CED-5/CED-12 (DOCK/ELMO) can promote and inhibit F-actin formation via distinct motifs that may target different GTPases

Coordinated activation and inhibition of F-actin supports the movements of morphogenesis. Understanding the proteins that regulate F-actin is important, since these proteins are mis-regulated in diseases like cancer. Our studies of C. elegans embryonic epidermal morphogenesis identified the GTPase CED-10/Rac1 as an essential activator of F-actin. However, we need to identify the GEF, or Guanine-nucleotide Exchange Factor, that activates CED-10/Rac1 during embryonic cell migrations. The two-component GEF, CED-5/CED-12, is known to activate CED-10/Rac1 to promote cell movements that result in the engulfment of dying cells during embryogenesis, and a later cell migration of the larval Distal Tip Cell. It is believed that CED-5/CED-12 powers cellular movements of corpse engulfment and DTC migration by promoting F-actin formation. Therefore, we tested if CED-5/CED-12 was involved in embryonic migrations, and got a contradictory result. CED-5/CED-12 definitely support embryonic migrations, since their loss led to embryos that died due to failed epidermal cell migrations. However, CED-5/CED-12 inhibited F-actin in the migrating epidermis, the opposite of what was expected for a CED-10 GEF. To address how CED-12/CED-5 could have two opposing effects on F-actin, during corpse engulfment and cell migration, we investigated if CED-12 harbors GAP (GTPase Activating Protein) functions. A candidate GAP region in CED-12 faces away from the CED-5 GEF catalytic region. Mutating a candidate catalytic Arginine in the CED-12 GAP region (R537A) altered the epidermal cell migration function, and not the corpse engulfment function. We interfered with GEF function by interfering with CED-5’s ability to bind Rac1/CED-10. Mutating Serine-Arginine in CED-5/DOCK predicted to bind and stabilize Rac1 for catalysis, resulted in loss of both ventral enclosure and corpse engulfment. Genetic and expression studies strongly support that the GAP function likely acts on different GTPases. Thus, we propose CED-5/CED-12 support the cycling of multiple GTPases, by using distinct domains, to both promote and inhibit F-actin nucleation.

We added an new Figure, Fig. 2A,B.The increased signal of ced-10::gfp is particularly striking.
Following the above point, the Figure 1E is not very supportive.How did the author measure the leading cell dynamics?Is the leading cell or the leading edge of a migrating cell?It seems that there is a small increase; however, it is indeed quite subtle.Can the author measure how much increase?How does this level increase correlate with increased F-actin amount?The author should present image frames from time-lapse movies to show the dynamics.

Response:
We have previously published on the F-actin dynamics of these cells.For example, Bernandskaya et al., 2012, Figure 3.We agree the description (and labeling) for Considering that fluorescence Arp-2/3 complex, WSP-1 and WAVE have already been deposited in the CGC, the author should examine if these actin nucleation factor also upregulated in the leading edge.
While not very necessary but definitely helpful, the biochemical analysis of the GEF or GAP activity should be presented or discussed.

Response:
We added in the Discussion, page 13, "To further investigate which GTPases are targets of the proposed GEF and GAP functions of CED-5/CED-12, it may be possible to test binding of the purified CED-5/CED-12 complex to GTP and GDP loaded versions of the three main GTPases, as we and others have done (Neukomm et al., 2011, Wallace et al, 2018).This will require working out the conditions for purifying the active complex….." The title is quite confusion: can the author specify when promote and when inhibit?If promotion is known, the author can emphasize the novel findings.

Response:
We are not sure what the suggestion is.Does the Reviewer want us to just emphasize the inhibition?We prefer to mention both, since we do not assume everyone knows what DOCK/ELMO does to F-actin levels.The data in our manuscript are clear: for corpse engulfment, loss of CED-5/CED-12 leads to less F-actin, while for ventral enclosure it leads to more F-actin ( and this happens in the same cells).
The writing is too lengthy.Many descriptions in the introduction have already been in the textbook.

Response:
A large effort was made in the Introduction to connect two fields that had not been connected before, branched F-actin regulation for corpse engulfment and for epidermal morphogenesis.We tried to trim the content, after adding all the suggested changes, and trimmed the text on pages: 2, 34 and 11.Please let us know about this textbook you mention.I will order it for my students.
The author should follow C. elegans gene/ protein nomenclature: it is CED-5.
Response: We call it CED-5, and are not sure where we may have been mistaken.

