Large-scale identification of plasma membrane repair proteins revealed spatiotemporal cellular responses to plasma membrane damage
- Okinawa Institute of Science and Technology Graduate University, Japan
Figures
Figure 1 with 1 supplement
Protein relocalization in response to SDS treatment.
(A) Schematic representation of screening methodology and images of Pkc1-GFP with or without 0.02% SDS treatment are shown. Scale bar, 5 μm. (B) Overlap of screening hits of C- and N-terminal libraries. p-Value for the significance of the overlap is indicated. Fisher’s exact test was performed. (C) Gene Ontology (GO) analysis of biological processes for the screening hits. Successfully observed proteins were used as the background protein sets. (D) Screening hits for six relocalization classes and the images of representative proteins in each class were shown. Numbers in parentheses indicate the number of proteins in the class. Scale bar, 2 μm.
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Figure 1—source data 1
Proteins whose localization changes in response to the SDS treatment.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Minor classes of localization changes in response to SDS treatment.
Representative images of GFP- or sfGFP-tagged proteins in minor classes with or without 0.02% SDS treatment are shown. Numbers in parentheses indicate the number of proteins in the class. Scale bar, 2 µm.
Figure 2
Laser damage assays identified 80 repair protein candidates.
(A) Schematic representation of screening methodology. Cells were imaged for 25 min in 30 s intervals after 405 nm laser damage. (B) 90 proteins change localization after laser damage. The classification of the repair protein candidates based on their localization changes, representative proteins, and representative biological processes in each category is shown. Scale bar, 2 μm. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. (C) Classification of repair protein candidates based on subcellular localization and domain. TMD+ represents the proteins that have transmembrane domains. The existence of transmembrane domains was predicted by TMHMM (Krogh et al., 2001). (D) Gene Ontology (GO) analysis of molecular functions for the repair protein candidates. The proteins whose localization changes in response to SDS treatment were used as background protein sets. (E) GO analysis of biological processes for the repair protein candidates. The proteins whose localization changes in response to SDS treatment were used as background protein sets. The ratio of the number of proteins associated with a specific GO term to the total number of proteins in the background is shown.
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Figure 2—source data 1
Repair protein candidates.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig2-data1-v1.xlsx
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Figure 2—source data 2
Quantification data of fluorescence signal of repair proteins.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig2-data2-v1.zip
Figure 3 with 3 supplements
The temporal order of the Pkc1 accumulation, polarized exocytosis, and clathrin-mediated endocytosis (CME) at the damage site.
(A) The representative images and normalized fluorescence intensity of Exo70-mNG (green) and Pkc1-mSc-I (purple) at the damage site after laser damage. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. n=10 cells. (B) The representative images and normalized fluorescence intensity of Pkc1-sfGFP (green) and Dnf1-mSc-I (purple) at the damage site after laser damage. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. n=8 cells. (C) Representative images, kymograph at the damage site, and fluorescence intensity at the damage site of Exo70-mNG (green) and Ede1-mSc-I (purple). n=10 cells. Yellow arrows show the damage site. White arrows show the coaccumulation of Exo70-mNG and Ede1-mSc-I at the damage site. (D) Representative images, kymograph at the damage site, and fluorescence intensity at the damage site of Dnf1-mNG (green) and Ede1-mSc-I (purple). n=9 cells. Yellow arrows show the damage site. White arrows show the coaccumulation of Dnf1-mNG and Ede1-mSc-I. Lines and shaded regions are the mean and the standard error of the mean, respectively. White scale bar, 2 μm.
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Figure 3—source data 1
Quantification data of fluorescence signal of respective repair proteins.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig3-data1-v1.zip
Figure 3—figure supplement 1
Summary of the accumulation time of repair proteins.
The repair protein candidates were grouped based on their functions. The box ranges from the accumulation times to the dispersion times of grouped proteins. See also Figure 2—source data 1.
Figure 3—figure supplement 2
Accumulation patterns of exocyst subunits.
Fluorescence intensity at the damage site of Exo70-mNG (green), (A) Exo84-mSc-I, (B) Sec3-mSc-I, (C) Sec5-mSc-I, (D) Sec6-mSc-I, (E) Sec8-mSc-I, (F) Sec10-mSc-I, and (G) Sec15-mSc-I (purple). Lines and shaded regions are the mean and the standard error of the mean, respectively. n=10 cells.
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Figure 3—figure supplement 2—source data 1
Quantification data of fluorescence signal of exocyst subunits.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig3-figsupp2-data1-v1.zip
Figure 3—figure supplement 3
Clathrin-mediated endocytosis (CME) continuously occurs at the damage site.
