Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this study, Seidenthal et al. investigated the role of the C. elegans Flower protein, FLWR-1, in synaptic transmission, vesicle recycling, and neuronal excitability. They confirmed that FLWR-1 localizes to synaptic vesicles and the plasma membrane and facilitates synaptic vesicle recycling at neuromuscular junctions, albeit in an unexpected manner. The authors observed that hyperstimulation results in endosome accumulation in flwr-1 mutant synapses, suggesting that FLWR-1 facilitates the breakdown of endocytic endosomes, which differs from earlier studies in flies that suggested the Flower protein promotes the formation of bulk endosomes. This is a valuable finding. Using tissue-specific rescue experiments, the authors showed that expressing FLWR-1 in GABAergic neurons restored the aldicarb-resistant phenotype seen in flwr-1 mutants to wild-type levels. In contrast, FLWR-1 expression in cholinergic neurons in flwr-1 mutants did not restore aldicarb sensitivity, yet muscle expression of FLWR-1 partially but significantly recovered the aldicarb-resistant defects. The study also revealed that removing FLWR-1 leads to increased Ca2+ signaling in motor neurons upon photo-stimulation. Further, the authors conclude that FLWR-1 contributes to the maintenance of the excitation/inhibition (E/I) balance by preferentially regulating the excitability of GABAergic neurons. Finally, SNG-1::pHluorin data imply that FLWR-1 removal enhances synaptic transmission, however, the electrophysiological recordings do not corroborate this finding.
Strengths:
This study by Seidenthal et al. offers valuable insights into the role of the Flower protein, FLWR-1, in C. elegans. Their findings suggest that FLWR-1 facilitates the breakdown of endocytic endosomes, which marks a departure from its previously suggested role in forming endosomes through bulk endocytosis. This observation could be important for understanding how Flower proteins function across species. In addition, the study proposes that FLWR-1 plays a role in maintaining the excitation/inhibition balance, which has potential impacts on neuronal activity.
Weaknesses:
One issue is the lack of follow-up tests regarding the relative contributions of muscle and GABAergic FLWR-1 to aldicarb sensitivity. The findings that muscle expression of FLWR-1 can significantly rescue aldicarb sensitivity are intriguing and may influence both experimental design and data interpretation. Have the authors examined aldicarb sensitivity when FLWR-1 is expressed in both muscles and GABAergic neurons, or possibly in muscles and cholinergic neurons? Given that muscles could influence neuronal activity through retrograde signaling, a thorough examination of FLWR-1's role in muscle is necessary, in my opinion.
We thank the reviewer for this suggestion. Indeed, the retrograde inhibition of cholinergic transmission by signals from muscle has been demonstrated by the Kaplan lab in a number of publications. We have now done the experiments that were suggested, see the new Fig. S3B: rescuing FLWR-1 in cholinergic neurons and in muscle did not perform any better in the aldicarb assay, while co-rescue in GABAergic neurons and muscle, like rescue in GABA neurons, led to a complete rescue to wild type levels. Thus, retrograde signaling from muscle to neurons does not contribute to effects on the E/I imbalance caused by the absence of FLWR1. The fact that muscle rescue can partially rescue the flwr-1 phenotype is likely due a cellautonomous effect of FLWR-1 on muscle excitability, facilitating muscle contraction.
Would the results from electrophysiological recordings and GCaMP measurements be altered with muscle expression of FLWR-1? Most experiments presented in the manuscript compare wild-type and flwr-1 mutant animals. However, without tissue-specific knockout, knockdown, or rescue experiments, it is difficult to separate cell-autonomous roles from non-cell-autonomous effects, in particular in the context of aldicarb assay results. Also, relying solely on levamisole paralysis experiments is not sufficient to rule out changes in muscle AChRs, particularly due to the presence of levamisole-resistant receptors.
We repeated the Ca2+ imaging in cholinergic neurons, in response to optogenetic activation, with expression of FLWR-1 in muscle, see Fig. 4E. This did not significantly alter the increased excitability of the flwr-1 mutant. Thus, we conclude that, along with the findings in aldicarb assays, the function of FLWR-1 in muscle is cell-autonomous, and does not indirectly affect its roles in the motor neurons. Also, cholinergic expression of FLWR-1 by itself reduced Ca2+ levels to those in wild type (Fig. 4E). In addition, we now also assessed the contribution of the N-AChR (ACR-16) to aldicarb-induced paralysis (Fig. S3C), showing that flwr-1 and acr-16 mutations independently mediate aldicarb resistance, and that these effects are additive. Thus, FLWR-1 does not affect the expression level or function of the N-AChR, as otherwise, the flwr1; acr-16 double mutation would not exacerbate the phenotype of the single mutants.
This issue regarding the muscle role of FLWR-1 also complicates the interpretation of results from coelomocyte uptake experiments, where GFP secreted from muscles and coelomocyte fluorescence were used to estimate endocytosis levels. A decrease in coelomocyte GFP could result from either reduced endocytosis in coelomocytes or decreased secretion from muscles. Therefore, coelomocytespecific rescue experiments seem necessary to distinguish between these possibilities.
We have performed a rescue of FLWR-1 in coelomocytes to address this, and found that this fully recovered the CC GFP signals to wild type levels. Therefore, the absence of FLWR-1 in muscles does not affect exocytosis of GFP. The data can be found in Fig. 5A, B.
