Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

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    Evaluation Summary:

    This manuscript is of interest to molecular neurobiologists studying Nicotinic acetylcholine receptors (nAChRs) or other membrane bound receptors. The paper highlights several different and complementary techniques relevant for studying membrane proteins in native conditions, which are relevant and useful to a wide audience.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (D α 5), Dα6, D α 7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.

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  1. Evaluation Summary:

    This manuscript is of interest to molecular neurobiologists studying Nicotinic acetylcholine receptors (nAChRs) or other membrane bound receptors. The paper highlights several different and complementary techniques relevant for studying membrane proteins in native conditions, which are relevant and useful to a wide audience.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Korona et al. aims to contribute to a better understanding of how insecticidal peptide toxins interact with Drosophila nAChR to eventually help combat resistance in target species. The authors use CRISPR/Cas9, larval-injections, pull-down assays, and proteomics to investigate Drosophila nAChR interactions with two peptide neurotoxins. They report interactions between Drosophila α5 (Dα5), Dα6 and Dα7 subunits and α-Btx. The authors also determine the localization of Dα6 during development of the Drosophila nervous system and show that there is prominent expression in Kenyon cells.

    The conclusions of the manuscript are supported by data. However, the introduction promises the reader a study that reaches wider than it does. Furthermore, there are several concerns that should be clarified or extended to help the reader understand the work.

    1. A major aim of the study is to help combat insecticide resistance towards neuropeptides in target species. As such, the authors should communicate how their results on neurotoxin binding to Drosophila nAChR translates to nAChRs in potential target species in the discussion to make the narrative of the paper more concise.

    2. Another aim of the study is to better understand the subunit composition of nAChRs and their distinctive binding properties (highlighted in both the introduction and discussion). However, the study does not provide information on this, e.g. what is the effect of the null-mutants on the subunit composition of present receptors and does the knock-out of one alter the expression levels of the others (Figure 5C)? Do Dα5, Dα6 and Dα7 interact with each other, or are they part of distinct receptors?

    3. Finally, a major aim of the study is also to identify which subunits interact with α-Btx. From the narrative of the manuscript, you get the impression that this study shows that Dα5, Dα6 and Dα7 interact with α-Btx (e.g. lines 120-122 and 164-183). Meanwhile, Landsell et al. has already shown this in the S2 expression system. The fact that the authors are confirming the interaction in a different expression system / endogenous system and the significance of this should be highlighted clearly.

    4. The authors highlight the need for studying the nAChR in a detergent-free system such as SMA to preserve protein-lipid interactions in near-native conditions in their introduction. However, it is not clear whether it is important to preserve protein-lipid interactions for the purpose of the pull-down assays or whether it might just as well have been carried out in detergent. One possibility is that it was important that the binding between the α-Btx resin and nAChR happened in detergent free conditions, but this is not explained. The authors should include a comment on why the detergent-free setup is important for their experiments.

    5. A major feat of the study is the proteomic analysis used for assessing to what extent the SMA copolymer solubilize nAChRs. This method (Figure 4) has a huge potential for analysis of solubilized proteins from endogenous sources in general, particularly when introducing new polymers for native nanodiscs. However, the approach seems excessive for what the authors wanted to achieve in this part of their study: to validate the use of SMA in solubilizing Drosophila membranes. It is well-established that SMA solubilizes membranes into native nanodiscs. This section breaks the narrative and confuses the reader, particularly after solubilization with SMA was already shown in Figure 3.

    6. nAChR subunit mutations with DsRED. The authors use DsRED under the control of the eye-specific 3xP3 promotor for screening of positive lines (Figure 1A, lines 135-138). The figure shows representative fruit flies of which two have red eyes. The description of the red fluorescent reporter protein in the eyes together with a picture with mixed eye phenotypes makes for a confusing piece of information, particularly for non-fly experts, and the authors should consider revising this.

    7. Analysis of SMALPS with TEM. The authors prepare membrane fractions from adult fly heads, solubilize with SMA, and subject the sample to TEM. The authors compare samples enriched for nAChRs with α-Btx coupled affinity resin with unenriched samples (Figure 3). Based on what seems to be purely visual comparison of two TEM images (Figure 3B and D), the authors conclude that α-Btx enrich for nAChR. This conclusion is solely based on the lack of nAChR "top views" (Figure 3E) in the unenriched sample. However, there are likely more than a single view represented in either of the two micrographs. As such, their conclusion is not supported by the analysis of their data.

    8. The authors conclude on an in vivo analysis of Dα6 localization and find that it localizes differently during development and that it is prominently expressed in Kenyon cells, a known target of α-Btx. However, as they have already shown that α-Btx is lethal to larvae (Figure 1), this section brings limited insight to non-fly experts without further discussion of the findings.

