Caenorhabditis elegans SEL-5/AAK1 regulates cell migration and cell outgrowth independently of its kinase activity

  1. Filip Knop
  2. Apolena Zounarová
  3. Vojtěch Šabata
  4. Teije Corneel Middelkoop
  5. Marie Macůrková

  • Curated by eLife

    eLife logo

    eLife assessment

    This useful study defines developmental roles for a protein kinase involved in endocytosis and reports a surprising finding that the kinase catalytic activity is unnecessary. However, several claims of the authors are only partially supported by the data. Although in its current form, this work is incomplete, it will be of broad interest to cell biologists and biochemists because this kinase was previously suggested to be a target of drug design efforts.

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

During Caenorhabditis elegans development, multiple cells migrate long distances or extend processes to reach their final position and/or attain proper shape. The Wnt signalling pathway stands out as one of the major coordinators of cell migration or cell outgrowth along the anterior-posterior body axis. The outcome of Wnt signalling is fine-tuned by various mechanisms including endocytosis. In this study, we show that SEL-5, the C. elegans orthologue of mammalian AP2-associated kinase AAK1, acts together with the retromer complex as a positive regulator of EGL-20/Wnt signalling during the migration of QL neuroblast daughter cells. At the same time, SEL-5 in cooperation with the retromer complex is also required during excretory canal cell outgrowth. Importantly, SEL-5 kinase activity is not required for its role in neuronal migration or excretory cell outgrowth, and neither of these processes is dependent on DPY-23/AP2M1 phosphorylation. We further establish that the Wnt proteins CWN-1 and CWN-2, together with the Frizzled receptor CFZ-2, positively regulate excretory cell outgrowth, while LIN-44/Wnt and LIN-17/Frizzled together generate a stop signal inhibiting its extension.

Article activity feed

  1. Version published to 10.7554/elife.91054 on eLife
  2. eLife

    Author response:

    Reviewer #2 (Public Review):

    (1) Some changes to statistical analyses are needed in this study.

    Fig. 1B, 1D, 2A, 3E, and 3F report the QL.d phenotype as a percentage of animals scored that were defective in migration. The methods make it clear this data is categorical rather than quantitative. Therefore, a t-test or any test designed for quantitative data is not appropriate. I suggest that the authors should investigate using a chi-squared or Fisher's exact test.

    For the reasons mentioned above, the calculation of standard deviation (as shown in error bars) is also not appropriate for Fig. 1B, 1D, 2A, 3E, and 3F. Of course, it is excellent that the authors scored multiple trials. For experiments with mutants, I suggest the authors might combine these trials or show separate results of each trial. For experiments using RNAi (Fig. 1B), each trial should be plotted separately because RNAi effectiveness can vary. If there is not enough space to show multiple trials, then I would ask that a representative trial be shown in the main figure and additional trials in a supplement.

    We thank the reviewer for pointing out the statistical mistake. For all figures assessing the QL.d migration phenotype (Fig.1B, 1D, 2A, 4A (former 3E), 4D (former 3F) and Fig.1 – figure supplement 1, Fig.2 – figure supplement 1, Fig.4 – figure supplement 2) the statistical significance was evaluated using Fisher’s exact test. For RNAi experiments (Fig. 1B) results from a representative experiment is shown and two additional trials are shown in Figure 1 – figure supplement 1. For experiments with mutants, results from separate trials were pooled and are presented in the main figures.

    In Fig. 1, 2, 3, and 5, it is not specified whether/how p-values were adjusted for multiple tests.

    We have applied Bonferroni correction for multiple testing in all Figures where it was relevant (Fig. 1, 2, 4, 5 and 6 and in their supplements) and this is now stated in all corresponding Figure legends.

