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. Author manuscript; available in PMC: 2023 Jan 12.
Published in final edited form as: Cold Spring Harb Protoc. 2016 Jan 4;2016(1):pdb.prot086504. doi: 10.1101/pdb.prot086504

Detecting Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule Degradation

Nicholas J Palmisano 1,2, Alicia Meléndez 1,2,*
PMCID: PMC9835150  NIHMSID: NIHMS1704424  PMID: 26729906

Abstract

Autophagy has been shown to play an active role during the early stages of embryogenesis in C. elegans (Melendez and Neufeld 2008; Zhang et al. 2009; Kovacs and Zhang 2010; Al Rawi et al. 2011; Sato and Sato 2011). Although their exact function is unknown, P granules are ribonucleoprotein particles thought to play a part in germ cell specification (Strome and Lehmann 2007). The localization of P granules is restricted to the germline precursor cells in wild-type embryos, as a result of their degradation in the somatic cell lineage (Hird et al. 1996). Autophagy was shown to be required for the degradation of P granules, since mutations in various autophagy genes result in the accumulation of the P granule components, PGL-1 and PGL-3 (termed PGL granules), in the somatic cells of C. elegans embryos (Zhang et al. 2009; Zhao et al. 2009; Tian et al. 2010).

SEPA-1 was discovered as an adaptor protein important for the degradation of PGL granules in somatic cells through its interaction with LGG-1, linking PGL granules to the autophagosome for removal (Zhang et al. 2009). In wild-type animals, SEPA-1 protein aggregates begin to form at the 16-cell stage embryo and peak at the 100-cell stage, but disappear significantly by the comma stage (Zhang et al. 2009). However, in autophagy mutants, SEPA-1 aggregates persist past the comma stage, confirming that SEPA-1 is targeted to the autophagosome for removal (Zhang et al. 2009; Tian et al. 2010; Lu et al. 2011).

sqst-1 encodes the C. elegans homolog of the adaptor protein p62/SQSTM1, which has also been investigated during embryogenesis (Tian et al. 2010; Lu et al. 2011). In mammals, p62 binds to both LC3 and poly-ubiquitinated proteins, linking them to the autolysosomal pathway (Kihara et al. 2001; Bjorkoy et al. 2005; Pankiv et al. 2007; Tian et al. 2010). SQST-1::GFP expression is diffuse in the cytoplasm throughout embryogenesis; however, in autophagy mutants, the SQST-1::GFP protein localizes to positive punctate structures, similar to that of SEPA-1 (Tian et al. 2010; Lu et al. 2011). Overall, SEPA-1, SQST-1, and PGL-1 fusion reporters have allowed for the identification of new autophagy genes, by screening for mutant animals that lacked the degradation of these autophagy substrates (Tian et al. 2010). Therefore, such reporters can be used to identify additional genes required for normal autophagy activity during embryogenesis.

Materials:

Reagents:

2%−3% agarose (2–3 grams of agarose filled to volume with 100ml of sterile deionized water)

M9 Minimal Medium buffer <R>:

NGM agar plates <R>:

25mM Sodium Azide (NaN3)

Equipment:

Fluorescent microscope (Zeiss AxioImager A1)

Microscope coverslips (1.22 mm X .13mm)

Microscope slides (75mm X 25 mm X .96mm)

Standard platinum wire pick

Strains:

bpIs151[Psqst-1::SQST-1::GFP, unc-76(+)] (Tian et al. 2010)

bpIs131[Psepa-1::SEPA-1::GFP, unc-76(+)] (Tian et al. 2010)

bnIs1[Ppie-1::GFP::PGL-1, unc-119(+)] (Tian et al. 2010)

RD210: unc-119(ed3); Is[Ppie-1::GFP::mCherry::LGG-1; unc-119(+)]

Graphic Flow Chart #2:

graphic file with name nihms-1704424-f0001.jpg

Method:

Standard Fluorescent Microscopy:

  1. Allow ∼50 gravid adults to lay eggs on a single plate for 10–15 minutes at the desired temperature, and then transfer the gravid adults to fresh plates. This can be repeated as desired. This will result in multiple plates containing tightly synchronized embryos.

