A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage
- Calico Life Sciences LLC, United States
- Department of Molecular and Cellular Biology, University of California, Berkeley, United States
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, United States
Figures
Figure 1 with 2 supplements
Identification of genes that prevent protein aggregation in maturing C. elegans oocytes.
(A) Diagram of the C. elegans germline. (B) Genome-wide RNAi-screen workflow. High-resolution images from the validation rounds are represented here. Scale bars, 10 µm; white arrows, GFP::RHO-1 …
Figure 1—figure supplement 1
Additional examples of phenotypes from the primary and the orthogonal screens, and the most-highly enriched GO-terms.
(A) RNAi clones causing GFP::RHO-1 aggregates in hermaphrodite oocytes (SA115), visualized at low magnification (×20 objective) during the primary screening. Scale bars, 20 µm; white arrows, …
Figure 1—figure supplement 2
Visualizing ER morphology and GFP::RHO-1 aggregates in very early oocytes.
ER morphology and GFP::RHO-1 localization in the first, newly formed oocytes from mCherry::SP12 expressing females (CF4557). Orange arrows, ER patches; white arrows, GFP::RHO-1 aggregates. Scale …
Figure 2 with 6 supplements
Endoplasmic reticulum (ER) morphology is regulated by sperm and correlates with protein aggregation.
(A) ER morphology in the germline of hermaphrodite (OCF15) and female (CF4542) animals, visualized using an mCherry::SP12 reporter. Orange arrows, ER patches. Scale bar, 10 µm. (B) GFP::RHO-1 …
Figure 2—figure supplement 1
Endoplasmic reticulum (ER) morphologies in hermaphrodite and female oocytes.
(A) Comparison of ER morphology in oocytes from hermaphrodites (OCF15) and females (CF4542) expressing the ER reporter mCherry::SP12 in the germline. Oocytes in the female gonad stack over time as …
Figure 2—figure supplement 2
Supplementary data related to Figure 2B.
Endoplasmic reticulum morphology and GFP::RHO-1 localization in the first, newly formed oocytes from mCherry::SP12-expressing hermaphrodites (CF4552). Scale bars,10 µm. Note that the GFP::RHO-1 …
Figure 2—figure supplement 3
Supplementary data related to Figure 2D.
Transmission electron micrographs from hermaphrodite (N2E) and female (CF4101) oocytes. (A) Female oocyte. Arrows, endoplasmic reticulum (ER) stacks. Left panel, magnification: ×1200, scale bar, 2 …
Figure 2—figure supplement 4
Simultaneous visualization of the alteration in endoplasmic reticulum (ER) morphology and clearance of aggregates in female oocytes (CF4560) as a function of time, following mating.
The earliest changes in the mCherry::SP12 signal (ER) were observed ~10 min after mating, whereas the first signs of aggregate (NMY-2::GFP) clearance appeared later, between 20 and 30 min. The ER …
Figure 2—figure supplement 5
Simultaneous visualization of oocyte ER morphology and protein aggregates in an unmated female (CF4560).
(A) The mCherry::SP12 and NMY-2::GFP signals in oocytes from CF4560 females visualized as in Figure 2—figure supplement 4 but not subjected to mating, to control for photobleaching. A single …
Figure 2—figure supplement 6
Changes in NMY-2::GFP signal following mating, as a function of time; and representative ER morphologies in assay-pool knockdowns.
(A) NMY-2::GFP signal skewness as a function of time in the proximal oocytes of CF4560 females, either subjected to mating with CB1490 males (white) or not exposed to males (pink). The mated females …
Figure 3 with 4 supplements
Sperm-dependent lysosomal localization of VHA-13.
(A) Distribution of GFP::VHA-13 in proximal oocytes of hermaphrodites (PHX1414) and females (CF4599). (B) LysoTracker-stained oocytes from GFP::VHA-13-expressing hermaphrodites and females for …
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Figure 3—source data 1
Skewness of oocyte GFP::VHA-13 signal as a function of time post mating.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Attempts to visualize endogenously tagged V-ATPase subunits in the germline.
Expression of endogenously tagged V-ATPase subunits from the V0 domain (A, B) and the V1 domain (C–E), and of a single-copy integrated germline transgene (F). None of the strains exhibited …
Figure 3—figure supplement 2
Investigating VHA-13 expression and function in the germline.
(A) Attempts to visualize endogenously tagged wrmScarlet::VHA-13 expression in the germline of PHX731 animals by dissecting gonads (n = 10 worms) to distance them from somatic tissues where it is …
Figure 3—figure supplement 3
GFP::VHA-13 localization in the presence and absence of sperm (Supplementary data related to Figure 3A).
Additional images showing the distribution of GFP::VHA-13 in proximal oocytes of (A) hermaphrodites (PHX1414) and (B) females (CF4599). Scale bars, 10 µm. (C) Changes in oocyte GFP::VHA-13 …
Figure 3—figure supplement 4
Supplementary data related to Figure 3B.