(A) Representative images of Sla1-GFP and Abp1-GFP. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. (B, C) Quantification of Sla1-GFP and Abp1-GFP signal at the damage site for three cells. Gray: the fluorescence intensity at the non-damaged site (control)+three times the standard deviation. (D) Fluorescence intensity at the damage site of Sla1-mNG (green) and Abp1-mSc-I (magenta) is shown. n=5 cells.
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Figure 3—figure supplement 3—source data 1
Quantification data of fluorescence signal of Sla1-GFP, Abp1-GFP, Sla1-mNG, Abp1-mSc-I.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig3-figsupp3-data1-v1.zip
Figure 4 with 3 supplements
Clathrin-mediated endocytosis (CME) proteins are required for polarized exocytosis at the damage site.
(A) The quantification results of fluorescence intensity of Myo2-sfGFP at the damage site and at the bud tip. n=13 for WT, n=12 for end3Δ, sla1Δ, rvs167Δ, and vrp1Δ. (B) The quantification results of the fluorescence intensity of Exo70-mNG at the damage site. n=18 for WT, n=12 for end3Δ, sla1Δ, and rvs167Δ, n=14 for vrp1Δ. Lines and shaded regions are the mean and standard error of the mean, respectively. The maximum fluorescence intensity at the damage site or fluorescence intensity changes at the bud tip were compared between the WT and each CME mutant using Dunnett’s multiple comparison test.
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Figure 4—source data 1
Quantification data of fluorescence signal of Pkc1-sfGFP and Exo70-mNG.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig4-data1-v1.zip
Figure 4—figure supplement 1
Growth screening of repair protein knockout mutants in plasma membrane (PM)/cell wall damage conditions.
(A) Schematic workflow and representative results of growth screening. The red square shows the sensitive mutants. Only the mutants that showed sensitivity to the stresses in two independent colonies were defined as sensitive to the stress. The results of the screening are shown in Figure 2—source data 1. (B) The overlap of the screening hits in the three PM/cell wall damage conditions is shown. (C) Growth assay of clathrin-mediated endocytosis (CME) mutants. A fourfold serial dilution of the yeast culture was prepared and spotted.
Figure 4—figure supplement 2
Representative images of Myo2-sfGFP and Exo70-mNG in clathrin-mediated endocytosis (CME) mutants.
(A) Representative images and whole-cell fluorescence quantification of Myo2-sfGFP in the indicated strains. The integrated density of whole-cell fluorescence of Myo2-sfGFP before laser damage is compared between WT and CME mutants using Dunnett’s test. n=13 for WT, n=12 for end3Δ, sla1Δ, rvs167Δ, and vrp1Δ. Scale bar, 2 µm. (B) Representative images and whole-cell fluorescence quantification of Exo70-mNG in the indicated strains. The integrated density of whole-cell fluorescence of Exo70-mNG before laser damage is compared between WT and CME mutants using Dunnett’s test. n=18 for WT, n=12 for end3Δ, sla1Δ, and rvs167Δ, n=14 for vrp1Δ. Scale bar, 2 µm. Yellow arrows show the damage site.
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Figure 4—figure supplement 2—source data 1
Quantification data of fluorescence signal of Pkc1-sfGFP and Exo70-mNG before laser damage.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig4-figsupp2-data1-v1.zip
Figure 4—figure supplement 3
Clathrin-mediated endocytosis (CME) proteins are not required for the degradation of Bni1-13xMyc and Sec3-GFP after SDS treatment.
(A) Immunoblotting of Bni1-13xMyc before and after SDS treatment (before (-), 1 hr, and 2 hr) in WT, rvs167∆, and end3∆. (B) Immunoblotting of Sec3-GFP before and after SDS treatment (before (-), 1 hr, and 2 hr) in WT, rvs167∆, and end3∆.
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Figure 4—figure supplement 3—source data 1
Original files of western blots.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
PDF files of western blots with sample labels.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig4-figsupp3-data2-v1.zip
Figure 5 with 3 supplements
Clathrin-mediated endocytosis (CME) at the bud tip directs repair proteins with transmembrane domains (TMDs) to the damage site.
(A) Representative images and the normalized fluorescence intensity at the bud tip of Dnf1-mNG and Ede1-mSc-I. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. Scale bar, 2 μm. (B) Kymograph of Dnf1-mNG and Ede1-mSc-I at the bud tip in WT and rvs167Δ. n=10 cells. (C–F) Max fluorescence intensity at the damage site and fluorescence intensity changes at the bud tip in WT and rvs167Δ. n=10 cells for Dnf1-mNG. n=11 cells for Slg1-sfGFP. n=12 cells for Sho1-GFP. n=10 cells for mNG-Snc1. Welch’s t-test was performed.