    (2) I felt the author's interpretation of the sel-5 mutant phenotypes in EXC, and the genetic interactions with Wnt signaling mutants, might be improved. The authors show convincing data that the sel-5 mutants display a shortened EXC outgrowth phenotype. Conversely, mutants with reduced Wnt signaling, such as the lin-17 or lin-44 mutants, displayed lengthened EXC outgrowth. The authors show that in double mutants, loss of sel-5 partially suppressed the EXC overgrowth defects of lin-17 or lin-44 mutants (Fig. 5). In my opinion, this data is consistent with a model where SEL-5 acts to inhibit Wnt signaling in EXC. An inhibitory role in a Wnt-receiving cell would be consistent with the known activity for human AAK1 in promoting negative feedback and endocytosis of LPR6. Interestingly, the authors mention in their discussion that a mutant of plr-1, which acts in the internalization of Frizzled receptors, has a shortened EXC phenotype similar to that of sel-5 mutants. These observations all seem consistent with an inhibitory role, yet the authors do not state this as their conclusion. A clarification of their interpretation is needed.

    We thank the reviewer for this feedback. Indeed, the above interpretation of the excretory cell migration data is plausible, however, we think that several lines of evidence argue against this possibility. First, measurements of the posterior canal length during L1/L2 larval stages show that LIN-44/LIN-17 signalling is not required for the early stages of excretory canal outgrowth, unlike SEL-5/VPS-29 (Fig. 5E, 6D). This suggests that SEL-5 and VPS-29 are required earlier than LIN-44 and LIN-17 and therefore should not act at the level of Wnt receptor internalization. Our new data with more mutant combinations revealed canal shortening in cwn-1; cfz-2 and cwn-2; cfz-2 mutants. This would rather suggest a positive role for SEL-5 and VPS-29 in Wnt pathway regulation. Either SEL-5/VPS-29 employ two different mechanisms of Wnt pathway regulation or alternatively, act prior to any Wnt-dependent step in the excretory canal outgrowth. The observed partial rescue of the lin-17 or lin-44 overgrowth defect by sel-5 could then be explained for example by a reduced speed of canal outgrowth in sel-5 mutants. Based on new findings about CWN-1, CWN-2 and CFZ-2 involvement we have also modified our model now presented in Fig.7.

    For changes to the Results section, see Response to Reviewer 1, point 4b. The Discussion part has been substantially rewritten and is presented below:

    LINE 428 “Our analysis of single Wnt and Frizzled mutants revealed that while loss of cwn-2 or cfz-2 expression resulted in a very mild shortening of the excretory canal, loss of lin-44 or lin-17 led to profound canal overgrowth (summarized in Fig. 7A). These findings suggested that two independent Wnt pathways could be employed to establish proper excretory canal length – one promoting canal extension and one generating the stop signal for growth termination. Further analyses of double mutants and other Wnt signalling components revealed that the extension-promoting pathway includes cwn-1 in addition to cwn-2 and cfz-2, while the stop-signal pathway encompasses lin-44, lin-17, dsh-1, mig-5 and mig-14. A similar repulsive role of LIN-44/LIN-17 complex has been described in the case of a posterior axon of C. elegans GABAergic DD6 motor neuron (Maro et al., 2009) or PLM, ALN and PLN neurons (Zheng et al., 2015). Loss of lin-44 or lin-17 expression promoted outgrowth of the posterior neurites of these neurons implicating that in wild type animals, LIN-44 serves as a repulsive cue. On the other hand, cwn-2 and cfz-2 were shown to positively regulate the posterior neurite outgrowth of RMED/V neurons with cwn-2 acting as an attractive cue (Song et al., 2010). The role of two other Wnt signalling components, egl-20 and mig-1, is less clear. No effect (mig-1) or only very mild overgrowth defect (egl-20) is observed in single mutants. However, both egl-20 and mig-1 significantly rescue the overgrowth phenotype of lin-17 mutants, while at the same time, mig-1 can suppress the shortening of canals in cfz-2 mutants. EGL-20-producing cells are localized around the rectum (Whangbo et al., 1999; Harterink et al., 2011), exactly where the excretory canals stop, while LIN-44 is expressed more posteriorly (Herman et al., 1995; Harterink et al., 2011). A possible explanation could thus be that while LIN-44 provides a general posterior repulsive signal, EGL-20 fine-tunes the exact stopping position of the growing canal. The role of different Wnts and Frizzleds in excretory canal outgrowth is summarized in Fig. 7B. Further investigation will be required to decipher the exact way how SEL-5 and the retromer crosstalk with Wnt signalling during excretory cell outgrowth. It is clear though that more than one mechanism is likely involved. First, sel-5 vps-29 mutants display canal shortening similarly to cwn-1; cfz-2 or cwn-2; cfz-2 suggesting a positive regulatory role. Mutants in lin-17 and lin-44 display canal overgrowth, yet sel-5 is partially able to suppress this phenotype. This would imply a negative regulatory role of sel-5 and be in agreement with the role of AAK1 in Wnt pathway regulation (Agajanian et al., 2019). However, sel-5 and vps-29 are required already during the initial larval outgrowth while the LIN-44/LIN-17 signal is required later. The observed rescue might thus also be explained by a delayed growth of the canal and not by a direct impact of sel-5 and vps-29 on LIN-44 or LIN-17 levels or localization.”

