LGG-1/GABARAP lipidation is not required for autophagy and development in Caenorhabditis elegans
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Abstract
The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans , but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.
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Referee #3
Evidence, reproducibility and clarity
In an interesting study, Leboutet et al. here make excellent use of CRISPR techniques to investigate the role of C-terminal di-glycine sequences important for lipidation in the two Atg8 orthologs LGG-1 and LGG-2 in the autophagy process and in C. elegans development. A main finding from the study is that the LGG-1(G116A) variant, except for visible LGG-1 punctae staining (likely representing autophagosomes), can perform all functions compared to WT LGG-1, suggesting that these can potentially be uncoupled from membrane conjugation, an unexpected concept.
To this end, an unaddressed concern in this study is that it has not been …
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Referee #3
Evidence, reproducibility and clarity
In an interesting study, Leboutet et al. here make excellent use of CRISPR techniques to investigate the role of C-terminal di-glycine sequences important for lipidation in the two Atg8 orthologs LGG-1 and LGG-2 in the autophagy process and in C. elegans development. A main finding from the study is that the LGG-1(G116A) variant, except for visible LGG-1 punctae staining (likely representing autophagosomes), can perform all functions compared to WT LGG-1, suggesting that these can potentially be uncoupled from membrane conjugation, an unexpected concept.
To this end, an unaddressed concern in this study is that it has not been ruled out if LGG-1(G116A) perhaps can still trigger an unspecified entity to associate with membranes. Specifically, the authors identify a lower band (referred to as an unexpected, minor band) in Fig 1C for G116A and G116AG117A, but do not investigate the nature of this band (noting, importantly, that these two mutants show normal development). Immuno-EM could be very useful here.
Importantly, all of the different LGG-1 mutants are not equally investigated (as done in Fig 1F-K), which is a missed opportunity for the study overall (eg G116AG117* in Fig. 2M and 4). In particular, comparative Western blots are missing for all of the different proteins.
Lastly, the study is missing a discussion of the ability of LGG proteins to dimerize (not mentioned at all), a deeper analysis of LGG-2 (later stages than 15 cells, Fig 5D, and of even more significance, EM of double mutants - are there really autophagosomes formed in these?), as well as a more in-depth investigation of ATG-4 interactions (atg-4 investigated only in Fig. 1N, but then never again), which could also help address possible mechanisms involving differentially interacting binding partners.
Other discussion points worth further elaboration includes how removal of paternal mitochondria in the absence of autophagosomes without LGG-1 and LGG-2 could take place (again, what does EM look like? This is a particularly important implication of this study, which warrants further study), as well as how the new study's finding possibly impact the use of transgenic C. elegans GFP::LGG-1 markers, including a G116A marker that the authors have published and used as a negative control.
Other relevant points:
Fig 2N and S2D are replicated.
Error bars are missing in Fig 3I.
Fig. 5K should be quantified over multiple repeats.
Fig 7 feels like almost 'walking' backwards, may be more efficiently integrated elsewhere in the manuscript (it is also not clear why lgg-2 RNAi is used here, instead of the mutants that are used everywhere else in the study?). Moreover, the authors may want to consider discussing Fig 3/development first (considering the reader has been informed that lgg-1 is an essential gene,- to this point, it is only later made clear that the lethal allele has 8% 'breakthroughs - are these the animals analyzed?) and Fig. 6/EM together with Fig. 1.
The yeast section is highlighted in the abstract whereas all data are in supplements; overall it could be better integrated. In particular, sequence alignments and Western blots are missing here.
Result section should be revisited for clarity and language, including written in past tense.
Significance
Insights into the role of a C-terminal di-glycine sequences in Atg8 is useful for our understanding of how especially Caenorhabtidis species may engage different precursor vs cleaved Atg8 isoforms for various biological functions. In particular, the interesting and novel concept proposed by the authors in this study is that LGG-1 possesses functions independently of membrane conjugation, which may have potential implications the use of lipidated Atg8 reporters as markers, but also how autophagosomes are formed and function more broadly. However, further evidences is needed to support this 'negative' finding, as commented on above.
