of-function mutant (isp-1(lf)) (C) and glp-1 loss-of-function mutant grown at 25°C (glp-1(lf)) (D). (E) Principal components analysis (PCA) of Lyso-IP replicates (IP) and flow-through controls (FT) in LMP-1 Lyso-IP of wild-type (WT), lipl-4 Tg, daf-2(lf), and isp-1(lf) worms. (F) PCA analysis of Lyso-IP replicates (IP) and flow-through controls (FT) in LMP-1 Lyso-IP of WT and glp-1(lf) worms grown at 25°C. (G) Venn diagram showing the overlap between the lysosome-enriched proteomes from WT and glp-1(lf) worms grown at 25°C. (H) Upset graph showing the overlap of lysosome-enriched proteins present in the long-lived worms but absent from WT worms.
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 hydrolases and transporter proteins from LMP-1 Lyso-IP proteomic analyses of wild-type (WT), lipl-4 Tg, daf-2(lf), and isp-1(lf) worms grown at 20°C and WT and glp-1(lf) worms grown at 25°C. (B) The lysosomal enrichment ratio (Lyso-IP vs. FT) for two homologs of AMP-activated protein kinase (AMPK) catalytic subunits, AAK-1 and AAK-2 in WT, lipl-4 Tg, daf-2(lf), isp-1(lf), and glp-1(lf) worms. (C) Reduction of AMPK using the loss-of-function mutant of aak-2, aak-2(lf) together with aak-1 RNAi knockdown decreases lifespan by 17% and 29% in the WT and lipl-4 Tg background, respectively. As a result, the lifespan extension caused by lipl-4 Tg is reduced from 72% to 48%. ***p < 0.001 by Log-rank test. The lifespan data are also in Supplementary file 8.
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 equality of proportions. (B) Heatmap showing the average levels of nucleoporin proteins NPP-6 and NPP-15 in Lyso-IP (IP) and flow-through (FT) samples from WT, lipl-4 Tg, daf-2(lf), and isp-1(lf) worms. Representative images of intestinal cells in WT, lipl-4 Tg (C), and daf-2(lf) (E) worms carrying LMP-1::RFP-3×HA and nucleus-enriched GFP, showing the accumulation of lysosomes around the perinuclear region in the lipl-4 Tg but not daf-2(lf) worms. Dashed lines circle intestinal cells and n marks the nucleus. Scale bar = 20 μm. Line graph showing the spatial distribution of lysosomes from the nuclear to peripheral region quantified by normalized regional RFP fluorescence signals in intestinal cells of WT, lipl-4 Tg (D), and daf-2(lf) (F) worms. N = 50 WT/33 lipl-4 Tg, 33 WT/28 daf-2(lf). Data are represented as mean ± standard deviation (SD). p values for (D) (from left to right): 1.23 × 10–7, 2.25 × 10–5, 0.00322, 0.368, 0.273, 0.0447, 0.00268, 1.20 × 10–5; p values for (F) (from left to right): 0.633, 0.0211, 0.00259, 0.0359, 0.767, 0.151, 0.106, 0.0671. lipl-4 Tg worms show lifespan extension compared to WT worms (G), which is fully suppressed by RNAi knockdown of npp-6 (H). ***p < 0.001, n.s. p > 0.05 by Log-rank test. daf-2(lf) worms show lifespan extension compared to WT worms (I), which is not affected by npp-6 RNAi knockdown (J). ***p < 0.001 by Log-rank test. The lifespan data are also in Supplementary file 8.
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 signals from the nuclear to the peripheral region in wild-type (WT), lipl-4 Tg (B), and daf-2(lf) (C) animals. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. p > 0.05 by Student’s t-test (unpaired, two-tailed) for each region. N = 50 WT/33 lipl-4 Tg, 33 WT/28 daf-2(lf). Data are represented as mean ± standard deviation (SD). p values for (B) (from left to right): 2.65 × 10−8, 3.19 × 10−8, 7.93 × 10−8, 3.62 × 10−7, 4.79 × 10−6, 2.98 × 10−5, 4.41 × 10−5; p values for (C) (from left to right): 0.357, 0.0529, 0.00611, 0.00246, 0.00985, 0.0261, 0.0423. isp-1(lf) worms show lifespan extension compared to WT worms (D), which is not affected by RNAi knockdown of npp-6 (E). ***p < 0.001 by Log-rank test. lipl-4 Tg worms show lifespan extension compared to WT worms (F), which is not affected by xpo-1 RNAi knockdown (G) and is partially suppressed by RNAi knockdown of ima-3 (H). ***p < 0.001 by Log-rank test.The lifespan data are also in Supplementary file 8. (I) The percentage of proteins with different subcellular localization is compared between lysosome-enriched proteomes from WT and daf-2 worms.