  3. eLife

    eLife assessment

    This useful study defines developmental roles for a protein kinase involved in endocytosis and reports a surprising finding that the kinase catalytic activity is unnecessary. However, several claims of the authors are only partially supported by the data. Although in its current form, this work is incomplete, it will be of broad interest to cell biologists and biochemists because this kinase was previously suggested to be a target of drug design efforts.

  4. eLife

    Reviewer #1 (Public Review):

    Recent work reported that the AP2-associated kinase 1 (AAK1) downregulates Wnt signaling by phosphorylating, thus activating, the µ-subunit of the AP2 complex (AP2M1), which recognizes an endocytic signal on the intracellular domain of the Wnt co-receptor LRP6 leading to its internalization (Agajanian, et al., 2018). It has also long been known that DPY-23/AP2M1 and the retromer complex, which controls trafficking between endosomes and the trans-golgi network and recycling from endosomes to the plasma membrane, regulate Wnt signaling in C. elegans, at least in part by modulating trafficking of the Wnt-secretion factor MIG-14/WLS (Pan, et al., 2008; Yan et al., 2008).

    Here the authors first set out to ask whether SEL-5/AAK1 plays a conserved role in Wnt signaling via phosphorylation of DPY-23/AP2M1 by assessing the function of SEL-5 in Wnt-regulated morphogenetic events; specifically, the well-characterized migration and polarization of several neurons and the less-understood process of excretory canal cell outgrowth.

    The authors found that the simultaneous removal of sel-5 and the retromer complex gene vps-29 resulted in synthetic neuronal and excretory canal outgrowth phenotypes, indicating that sel-5 and the retromer complex function in parallel in these processes. Genetic interactions between sel-5 and Wnt pathway components were also examined, and for QL neuroblast migration, loss of sel-5 exacerbated phenotypes caused by loss of the Wnt receptor LIN-17/FZD, but not those caused by loss of a different receptor, MIG-1/FZD. The authors assessed the site of sel-5 function in neuronal migration defects via tissue-specific rescue and identified the hypodermis, a known source of Wnt ligands, and muscles as sites where sel-5/AAK1 activity is required.

    The novelty in this work comes from the discovery of a function for sel-5/AAK1 and the retromer complex in excretory canal outgrowth, identified by phenotypes caused by simultaneous loss of sel-5 and retromer components. This synthetic phenotype is rescued by restoring sel-5 to either the excretory canal cell or the hypodermis, suggesting autonomous and non-autonomous functions for sel-5 in canal outgrowth. The authors also confirmed previous results showing that loss of LIN-17/FZD results in excretory canal overgrowth, and by carrying out an extensive survey of Wnt-pathway mutants they discovered that LIN-44/Wnt is likely the ligand that functions via LIN-17 as a "stop" signal in canal outgrowth. They also implicate a CWN-1/Wnt-CFZ-2/FZD pathway as required for canal outgrowth and find genetic interactions between sel-5/AAK1 and the lamellipodin ortholog mig-10, suggesting that these genes function in parallel to promote excretory canal outgrowth.