Additional images of the LysoTracker-stained oocytes from GFP::VHA-13-expressing (A) hermaphrodites (PHX1414) and (B) females (CF4599) for visualization of acidic lysosomes. The two signals …
Figure 4 with 1 supplement
Lysosome acidification and GFP::VHA-13 localization following knockdown of genes identified by the screen.
(A) LysoTracker staining in hermaphrodites (SA115) subjected to RNAi. The LysoTracker signal is represented as the ratio of proximal oocyte to distal germline staining. Staining ratios obtained from …
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Figure 4—source data 1
LysoTracker signal intensity ratios (maturing oocytes/distal germline) in assay pool knockdowns.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig4-data1-v1.xlsx
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Figure 4—source data 2
Skewness of oocyte GFP::VHA-13 signal in assay pool knockdowns.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Lysosomal acidification and GFP::VHA-13 localization in the germline following knockdowns of orphan genes.
(A) Analysis of germline LysoTracker staining in hermaphrodites (SA115) subjected to RNAi for the orphan genes. The LysoTracker signal is represented as the ratio of proximal oocyte to distal …
Figure 5 with 3 supplements
Analysis of mitochondrial membrane potential and ESCRT-complex subunits.
(A) Analysis of oocyte ΔΨ using DiOC6(3) staining in RNAi-treated hermaphrodites (OCF15), represented as the ratio of proximal oocyte to distal germline staining. Staining ratios obtained from N2E …
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Figure 5—source data 1
DiOC6(3) signal intensity ratios (maturing oocytes/distal germline) in assay pool knockdowns.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig5-data1-v1.xlsx
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Figure 5—source data 2
LysoTracker signal intensity ratios (maturing oocytes/distal germline) in ESCRT subunit knockdowns.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig5-data2-v1.xlsx
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Figure 5—source data 3
BioTracker ATP-Red staining of oocytes.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig5-data3-v1.xlsx
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Figure 5—source data 4
Skewness of oocyte GFP::VHA-13 signal in ESCRT subunit knockdowns.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig5-data4-v1.xlsx
Figure 5—figure supplement 1
Analysis of germline mitochondrial membrane potential; and effect of Krebs cycle gene knockdowns on GFP::RHO-1 aggregates.
(A) Analysis of germline DiOC6(3) staining in hermaphrodite animals (OCF15) subjected to RNAi for the orphan genes, represented as the ratio of proximal oocyte to distal germline staining. Staining …
Figure 5—figure supplement 2
Analysis of germline ATP levels.
(A) BioTracker ATP-Red stained germlines of dye-fed animals also subjected to knockdowns of electron transport chain (ETC) subunits; proximal germlines, white outlines. Scale bars, 20 µm. (B) …
Figure 5—figure supplement 3
Consequences of ESCRT gene knockdown on GFP::RHO-1 aggregates and GFP::VHA-13 localization.
(A) GFP::RHO-1 aggregation and LysoTracker signal in hermaphrodites (SA115) subjected to ESCRT-complex RNAi. The proximal oocyte/distal germline ratio of LysoTracker intensity is indicated for each …
Figure 6 with 4 supplements
Analysis of protein aggregation and lysosome acidification in somatic tissues.
(A) Representative images of CF4609 animals expressing KIN-19::split-wrmScarlet11 and somatic split-wrmScarlet1-10 at days 1, 4, 7, and 11 of adulthood. Maximum intensity projections of 3D stacks …
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Figure 6—source data 1
Number of KIN-19 aggregates per animal.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig6-data1-v1.xlsx
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Figure 6—source data 2
Skewness of intestinal LysoTracker signal.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig6-data2-v1.xlsx
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Figure 6—source data 3
Quantitative RT‐PCR analysis of mRNA levels in ESCRT subunit knockdowns.
- https://cdn.elifesciences.org/articles/62653/elife-62653-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
Supplementary data related to Figure 6B.
The same image sets of KIN-19 aggregates used in Figure 6B were scored again, blindly, by three additional investigators (A–C). Mann–Whitney test was used to calculate the statistical significance, …
Figure 6—figure supplement 2
LysoTracker staining in the intestines of animals subjected to gene knockdowns that caused KIN-19::split-wrmScarlet aggregation in the head.
Due to variability in dye uptake and staining, the actual LysoTracker signal intensities within the intestines were not evaluated. Instead, the mean skewness values, s, indicative of inhomogeneity …
Figure 6—figure supplement 3
Q35::YFP aggregation in body-wall muscle with age; and following assay-pool knockdowns.
(A) Representative images of AM140 animals expressing the polyQ reporter Q35::YFP in body wall muscle cells imaged at days 1, 2, and 3 of adulthood, with appearance of age-associated aggregates on …
Figure 6—figure supplement 4
Investigating the contribution of ESCRT genes in preventing KIN-19 aggregation.
(A) Representative images of CF4609 animals expressing KIN-19::split-wrmScarlet11 and somatic split-wrmScarlet1-10 subjected to ESCRT-complex RNAi or empty-vector negative control from the L1 stage, …
Figure 7
Model: biological processes implicated in the clearance of protein aggregates during oocyte maturation.