  2. Calculate the time that recently laid embryos will require to reach the desired stage of embryogenesis (i.e. at 20°C, for ∼100 cell stage embryos, wait an additional ∼200 minutes). The amount of time required to reach the desired stage of embryogenesis will vary according to temperature.

  3. Once the stage of interest has been reached, imaging of the embryos can begin. Apply a single drop of molten 2%−3% agarose onto a microscope slide. Apply another microscope slide on top of the slide containing the drop of agarose to form a thin agarose pad.

  4. Take ∼2.5 uL of M9 buffer and place it in the center of the agarose pad.

  5. Using a platinum wire, first dab the pick into bacteria and then GENTLY pick embryos and submerge them into the M9 buffer on the slide.

  6. Use 63X or 100X magnifications to quantify and/or take images of the fluorescent positive puncta found within the embryos. Embryos should not be kept on slides for more than 15 minutes.

Data Analysis:

Changes in autophagic activity during embryogenesis are usually visualized by monitoring changes in the expression pattern of a fluorescent reporter relative to the control. Such changes may be easily observed since, under wild-type conditions, some reporters may have a diffuse cytoplasmic expression, whereas, under conditions of defective autophagy, the reporter may accumulate into aggregate-like structures or positive punctate structures. An example of this can be found by comparing wild-type animals and autophagy defective embryos expressing SQST-1::GFP or GFP::LGG-1 (Tian et al. 2010; Lu et al. 2011). Other changes may not be so prominent and may include an increase in the number or size of pre-existing protein aggregates, as is found when comparing wild-type animals and autophagy mutant embryos treated with anti-LGG-1 primary antibodies (Tian et al. 2010; Lu et al. 2011).

To determine whether subtle changes in the expression of a reporter protein are significant will require additional methods. One method is to quantify the positive punctate structures, however, manual quantification of aggregates within embryos can be extremely difficult as autofluorescence is usually too high and the number of positive puncta too great. Image analysis programs may provide better quantification abilities; however, even with image analysis programs, one may not distinguish true punctate structures from autofluorescence. One option for measuring changes in the number of autophagosomes, or active autophagy, during embryogenesis, is to measure the fluorescence intensity of the autophagy reporter used (Morselli et al. 2011). Such changes in fluorescent intensity can be used to determine whether there is elevated or decreased expression of the reporter, which may be indicative of increased or decreased autophagy.

Troubleshooting:

Problem: Embryos have not reached the expected stage from the time of egg lay (step 2).

Solution: It is possible that the particular strain being used has a delay in development compared to wild-type animals. Calculate the time required to reach the developmental stage of interest.

Problem: Difficulties arise when attempting to separate embryos from the platinum wire pick (Step 5).

Solution: CAREFULLY press the platinum wire against the agarose pad several times, without tearing it, to release embryos.

Problem: Positive puncta found within embryos are too large to quantify (step 6)

Solution: Image processing and analysis software, such as ImageJ or MetaMorph, can be used to quantify puncta number and/or fluorescent intensity by following the instructions provided by the software.

Related Techniques:

Immunofluorescence with anti-LGG-1, anti-SQST-1, and anti-SEPA-1 antibodies

Discussion:

When evaluating reporter expression, to determine if a gene of interest plays a role in autophagy during embryogenesis, changes in the expression pattern of the reporter may not be apparent compared to controls. As a result, additional methods, such as image processing software, will be required to ensure that such changes are significant. If choosing to monitor GFP::LGG-1 or SQST-1::GFP expression, care should also be taken. In mammals, p62 can form cytoplasmic inclusions unrelated to autophagosomes (Zatloukal et al. 2002; Bjorkoy et al. 2005). Furthermore, in mammals, LC3 can become incorporated into p62 cytosolic aggregates through its direct interaction with p62 (Shvets et al. 2008). Although, this has not been formally shown in C. elegans, if visualizing SQST-1::GFP or GFP::LGG-1 positive structures, additional reporters (Table 2) may be required to determine whether such aggregates are true autophagosomes or membrane free inclusion bodies. Overall, when analyzing autophagy activity in embryos, careful interpretation of results may require additional methods and/or techniques.