    The most intriguing claim in this work is the suggestion that neither DPY-23 phosphorylation nor SEL-5 kinase activity is required for their function in Wnt signaling. However, the tools used to support these conclusions are not well-characterized. First, a new dpy-23 phosphorylation site-mutant is not genetically characterized, thus it is difficult to interpret the negative results obtained with this allele. Second, although the mutations introduced into SEL-5 are expected to abolish kinase activity, this is not demonstrated biochemically, nor are the effects, if any, of mutations on protein stability/localization assessed. Finally, experiments testing the function of SEL-5 kinase mutants are reported using only one multi-copy extrachromosomal array per construct. Because these types of transgenes vastly overexpress proteins, it is likely that even proteins with reduced function will rescue, raising concerns regarding the conclusion that kinase activity is not necessary for SEL-5 function.

    In conclusion, it is not clear that the findings presented here will be of great general interest, as they mostly support previously-known functions for SEL-5/AAK1, DPY-23/AP2M1, and the retromer complex in Wnt-mediated signaling. Thus, this work will mainly be of interest to researchers studying Wnt-mediated cell outgrowth, and more specifically to those studying the C. elegans excretory canal. Moreover, the study lacks coherence: initially, there is a clear hypothesis testing a role for SEL-5/AAK1 in DPY-23/AP2M1 phosphorylation and how this impinges on Wnt signaling. This model appears to be refuted (although, as noted above the tools used to do this need to be better validated), but the authors do not explore alternative targets or functions for SEL-5/AAK1, nor do they directly assess how SEL-5 or the retromer complex impinge on Wnt signaling in excretory canal outgrowth. Thus, there is little mechanistic insight provided by this work.

  5. eLife

    Reviewer #2 (Public Review):

    Summary
    This study by Knop, et al. defines two different developmental roles for the conserved SEL-5/AAK1 protein kinase in Caenorhabditis elegans. In other organisms, AAK1 was known to promote the recycling of the Wntless sorting receptor and endocytosis of Wnt receptors. This study establishes that SEL-5 acts in two roles in C. elegans: in Wnt-producing cells, a role that promotes migration of a neuroblast termed QL.d, and in Wnt-receiving cells, a role that promotes outgrowth of the excretory cell (EXC). Before this study, SEL-5/AAK1 was thought to regulate endocytosis through phosphorylation of AP2M1 and other endocytic adaptor proteins. This study shows convincing data that the SEL-5 makes a partial contribution to AP2M1 phosphorylation, and more surprisingly, that its roles in Wnt-producing and Wnt-receiving cells of C. elegans do not require SEL-5 catalytic activity. Human AAK1 was previously suggested to be a target of drug design efforts due to its roles in neuropathic pain, viral infection, and Alzheimer's disease. The discovery that some roles for SEL-5/AAK1 are independent of catalytic activity will be of broad interest to cell biologists and biochemists.

    Strengths
    (1) The data establishing the requirement for SEL-5 in QL.d migration and EXC outgrowth (Fig. 1 and Fig. 4) is rigorous and convincing. My assessment of the rigor is based on the following: First, the authors show that two independently derived sel-5 deletion mutations result in defects in QL.d and EXC. Second, the authors show that providing wild-type, GFP-tagged SEL-5 results in significant rescue of both phenotypes. Importantly, they use tissue-specific transgenes to show that the requirement for SEL-5 in QL.d migration is non-cell-autonomous, and the requirement for SEL-5 in EXC outgrowth is cell-autonomous (Fig. 2). For rescue experiments, they show that each tissue-specific transgene is indeed expressed strongly in the tissue of interest. This establishes the roles for SEL-5 in two different roles, in Wnt-producing and Wnt-receiving cells.

    (2) The authors present three lines of convincing biochemical and genetic evidence that SEL-5 kinase catalytic activity is not important for its roles in Wnt-producing and Wnt-receiving cells.