Tables
Table 1
Functional gene categories from the proteostasis screen.
| Biological process | RNAi target | Gene code | Normal ER architecture | GFP::VHA-13 puncta | Lysosome acidification | Reduction in ΔΨ |
|---|---|---|---|---|---|---|
| Protein degradation | pbs-7 | F39H11.5 | No | No | No | No |
| rpn-6.1 | F57B9.10 | Mixed | No | No | No | |
| Protein synthesis (translation) | rps-20 | Y105E8A.16 | No | No | No | No |
| rpl-3 | F13B10.2 | No | No | No | No | |
| Protein folding (chaperones) | cct-1 | T05C12.7 | No | Reduced | No | No |
| cct-5 | C07G2.3 | No | Reduced | No | No | |
| Cytoskeleton-associated process | act-1 | T04C12.6 | No | No | No | No |
| pat-3 | ZK1058.2 | No | No | No | No | |
| ani-2 | K10B2.5 | No | No | No | No | |
| ER protein homeostasis | hsp-4 | F43E2.8 | Yes | No | No | No |
| spcs-1 | C34B2.10 | Yes | No | No | No | |
| srpa-68 | F55C5.8 | Yes | No | No | No | |
| Vesicle-mediated transport (trafficking) | copb-2 | F38E11.5 | No | No | No | No |
| sec-24.1 | F12F6.6 | Inconclusive | No | No | No | |
| sar-1 | ZK180.4 | Mixed | No | No | No | |
| Lysosome acidification (V-ATPase) | vha-13 | Y49A3A.2 | Yes | No signal | No | No |
| vha-12 | F20B6.2 | Yes | No | No | No | |
| vha-2 | R10E11.2 | Yes | No | No | No | |
| ATP synthase | atp-3 | F27C1.7 | Yes | Yes | Yes | No |
| atp-2 | C34E10.6 | Yes | Yes | Yes | No | |
| RNA processing | ess-2 | F42H10.7 | No | Reduced | No | No |
| cgh-1 | C07H6.5 | Mixed | No | No | No | |
| let-711 | F57B9.2 | Mixed | Reduced | No | No | |
| Calcium ion transport | itr-1 | F33D4.2 | No | Reduced | No | No |
| sca-1 | K11D9.2 | Yes | Reduced | No | No | |
| Orphans | kin-2 | R07E4.6 | Mixed | No | No | No |
| ttr-14 | T05A10.3 | Mixed | No | No | No | |
| par-5 | M117.2 | Yes | No | No | No | |
| xpo-1 | ZK742.1 | Yes | No | No | No | |
| srab-17 | T11A5.2 | Yes | Reduced | No | No | |
| rmd-2 | C27H6.4 | Yes | No | No | No | |
| dlg-1 | C25F6.2a.1 | No | No | No | No | |
| lgc-46 | Y71D11A.5 | Yes | Reduced | No | No | |
| aco-2 | F54H12.1 | Yes | No | No | No | |
| imb-1 | F28B3.8 | Inconclusive | No | No | No | |
| ESCRT subunits* | vps-20 | Y65B4A.3 | Not tested | Yes | Yes | Not tested |
| vps-28 | Y87G2A.10 | Not tested | Yes | Yes | Not tested | |
| vps-37 | CD4.4 | Not tested | Yes | Yes | Not tested | |
| vps-54 | T21C9.2 | Not tested | Yes | Yes | Not tested | |
| List of representative genes from each functional category that were selected for the assay pool and their effects on ER morphology, lysosome acidification, GFP::VHA-13 localization, and mitochondrial membrane potential. *The ESCRT complex subunits were not derived from the original screen but from subsequent candidate testing. ER: endoplasmic reticulum. | ||||||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| RNAi reagents (Caenorhabditis elegans) | Multiple | C. elegans RNAi Collection (Source BioScience) | RRID:SCR_017064 | Supplementary file 1 |
| Strains, (Caenorhabditis elegans) | Multiple | Caenorhabditis Genetics Center (CGC) | RRID:SCR_007341 | Supplementary file 2 |
| Software, algorithm | GraphPad Prism 9.0.1 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | ImageJ/Fiji | Fiji | RRID:SCR_002285 |
Additional files
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Supplementary file 1
Germline proteostasis candidates captured in the screen.
List of candidates obtained from primary screening and validation rounds with GFP::RHO-1. The phenotypic strengths and fertility status are indicated for each. Candidates that were discarded on the basis of their known functions, possible off-target effects, or the failure to pass orthogonal verification with NMY-2::GFP are indicated accordingly.
- https://cdn.elifesciences.org/articles/62653/elife-62653-supp1-v1.xlsx
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Supplementary file 2
List of C. elegans strains.
Description of all strains used in the study, including the corresponding genotypes and sources.
- https://cdn.elifesciences.org/articles/62653/elife-62653-supp2-v1.xlsx
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Supplementary file 3
Candidates from primary screen that did not pass validation.
This list is included because some of these candidates could be false negatives, as was the case for the ESCRT-complex genes. Also included are the gene ontology terms (biological process) they represent.
- https://cdn.elifesciences.org/articles/62653/elife-62653-supp3-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62653/elife-62653-transrepform-v1.docx