Reviewer #2:
The small GTPase Rac/CED-10 is known to be required for ventral epidermal cell migration during embryonic morphogenesis, but the Guanine Nucleotide Exchange Factor (GEF) activating it has not been characterized so far.The authors ask whether the bipartite Rac-GEF CED-5/CED-12 could fulfil this role.They find that strong mutations in those proteins affect the speed of ventral enclosure and lead to an increase in actin dynamics at the leading edge, arguing that they do present the hallmarks of proteins being required for ventral enclosure.However, those ced-5 or ced-12 mutations do not enhance the lethality of a partial ced-10 mutation.In search for an explanation for this conundrum, they remark that CED-12 presents some discreet homology to the active site of Rho GTPase Activating Proteins (RhoGAP).They find that mutating a critical Arg residue in this putative CED-12 GAP motif behaves as a partial suppressor of the RhoA effector Rho-kinase/LET-502, and that null mutations in ced-5 or ced-12 enhance a let-502 hypomorph.Likewise, the CED-12 GAP mutation suppresses a mutation in the Cdc42 effector WSP-1, suggesting that CED-5/CED-12 might act through Rho or Cdc42.Finally, they find that the mutation in the putative CED-12 GAP domain enhances actin level at the leading edge of epidermal cells during ventral enclosure, whereas a mutation in CED-5 within a domain with partial homology to the Rac-binding domain of the CED-5 vertebrate homolog DOCK2 removes actin from the leading edge.They propose that CED-5/CED-12 would act as a bona fide RACGEF in apoptosis, but not during ventral enclosure.Instead, they argue that it acts as a GAP for Cdc42 and Rho to limit the amount of linear actin.
The genetic results are in general compelling, although the differences are sometimes limited such as the enhancement of wsp-1(gm324) lethality by ced-5/12, or only double lethality such as the effect on let-502(sb118), both of which are slightly indirect and the change in lethality might be significant only at another stage than ventral enclosure; the interactions with Rho-1(RNAi) or Cdc42(RNAi) are also contradictory as to the role of CED-5 vs CED-12 on RhoA.So, I take positively their genetic arguments, but I am not yet convinced by the broader conclusions.The authors argue that the putative CED-12 GAP domain is unlikely to come in contact with Rac/CED-10 owing to its position in the structure.Given the homology between Rac, Cdc42 and RhoA, how would CED-5/CED-12 be more prone to regulated Cdc42 and RhoA?Additionally, isn't it surprising if the putative CED-5/12 GAP domain regulates filopodia inducing GTPases that there aren't filopodia visible during ventral enclosure?
Response: These comments by the Reviewer were extremely helpful for revising the manustcript.We have tried to address these all.To respond to each of these points: a. Change in lethality might be significant only at another stage: The Reviewer raises an excellent point that is easy to address, as we have explained in our previous work ( Second, the structure shows that in the open and closed conformation the GAP domain faces away from the Rac binding domain and is not buried in other regions.This simply says it is available to act as a GAP in either conformation.Revised Figure 4 (Fig. 5) adds a light blue "membrane" and the membrane attachment by the DHR1 AAs to help orient readers about how the closed complex is proposed to attach to the membrane.
Finally, the evidence we provide that the GAP domain may act on RHO-1 or CDC-42 is functional (based on the genetics and the imaging), not structural.Genetics experiments strongly suggest the GAP function affects both RHO-1 and CDC-42 pathways (Revised Figure 7B).We have changed the Title and Abstract to show that interactions with RHO-1 and CDC-42 are supported but not proven by our data.

c.Why don't we see filopodia?
We never say that there are no filopodia, but agree the images did not illustrate the protrusions well.We have added images of close ups of the cells to illustrate the protrusions [ Fig. 2C].In fact, we use the term "protrusions" which can include lamellipodia and filopodia (added, page 4, "We use the term "protrusion" here and throughout, since the leading cells make membrane extensions that encompass both lamellipodia and filopodia.")Imaging F-actin dynamics shows that all of the cells, the leading cells and the pocket cells, make sharp thin protrusions that may be filopodia (Fig. 2C).When we removed arp-2 by RNAi, thus strongly reducing branched actin, we see all of the cells making thin protrusions that may be called filopodia (Patel et al., 2008), presumably because formins are still active.So, to answer this query: yes, CED-5/12 GAP domain may be regulating protrusions that include filopodia.

Reviewer #3:
Knowing that CED-12 and its partner CED-5 function as a GEF for CED-10/Rac1 to promote corpse engulfment of dying cells during embryogenesis and distal tip cell migration in C. elegans, Venkatachalam et al. characterize the role of CED-5/CED-12 during embryonic epidermal cell migration.The authors found that inhibition of CED-5/CED-12 led to some embryonic lethality but, counterintuitively, resulted in increased F-actin levels and cell migration velocity.To address how CED-12/CED-5 could inhibit F-actin in this cellular context, they investigated the role of a GAP domain that has not been described previously.Mutating a candidate catalytic Arginine in the CED-12 GAP region (conserved and usually mutated to inhibit other GAPs) resulted in decreased embryonic lethality when compared to the null cdc12 mutant, and specifically increased F-actin levels in epidermal migrating cells but not in engulfing corpses.Mutating a candidate GEF region on CED-5 predicted to bind and stabilize Rac1 for GTP transfer, resulted in apparent failure in both corpse engulfment and epidermal cell migration.By doing genetic experiments and assessing levels of reporters for 3 GTPases, the authors propose a model where CED-5/CED-12 function both as a GEF and a GAP, supporting the cycling of multiple GTPases that may vary depending on the cellular context.
Although the main findings are interesting and novel, the manuscript and experimental work therein could be more rigorous, more thorough and better explained.It is my opinion that the manuscript should be significantly improved before considering it for publication in PLoS Genetics.
Below follows a list of comments/suggestions for improvement.
Major comments: The mutants that were generated were based on predictions.What validates that they really correspond to a CED-12 GAP mutant and a CED5 GEF mutant?