    Taking a biochemical approach, they use quantitative Westerns to assess the degree of AP2M1 phosphorylation in sel-5 mutants (Fig. 3). Their results show that AP2M1 phosphorylation is diminished, but not absent in mutants. Their results are convincing because they make use of GFP-tagged AP2M1 to probe for total and phospho-AP2M1. I note that they included uncropped Western blots in supplemental data. Furthermore, they make use of a GFP-tagged AP2M1 mutant (T160A) to confirm which residue is phosphorylated. Their results suggest that some mechanism other than AP2M1 phosphorylation may account for the sel-5 mutant phenotypes.

    Taking a genetic approach, they make use of a unique allele, dpy-23(mew25), that alters the known AP2M1 phosphorylation site. They show that animals carrying this allele do not display the QL.d and EXC phenotypes (Fig. 3 and Fig. 5). Finally, in a more direct test of whether SEL-5 requires catalytic activity, they make use of GFP-tagged SEL-5 forms mutated at either the active site or the ATP-binding site of the SEL-5 kinase domain. They show that either SEL-5 mutant form successfully rescues the QL.d and EXC defects seen in sel-5 mutants (Fig. 3), suggesting that SEL-5 catalytic activity is unnecessary.

    (3) The authors have produced an elegant GFP knock-in allele of the sel-5 gene, allowing analysis of expression and localization in living animals (Fig. 2).

    (4) The authors make use of genetic interactions with Wnt signaling mutants to show that SEL-5 acts in a role that promotes Wnt signaling for the QL.d cell (Fig. 1) and counteracts Wnt signaling for the EXC (Fig. 5).

    Weaknesses
    (1) Some changes to statistical analyses are needed in this study.

    Fig. 1B, 1D, 2A, 3E, and 3F report the QL.d phenotype as a percentage of animals scored that were defective in migration. The methods make it clear this data is categorical rather than quantitative. Therefore, a t-test or any test designed for quantitative data is not appropriate. I suggest that the authors should investigate using a chi-squared or Fisher's exact test.

    For the reasons mentioned above, the calculation of standard deviation (as shown in error bars) is also not appropriate for Fig. 1B, 1D, 2A, 3E, and 3F. Of course, it is excellent that the authors scored multiple trials. For experiments with mutants, I suggest the authors might combine these trials or show separate results of each trial. For experiments using RNAi (Fig. 1B), each trial should be plotted separately because RNAi effectiveness can vary. If there is not enough space to show multiple trials, then I would ask that a representative trial be shown in the main figure and additional trials in a supplement.

    In Fig. 1, 2, 3, and 5, it is not specified whether/how p-values were adjusted for multiple tests.

    (2) I felt the author's interpretation of the sel-5 mutant phenotypes in EXC, and the genetic interactions with Wnt signaling mutants, might be improved. The authors show convincing data that the sel-5 mutants display a shortened EXC outgrowth phenotype. Conversely, mutants with reduced Wnt signaling, such as the lin-17 or lin-44 mutants, displayed lengthened EXC outgrowth. The authors show that in double mutants, loss of sel-5 partially suppressed the EXC overgrowth defects of lin-17 or lin-44 mutants (Fig. 5). In my opinion, this data is consistent with a model where SEL-5 acts to inhibit Wnt signaling in EXC. An inhibitory role in a Wnt-receiving cell would be consistent with the known activity for human AAK1 in promoting negative feedback and endocytosis of LPR6. Interestingly, the authors mention in their discussion that a mutant of plr-1, which acts in the internalization of Frizzled receptors, has a shortened EXC phenotype similar to that of sel-5 mutants. These observations all seem consistent with an inhibitory role, yet the authors do not state this as their conclusion. A clarification of their interpretation is needed.

    Impact/significance
    (1) Among researchers using C. elegans, this study provides a foundation for further investigation of the role of endocytosis, SEL-5, and the retromer, in Wnt trafficking. It is particularly useful that the authors define two different phenotypes that arise from Wnt-producing and Wnt-receiving cells.

    (2) Among a broader community of cell biologists and biochemists, this study will be of interest in its finding that SEL-5/AAK1 kinase catalytic activity is unnecessary for the regulation of Wnt signaling.

  6. Version published to 10.1101/2023.03.29.534638v2 on bioRxiv
  7. Version published to 10.1101/2023.03.29.534638v1 on bioRxiv