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Referee #2
Evidence, reproducibility and clarity
Leboutet et al. use a clever strategy to test the role of LC3 modifications in animal cells. They generate an allelic series of cleavage site mutants of the major LC3 isoform in C. elegans, LGG-1. They convincingly demonstrate that a non-cleavable precursor form of LC3(AA) is unable to localize or function during various forms of macroautophagy, embryonic development, adult survival, or cell death/corpse clearance. A pre-cleaved intermediate form of LC3(A*) is also unable to localize or function during various forms of macroautophagy and has neomorphic characteristics visualized by EM and corpse clearance, but fully functions to …
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Referee #2
Evidence, reproducibility and clarity
Leboutet et al. use a clever strategy to test the role of LC3 modifications in animal cells. They generate an allelic series of cleavage site mutants of the major LC3 isoform in C. elegans, LGG-1. They convincingly demonstrate that a non-cleavable precursor form of LC3(AA) is unable to localize or function during various forms of macroautophagy, embryonic development, adult survival, or cell death/corpse clearance. A pre-cleaved intermediate form of LC3(A*) is also unable to localize or function during various forms of macroautophagy and has neomorphic characteristics visualized by EM and corpse clearance, but fully functions to promote embryonic development. Surprisingly, mutating the predicted cleavage site of LC3(AG) results in defects in localization, but only a mild delay in autophagic flux. Similarly, LC3(AG) mutants show no defects in viability or embryonic development, which the authors show is partially due to the function of the other LC3 isoform, LGG-2.
Major comments:
What is the new LGG form * in Fig. 1C? Does the Mass Spec data give any hints? The authors imply that this is not lipidated, but show no direct evidence for this statement. There are reports of LC3 conjugation to lipids beside PE, such as PS. Could this represent a switch form LC3-PE to LC3-PS? Or simply cleavage and lipidation at G117? The lack of localization to autophagosomes convincingly demonstrates that this form * does not act like the classic form II, which was thought to be the functional form of LC3, but more information about this isoform would be needed to convincingly make the author's conclusions about lipidation.
The text compares the number of omegasomes vs phagophores vs autophagosomes and refers to Fig. 7E-G, but these graphs do not clearly identify the number of double-positive and single-positive populations, making it impossible to interpret this data. A graph similar to Fig. S5A should replace 7E-G to clearly convey this data.
Fig. 7E vs 7P - Why are there twice as many ATG-18 dots in 7P controls? Is one OP50-fed and the other HT115-fed? Or are the strains different? Why this is different isn't clear from the methods and is missing from the worm strain list.
Fig. S4F - I'm not sure of the utility of the LGG-1 rescue experiments in yeast. WT LGG-1 expression doesn't appear to significantly rescue atg8∆ mutants and it's not clear that there is any significant difference between different LGG-1 isoforms, especially given the broken y-axis. Also showing n=1 and missing statistics. The other yeast experiments are more interpretable and these findings do not significantly add to the paper.
Minor comments:
First half of the first paragraph of the introduction is under-referenced. Please cite relevant review articles. Introduction could also be shortened and more to the point.
Missing statistics in Fig. 1L right. Can't conclude it's increased if not significant.
Fig. 1N is not discussed in the manuscript.
Fig. 3 would be improved by maintaining the color scheme from Fig. 2
Fig. 3H and Fig. 4D are showing similar data in opposite ways (viability vs. lethality). For your reader's sake, please use the same measure for the same assay.
There is no 5-cell stage. C. elegans early embryonic stages are 1, 2, 3, 4, 6, 7, 8, 12, 14, 15.
The relative prevalence of LGG-2-I vs LGG-2-II should be presented in Fig. 5K, similar to the analysis of LGG-1 isoforms in Fig. 1C. It appears that LGG-2 conjugation is being altered in various lgg-1 alleles.
Fig. 6H - EM counts are typically represented as number per section area, not section. The size of cell sections can vary by a large amount.
The authors refer to G116AG117* as gain-of-function, but this is confusing given all the LGG-1 functions lost. A more accurate term could be neomorphic, although the authors haven't performed the genetics to test whether the allele is antimorphic (i.e. G116AG117*/null).
Why wasn't the double alanine mutant used in any assays past Fig. 3?
Fig. 7R right model - Phagophore membranes need to be connected at the ends - What are the light green circles representing? - Why does the blue G116A mutant localize to the cargo in the model? The author's said they didn't observe any localization.
Why is Fig. 2N identical to Fig. S3D? There's no need to include the same data twice. Also, both contain an error on the y-axis (15 instead of 5).
Discussion - P. 12 - "Our genetic data indicate that form I of LGG‐1 is sufficient for initiation, elongation and closure of autophagosomes". Indicate is an overstatement. The authors do not perform assays for initiation, elongation or closure.
Discussion - P. 12 - "paternal mitochondria could be degraded by autophagosomes devoid of both LGG‐1 and LGG‐2 " - I couldn't find data in this paper where paternal mitochondria are shown to never have LGG-1 or LGG-2 on them. A single time point analysis isn't sufficient to demonstrate that for molecules that dynamically associate and disassociate with membranes.
Significance
This study shatters our existing models of LC3 function. That LC3 could show any function without localizing to autophagosomes or other structures goes against our current understanding of LC3 function, making this study incredibly important for the autophagy field and the myriad autophagy-relevant clinical fields.