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Figure 5—source data 1
Quantification data of fluorescence signal of repair proteins at the bud tip.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig5-data1-v1.zip
Figure 5—figure supplement 1
Fluorescence intensity changes of clathrin-mediated endocytosis (CME) proteins at the bud tip.
(A) Representative images of Apl1-GFP, Ede1-GFP, and Ent1-GFP. (B) The ratio of normalized fluorescence intensity at 10 min at the bud tip to the normalized fluorescence intensity at 0 min at the bud tip for all repair protein candidates. Red: CME proteins. The list of CME proteins is adapted from Goode et al., 2015. The p-value was calculated by the two-sided Mann-Whitney U test.
Figure 5—figure supplement 2
Time course fluorescence intensity changes of Dnf1-mNG and Ede1-mSc-I at the bud tip in rvs167Δ.
Representative images of Dnf1-mNG and Ede1-mSc-I in rvs167Δ and normalized fluorescence intensity of Dnf1-mNG (green) and Ede1-mSc-I (purple) at the bud tip in rvs167Δ. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. Scale bar, 2 µm.
Figure 5—figure supplement 3
Time course fluorescence intensity changes of repair proteins with transmembrane domains (TMDs).
(A) Representative images of Slg1-sfGFP and the normalized fluorescence intensity at the damage site and at the bud tip in WT and rvs167Δ. Scale bar, 2 µm. (B) Representative images of Sho1-GFP and the normalized fluorescence intensity at the damage site and at the bud tip in WT and rvs167Δ. Scale bar, 2 µm. (C) Representative images of mNG-Snc1 and the normalized fluorescence intensity at the damage site and at the bud tip in WT and rvs167Δ. Scale bar, 2 µm. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. Scale bar, 2 µm.
Figure 6
mNG-Snc1 is recovered from the damage site to the bud tip after plasma membrane (PM) repair.
(A) Schematic of transient expression induction of mNG-Snc1 by Gal1 promoter and representative images of mNG-Snc1 and Ede1-mSc-I. After transcription activation of the Gal1 promoter by adding 3% galactose, we stop the expression by transferring the cells to glucose media. The cells were incubated for at least 3 hr before the laser damage assay. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. Scale bar, 2 μm. (B) Quantification of mNG-Snc1 (green) and Ede1-mSc-I (purple) at the damage site. (C) Quantification of mNG-Snc1 at the bud tip (green) and at the damage site (tomato). (D) The changes in the normalized mNG-Snc1 signal at the bud tip. n=8 cells.
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Figure 6—source data 1
Quantification data of fluorescence signal of mNG-Snc1 and Ede1-mSc-I.
- https://cdn.elifesciences.org/articles/108585/elife-108585-fig6-data1-v1.zip
Figure 7
Model of spatiotemporal cellular responses to plasma membrane (PM) damage in budding yeast.
We showed the hypothetical model of spatiotemporal PM damage responses in budding yeast. First, the degradation of Sec3 and Bni1 resolved the polarity competition between the bud tip and the damage site (Kono et al., 2012). Within 10 min after laser damage, clathrin-mediated endocytosis (CME) directs repair proteins with transmembrane domains (TMDs) to the damage site from the bud tip. At the damage site, polarized exocytosis and CME simultaneously occur, with exocytosis predominating approximately within 20 min and with CME predominating approximately 20 min after laser damage. CME targets repair proteins with TMDs from the bud tip to the damage site. The endocytosed PM proteins are retargeted to the bud tip again after the PM repair is finished. The retargeted PM proteins may be involved in the resumption of cell growth after PM repair. The numbers represent the temporal order of events.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Saccharomyces cerevisiae) | Budding yeast BY4741 background | Supplementary file 1 | ||
| Antibody | Anti GFP (Mouse monoclonal) | Roche/Merck | RRID:AB_390913 | WB 1:500 |
| Antibody | Anti Myc (Mouse monoclonal) | Santa Cruz Biotechnology | RRID:AB_627268 | WB 1:250 |
| Antibody | Anti-α-Tubulin (Rat monoclonal) | Bio-Rad | RRID:AB_325005 | WB 1:5000 |
| Software, algorithm | Fij | https://doi.org/10.1038/nmeth.2019 | RRID:SCR_002285 |
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://cdn.elifesciences.org/articles/108585/elife-108585-supp1-v1.xlsx
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Supplementary file 2
Yeast libraries used in this study.
- https://cdn.elifesciences.org/articles/108585/elife-108585-supp2-v1.xlsx
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Supplementary file 3
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/108585/elife-108585-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108585/elife-108585-mdarchecklist1-v1.docx
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Large-scale identification of plasma membrane repair proteins revealed spatiotemporal cellular responses to plasma membrane damage
eLife 14:RP108585.
https://doi.org/10.7554/eLife.108585.3