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 between lysosome-enriched proteomes using LMP-1 Lyso-IP and CTNS-1 Lyso-IP. (C) Pie chart showing subcellular location categories of lysosome-enriched proteins. (D) The proportion of candidates with lysosomal localization annotation in different candidate groups. ‘LMP-1 all’ and ‘CTNS-1 all’, all candidates from LMP-1 Lyso-IP and CTNS-1 Lyso-IP, respectively; ‘LMP-1 only’ and ‘CTNS-1 only’, candidates only identified from LMP-1 Lyso-IP or CTNS-1 Lyso-IP, respectively. (E) Normalized protein levels (z-score across samples) of autophagy-related components and mTORC1 signaling factors from CTNS-1 Lyso-IP proteomic analyses of wild-type (WT) worms and LMP-1 Lyso-IP proteomic analyses of WT and lipl-4 Tg worms. (F) Representative muscle images in the wrmScarlet::LMTR-3 knock-in line crossed with either LMP-1::mNeonGreen knock-in line or CTNS-1::mNeonGreen knock-in line. Scale bar = 20 μm. (G) Normalized protein levels (z-score across samples) of previously annotated lysosomal proteins from LMP-1 Lyso-IP proteomic analyses of WT and lipl-4 Tg worms and CTNS-1 Lyso-IP proteomic analyses of WT worms.
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 of CTNS-1 Lyso-IP (IP) and Flow-through (FT). Example images of knock-in lines with wrmScarlet::LMTR-3 and CTNS-1::mNeonGreen (C) and LMP-1::mNeonGreen (D) in hypodermis and intestine. Scale bar = 20 μm.
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 RNAi (E). Scale bar = 50 μm. RNAi knockdown of unc-32 or vha-5 decreases the lysosome number (****p < 0.0001) (F) but increases the lysosome size (****p < 0.0001, ***p < 0.001) (G). The average lysosome number and size per pair of intestinal cells were quantified. Data are shown as mean ± standard deviation (SD). Student t-test (unpaired, two-tailed) was performed between the empty vector and RNAi-treated groups. At least three independent experiments with ~10 worms in each were performed for each condition. n.s. p > 0.05, (H) RNAi knockdown of R144.6 and unc-32 (***p < 0.001) increase and decrease lysosomal pH, respectively. Lysosomal pH was calculated based on LysoSensor’s lifetime measured by Fluorescence Lifetime Microscopy. Data are shown as mean ± SD. Student’s t-test (unpaired, two-tailed) was performed between the empty vector and RNAi-treated groups. Two independent experiments with at least five worms in each were performed in R144.6 RNAi and unc-32 RNAi conditions. The vha-5 and slc36.2 RNAi knockdown did not show significant changes in one replicate and were not retested with another replicate. n.s. p > 0.05. (I) The structure of the R144.6 protein predicted by AlphaFold2 supports it as a solute carrier family transporter. (J) Confocal fluorescence microscopy images show that mNeonGreen signals from endogenously tagged R144.6 colocalize with LysoTracker Red signals in the hypodermis. Scale bar = 10 μm.
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. The relative LysoSensor changes were quantified in (F). ~10 worms were quantified in each condition. Data are shown as mean ± standard deviation (SD). Student’s t-test (unpaired, two-tailed) was performed between the empty vector and RNAi-treated groups (**p < 0.01, ****p < 0.0001).
Additional files
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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
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Supplementary file 2
Lysosome-enriched proteome exhibits tissue specificity.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp2-v2.xlsx
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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
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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
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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
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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
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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
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Supplementary file 8
Summary of lifespan analyses.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp8-v2.xlsx
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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
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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
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Supplementary file 11
File list of mass spectrum samples.
- https://cdn.elifesciences.org/articles/85214/elife-85214-supp11-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85214/elife-85214-mdarchecklist1-v2.docx
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Source code 1
Matlab code for lysosome distribution quantification.
- https://cdn.elifesciences.org/articles/85214/elife-85214-code1-v2.zip
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Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity
eLife 13:e85214.
https://doi.org/10.7554/eLife.85214