My expertise is C. elegans genetics, embryonic development, membrane trafficking, and non-canonical autophagy.
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Referee #1
Evidence, reproducibility and clarity
The manuscript by Laboutet et al., titled: "LGG-1/GABARAP lipidation is dispensable for autophagy and development in C. elegans," describes the potential function of a nonlipidated LGG-1 mutant containing a G116A mutation. Comparison of a G116A missense mutation to the lgg-1 null mutation or a lgg-1(G116A>G117*) suggests that there is some function retained in the G116A missense mutation. The authors claim that no foci form in the lgg-1(G116A) mutants and take this to mean that there is no lipidation. Assays for autophagy function are carried out, such as the degradation of paternal mitochondria in the 1-cell and 15-cell embryo, …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript by Laboutet et al., titled: "LGG-1/GABARAP lipidation is dispensable for autophagy and development in C. elegans," describes the potential function of a nonlipidated LGG-1 mutant containing a G116A mutation. Comparison of a G116A missense mutation to the lgg-1 null mutation or a lgg-1(G116A>G117*) suggests that there is some function retained in the G116A missense mutation. The authors claim that no foci form in the lgg-1(G116A) mutants and take this to mean that there is no lipidation. Assays for autophagy function are carried out, such as the degradation of paternal mitochondria in the 1-cell and 15-cell embryo, survival after L1 starvation, normal lifespan, and the presence of apoptotic corpses. In all cases, the lgg-1(G116A) mutant clearly shows function. However, how can we be sure that there is no lipidated form? The authors state that not seeing LGG-1 positive dots in the embryos with an LGG-1 antibody is enough to state that this is not a lipidated form of LGG-1. However, this should be confirmed biochemically. If there were absolutely no lipidated form, the authors also would have to confirm that the function that they see in their assays, for example in survival after starvation, or in degradation of paternal mitochondria is indeed autophagy-dependent. Double mutants with the lgg-1(G116A) and a degradation mutant, like epg-5, should eliminate the activity seen in their assays. Otherwise, this activity may be due to another function of LGG-1 that is not autophagy-dependent.
Major questions:
1.Can we be sure that there is no lipidated form? What if another amino acid can be lipidated to a lower extent? If it is not lipidated, how do the authors propose that this LGG-1 mutant is functioning? In the G116A mutants, and G116AG117* mutant, a new band shows in between the LGG-1 I and LGG-1 II forms, does this band have any activity?
2.In Fig. 1, functional assays for LGG-1 dependent autophagy function in the manuscript are the degradation of paternal mitochondria in the 1-cell and 15-cell embryo, survival after L1 starvation, normal lifespan, and the presence of apoptotic corpses. In Fig. 4, the authors show that most of this activity may be due to redundancy with LGG-2, as the starvation survival of lgg-1(G116A) mutants is mostly abolished by the lgg-2 null mutation. Three assays are done to compare the lgg-1(G116A) single to the lgg-1(G116A); lgg-2 null double, and in 2/3 assays there is still activity conferred by the lgg-1(G116A) mutant observed in the double mutants. What if this activity is not autophagy-dependent?
3.In Figures 1F (100 cell embryo) with lgg-1(G116A) mutant, there are light foci visible, clearly not as bright as in the wild-type, but could these be some less lipidated form of LGG-1 with some remaining function? Again, in figure 4J with the lgg-1(G116A); lgg-2 null (15 cell embryo), very light foci accumulate. What are these?
4.In Figure S4F there is a small difference between the atg8 +empty vector and the atg8 +LGG-1(G116A), however there are no statistics shown.
5.There is evidence that the efficiency of degradation by autophagy in aggrephagy is modulated by the composition of the aggregates (Zhang et al 2017). A model has been proposed where PGL-1, PGL-3 and SEPA-1 are mainly degraded via an EPG-2 mediated pathway, however an EPG-2 independent pathway also exists. Which pathway is being used in the LGG-1(G116A) mutant?
Minor points:
- The manuscript would benefit from some language editing. In page 2, line 5, it reads: "The general scheme is successive recruitment of a series of protein complexes involved in the dynamic of the process through several steps implicating the phosphorylation of lipids..." Here, it should read "dynamics." The authors use this term often and they should refer to "dynamics".
- The label "1-cell" are missing in Fig. 1B showing the lgg-1() mutant on the left.
Significance
The manuscript reports a truly novel finding and could be potentially interesting to the autophagy research community. Because the authors make a claim that something is not required, the burden of proof is higher and the authors have to unequivocally show that the LGG-1(G116A) mutant is not lipidated.
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