Response:
The validation presented in this study is functional, not biochemical.The GAP mutation is doing precisely what one would expect: rescuing partial loss of the GTPase.The GEF mutation, as we explain in the text, likely interferes with more than GEF function so it has been renamed Rac binding mutant, and less emphasis is made on its interactions with the three GTPases.There is no simple amino acid change that has been proposed to cleanly mutate GEF function, unlike the well supported use of mutating the catalytic Arginine (R) to block GAP function.
To mutate the GEF function, we tried mutating the PPP motif that suggested was needed for GEF function (Gumienny et al., 2001;Lu et al., 2005), and got a mild, partial phenotype.More recent work suggests the PPP is not essential for GEF function.We used the new structures to design a mutation that would interfere with GEF function.Blocking Rac1 binding seemed a good way to interfere with GEF function.However, as we clarify in the revised draft, page 9, this mutant is different from simple loss of function, since it reduced epidermal F-actin, the opposite of the ced-5 null.We added on page 9, "We refer to this mutant as the ced-5(pj81) Rac1/CED-10 Binding mutant." An immunoblot to show protein levels in the several mutants could be shown.If the mutated protein is not expressed at wild-type levels, care has to be taken when taking conclusions.

Response:
We do not have antibodies to do this experiment.
Figure 5B: Explain to the reader why would the results obtained with a GAP null be different from a GAP point mutant.

Response:
We assume the Reviewer meant why a null mutation in ced-5 or ced-12 is different from the ced-12 GAP mutant?We say on page 10, "These results suggested the GAP activity of CED-12 acts on RHO-1, while the GEF function acts in parallel to RHO-1." Figure 5B: Should add the GEF point mutant (ced5(pj76)) in addition to the ced5 null.

Response:
We did not pursue this since we are no longer calling it the GEF mutant.The results would be hard to interpret.Response: We added measurements of epidermal cells.For CDC-42 we measured epidermal cells, just as migration is set to start.For GFP::WVE-1, which is very faint, we show epidermal seam cells in 1.5 fold embryos.The migrating epidermal cells from the ventral view are quite thin, so measuring their levels and not measuring the signal underneath them was problematic.The epidermal signal we show, and the fact that the signal is either decreased (ced-12 GAP) or unchanged (ced-5 Rac binding mutant), supports the data presented.The drop in gfp::wve-1 for ced-12 GAP mutant was seen in all tissues measured.We try to address this result in the Discussion.
Opposite messages: In the text it says that "…while depleting rho-1 in ced-12(pj74) GAP mutant reduced lethality to 8%, and this drop was significant (Table 3)."In table 3: ced-12(pj74); rho-1 RNAi 0.76 lethality" Response: Thank you, this was a typo.Table 3 should be 0.076, i.e. 0.08 = 8%.This has been fixed.Table 3 is not needed since we show the main results in Fig. 7B., but this can be included in the submitted materials.
"cross the candidate GAP to hypomorphic alleles, or partial loss of function, of the target GTPases.The prediction is that loss of a GAP in combination with a hypomorphic allele (ADD of the small GTPAse) will rescue the loss of function phenotype… loss of a GEF in combination with a hypomorphic allele (ADD of the small GTPAse) will synergistically enhance the loss of function phenotype." Response: Thank you.Fixed, bottom of page 9.
-However, loss of let-502 or wsp-1 brought embryonic lethality back to the mutants alone and did not improve it further.Explain.
Fig. 1E could be improved.Therefore we have added a separate Figure, new Figure 2C, showing the leading edge dynamics and also showing larger frames from time-lapse movies.

Figure 5C :
Figure 5C: Effect of AHPH Rho biosensor should be assessed during epidermal cell migrationwhy looking at pharynx and buccal activity?
It is true that the molecular interpretation of these changes is open to interpretation.That is why we also tried to measure effects on the RHO-1 and CDC-42 biosensors.b.Given the homology between Rac, Cdc42 and RhoA, how would CED-5/CED-12 be more prone to regulate(d) Cdc42 and RhoA?This important question involves two distinct points.First, it is not fully understood how GEFs can distinguish between different GTPases.However, to date, DOCK GEFs are known to act as GEFs on Rac and CDC-42 and never on RHO-1.Structural studies provide arguments to explain this(Kulkarni et al., 2011 DOI 10.1074/jbc.M111.236455;Reviewed in Boland et al., 2023 doi: 10.1002/1873-3468.14523).
for example,Bernadskaya et al., 2012).We typically compare three categories of dying embryos: Early arrest (no differentiation), Ventral Enclosure arrest (tissue differentiate, but no epidermal migration = Gex phenotype,Soto et al., 2001); Elongation arrest (epidermis encloses but elongation fails).These categories were scored by the first student to work on this project, author Sushma Mannimala.We have added her data to Table1.We also add the causes for lethality throughout the text.