  3. Reviewer #2 (Public Review):

    The authors tried to identify the primary target of two peptide toxins, Hv1a and α-Btx, in Drosophila melanogaster. They first analysed candidate targets of these toxins by testing them on wild type and mutant Drosophila larvae lacking nicotinic acetylcholine receptor (nAChR) and found that α4- and β2-null mutants were resistant to Hv1a, whereas α5, α6 and α7 mull mutants were resistant to α-Btx. To confirm this, they employed SMALP technology combined with affinity beads to enrich the target peptides without any solvents to remove lipids surrounding membrane-bound proteins. The LCMS analysis identified target candidate peptides and associated proteins. Further, they investigated potential glycosylation sites in the target nAChR subunits possibly underlying toxin binding, and observed α6 peptide localisation in the nervous system. All these results support that the two toxins differentially target nAChRs in Drosophila. The strength of this study is that they analysed the toxin-target nAChR interactions by using membrane proteins without using any solvents to ensure that proteins remain intact or nearly intact. The weakness of this study is that they did not analyse gene expression changes in response to the deletion of the nAChR subunit gene, which may strongly affect the nAChR components in the nervous system. Also, it is unfortunate that they did not confirm the conclusion by testing the toxins on recombinant nAChRs, which is now possible to express in cell lines or Xenopus laevis oocytes. The impact of the results is limited, but the method, notably SMALP, is influential to scientists studying membrane proteins.

  4. Reviewer #3 (Public Review):

    The authors aimed to use genome editing techniques to allow a thorough investigation of the interaction of peptide toxins with specific nicotinic acetylcholine receptor subunits. They also aimed to support the toxicity data by showing an interaction of the protein with the ligand and developed a technique to do this. The study has generated a set of mutants in the Drosophila nAChR subunit genes by disrupting an exon close to the N-terminal of the protein. This allowed the authors to examine the involvement of nine of these subunits in the response of D. melanogaster to two peptide toxins, alphaBtx and Hv1a, while as previously reported the Dbeta1 disruption was homozygous lethal and was not tested.

    The final genetic background of the mutant flies should be made clearer. The statement on line 132 (p5) that the mutations were generated in virtually identical genetic backgrounds is accurate, however based on the crossing scheme described it appears that these were then crossed to a different background, w1118 (line 519, p22) and then balanced? The strain used to balance these could also be detailed and which chromosomes are in the different strains specified.

    In particular, it is clear that the X-chromosome strains generated (alpha7 and alpha3) both have red eyes in figure 1A. There also appear to be phenotypic differences in body color for the Dalpha2 and Dalpha6 mutant strains. This is important with respect to at least two elements of the study.

    1. The authors proposed that these mutant strains are virtually the same genetic background, and this was a benefit to those generated in a prior study. I would expect this to mean that the only difference is the insertion of the construct used to disrupt each gene.

    2. There could be some confounding effects from using strains with different genetic backgrounds for behavioral assays, this could be particularly true for negative geotaxis assays. If the backgrounds are not precise, this potential confounding factor should be noted and discussed.

    A real strength of the study is the support of toxicological data with their proteomic analysis. Nine of the mutant strains were tested for responses to alphaBtx and Hv1a using injection of larvae and results from their study support the claim that the Hv1a and α-Btx toxins do not share the same target. Not only from different effects on larvae when injected for each of the subunit mutants tested but also from the proteomics data where use of alphaBtx conjugated beads predominantly isolated Dalpha5/Dalpha6/Dalpha7 subunits.

    In discussing the targets of Hv1a, further discussion of the potential for the Dbeta1 subunit to be involved, given it was not tested, would be valuable. This is particularly relevant given the results reported in Ihara, et al., 2020 of the importance of the Dbeta1 subunit in formation of functional receptors in their oocyte expression system.

    Do homozygous Dbeta1 mutants generated in this study survive beyond eclosion? A recent study has shown that a mutant strain harboring a deletion of the Dbeta1 gene is not able to be maintained as a homozygous stock, however it does survive to adulthood, albeit with a significantly shortened life-span and also locomotory/mating defects. It would be useful to include further details on the Dbeta1 strain created in this study to determine if it has a more severe phenotype.

    Another significant contribution of this study is the method reported for isolating and enriching nAChR subunits using SMALPs, which will help facilitate the study of native nAChR complexes. This technique was well validated using an analysis of the enrichment of transmembrane proteins in the presence and absence of styrene maleic acid copolymer. This method allowed Korona and colleagues to determine through proteomic analysis that there was significant binding of three nAChR subunits (Dalpha5/Dalpha6/Dalpha7) to alphaBtx. Further evidence was provided through use of different mutant backgrounds carrying disruptions in specific nAChR subunits. Hence they provide compelling evidence that Dalpha5/Dalpha6/Dalpha7 bind alphaBtx. They also identified glycosylation sites of Dalpha5/Dalpha6/Dalpha7 (and other subunits) adding to the evidence in the literature that glycosylation may be involved in the binding of alphaBtx to nAChR subunits.

    This study makes a valuable contribution to the field of insecticide research, provides useful tools and methods for studying the biology of nicotinic acetylcholine receptors and has the potential to contribute to wider studies of membrane proteins.