Recipes:

M9 Minimal Medium buffer:

22mM KH2PO4

22mM Na2HPO4

85mM NaCl

1mM MgSO4

Autoclave for 15 minutes on liquid cycle

NGM agar plates (per 4L):

68g Bacto Agar powder

12g NaCl

10g Bacto peptone

4ml 5mg/ml of Cholesterol in 100% EtOH

Add 3.9L deionized H2O

Autoclave for 70 minutes on liquid cycle, let cool, and add the following:

4ml 1M CaCl2

4ml 1M MgSO4

100ml 1M KPO4

References

  1. Al Rawi S, Louvet-Vallee S, Djeddi A, Sachse M, Culetto E, Hajjar C, Boyd L, Legouis R, Galy V. 2011. Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission. Science 334: 1144–1147. [DOI] [PubMed] [Google Scholar]
  2. Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H, Johansen T. 2005. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtininduced cell death. J Cell Biol 171: 603–614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hird SN, Paulsen JE, Strome S. 1996. Segregation of germ granules in living Caenorhabditis elegans embryos: Celltype-specific mechanisms for cytoplasmic localisation. Development 122: 1303–1312. [DOI] [PubMed] [Google Scholar]
  4. Kihara A, Noda T, Ishihara N, Ohsumi Y. 2001. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J Cell Biol 152: 519–530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kovacs AL, Zhang H. 2010. Role of autophagy in Caenorhabditis elegans. FEBS Lett 584: 1335–1341. [DOI] [PubMed] [Google Scholar]
  6. Lewis JA, Fleming JT. 1995. Basic culture methods. Methods Cell Biol 48: 3–29. [PubMed] [Google Scholar]
  7. Lu Q, Yang P, Huang X, Hu W, Guo B, Wu F, Lin L, Kovacs AL, Yu L, Zhang H. 2011. The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes. Dev Cell 21: 343–357. [DOI] [PubMed] [Google Scholar]
  8. Manil-Ségalen M, Culetto E, Legouis R, Lefebvre C. 2014. Interactions between endosomal maturation and autophagy: Analysis of ESCRT machinery during Caenorhabditis elegans development. Methods Enzymol 534: 93–118. [DOI] [PubMed] [Google Scholar]
  9. Meléndez A, Neufeld TP. 2008. The cell biology of autophagy in metazoans: A developing story. Development 135: 2347–2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Morselli E, Marino G, Bennetzen MV, Eisenberg T, Megalou E, Schroeder S, Cabrera S, Benit P, Rustin P, Criollo A, et al. 2011. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. J Cell Biol 192: 615–629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Overvatn A, Bjorkoy G, Johansen T. 2007. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282: 24131–24145. [DOI] [PubMed] [Google Scholar]
  12. Sato M, Sato K. 2011. Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos. Science 334: 1141–1144. [DOI] [PubMed] [Google Scholar]
  13. Shvets E, Fass E, Scherz-Shouval R, Elazar Z. 2008. The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes. J Cell Sci 121: 2685–2695. [DOI] [PubMed] [Google Scholar]
  14. Strome S, Lehmann R. 2007. Germ versus soma decisions: Lessons from flies and worms. Science 316: 392–393. [DOI] [PubMed] [Google Scholar]
  15. Tian Y, Li Z, Hu W, Ren H, Tian E, Zhao Y, Lu Q, Huang X, Yang P, Li X, et al. 2010. C. elegans screen identifies autophagy genes specific to multicellular organisms. Cell 141: 1042–1055. [DOI] [PubMed] [Google Scholar]
  16. Zatloukal K, Stumptner C, Fuchsbichler A, Heid H, Schnoelzer M, Kenner L, Kleinert R, Prinz M, Aguzzi A, Denk H. 2002. p62 is a common component of cytoplasmic inclusions in protein aggregation diseases. Am J Pathol 160: 255–263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zhang Y, Yan L, Zhou Z, Yang P, Tian E, Zhang K, Zhao Y, Li Z, Song B, Han J, et al. 2009. SEPA-1 mediates the specific recognition and degradation of P granule components by autophagy in C. elegans. Cell 136: 308–321. [DOI] [PubMed] [Google Scholar]
  18. Zhao Y, Tian E, Zhang H. 2009. Selective autophagic degradation of maternally-loaded germline P granule components in somatic cells during C. elegans embryogenesis. Autophagy 5: 717–719. [DOI] [PubMed] [Google Scholar]

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