The manuscript states that GCaMP was used to estimate Ca2+ levels at presynaptic sites. However, due to the rapid diffusion of both Ca2+ and GCaMP, it is unclear how this assay distinguishes Ca2+ levels specifically at presynaptic sites versus those in axons. What are the relative contributions of VGCCs and ER calcium stores here? This raises a question about whether the authors are measuring the local impact of FLWR-1 specifically at presynaptic sites or more general changes in cytoplasmic calcium levels.
We compared Ca2+ signals in synaptic puncta versus axon shafts, and did not find any differences. The data previously shown have been replaced by data where the ROIs were restricted to synaptic puncta. The outcome is the same as before. These data are provided in Fig. 4A, B, E, F. We thus conclude that the impact of FLWR-1 is local, in synaptic boutons.
The experiments showing FLWR-1's presynaptic localization need clarification/improvement. For example, data shown in Fig. 3B represent GFP::FLWR-1 is expressed under its own promoter, and TagRFP::ELKS-1 is expressed exclusively in GABAergic neurons. Given that the pflwr-1 drives expression in both cholinergic and GABAergic neurons, and there are more cholinergic synapses outnumbering GABAergic ones in the nerve cord, it would be expected that many green FLWR-1 puncta do not associate with TagRFP::ELKS-1. However, several images in Figure 3B suggest an almost perfect correlation between FLWR-1 and ELKS-1 puncta. It would be helpful for the readers to understand the exact location in the nerve cord where these images were collected to avoid confusion.
Thank you for making us aware that the provided images may be misleading. We have now extended this Figure (Fig. 3A-C) and provided more intensity profiles along the nerve cords in Fig. S4A-C. The quantitative analysis of average R2 for the two fluorescent signals in each neuron type did not show any significant difference between the two, also after choosing slightly smaller ROIs for line scan analysis. We also highlighted the puncta corresponding to FLWR-1 in both neurons types, as well as to ELKS-1 in each specific neuron type, to identify FLWR-1 puncta without co-localized ELKS-1 signal. Also, we indicated the region that was imaged, i.e. the DNC posterior of the vulva, halfway to the posterior end of the nerve cord.
The SNG-1::pHluorin data in Figure 5C is significant, as they suggest increased synaptic transmission at flwr-1 mutant synapses. However, to draw conclusions, it is necessary to verify whether the total amount of SNG-1::pHluorin present on synaptic vesicles remains the same between flwr-1 mutant and wild-type synapses. Without this comparison, a conclusion on levels of synaptic vesicle release based on changes in fluorescence might be premature, in particular given the results of electrophysiological recordings.
We appreciate the comment. We now added data and experiments that verify that the basal SNG-1::pHluorin signal in the plasma membrane, measured at synaptic puncta and in adjacent axonal areas, is not different in flwr-1 mutants compared to wild type in the absence of stimulation. This data can be found in Fig. S5A. In addition, we cultured primary neurons from transgenic animals to compare total SNG-1::pHluorin to the vesicular fraction, by adding buffers of defined pH to the external, or buffers that penetrate the cell and fix intracellular pH. These experiments (Fig. S5B, C) showed no difference in the vesicle fraction of the pHluorin signal in wild type vs. flwr-1 mutant cells, demonstrating that flwr-1 mutants do not per se have altered SNG-1::pHluorin in their SV or plasma membranes.
Finally, the interpretation of the E74Q mutation results needs reconsideration. Figure 8B indicates that the E74Q variant of FLWR-1 partially loses its rescuing ability, which suggests that the E74Q mutation adversely affects the function of FLWR-1. Why did the authors expect that the role of FLWR-1 should have been completely abolished by E74Q? Given that FLWR-1 appears to work in multiple tissues, might FLWR-1's function in neurons requires its calcium channel activity, whereas its role in muscles might be independent of this feature? While I understand there is ongoing debate about whether FLWR1 is a calcium channel, the experiments in this study do not definitively resolve local Ca2+ dynamics at synapses. Thus, in my opinion, it may be premature to draw firm conclusions about calcium influx through FLWR-1.
Thank you for bringing this up. We did not expect E74Q to necessarily abolish FLWR-1 function, unless it would be a Ca2+ channel. Of course the reviewer is right, FLWR-1 might have functions as an ion channel as well as channel-independent functions. Yet, we are quite confident that FLWR-1 is not an ion channel. Instead, we think that E74Q alters stability of the protein (however, in the absence of biochemical data, we removed this conclusion), and that this impairs the function of FLWR-1 as a modulator, or possibly even, accessory subunit of the PMCA MCA-3. This interaction was indicated by a new experiment we added, where we found that FLWR-1 and MCA-3 must be physically very close to each other in the plasma membrane, using bimolecular fluorescence complementation (see new Fig. 9A, B). This provides a reasonable explanation for findings we obtained, i.e. increased Ca2+ levels in stimulated neurons of the flwr-1 mutant. If FLWR-1 acts as a stimulatory subunit of MCA-3, then its absence may cause reduced MCA-3 function and thus an accumulation of Ca2+ in the synaptic terminals. In Drosophila, hyperstimulation of neurons led to reduced Ca2+ levels (Yao et al., 2017, PLoS Biol 15: e2000931), suggesting that Flower is a Ca2+ channel. Based on our findings, we suggest an alternative explanation. Based on proteomics, the PMCA is a component of SVs (Takamori et al., 2006, Cell 127: 831-846). Increased insertion of PMCA into the plasma membrane during high stimulation, along with impaired endocytosis in flower mutants, would increase the steadystate levels of PMCA in the PM. This could lead to reduced steady state levels of Ca2+. This ‘g.o.f.’ in Flower may also impact on Ca2+ microdomains of the P/Q type VGCC required for SV fusion, which could contribute to the rundown of EPSCs we find during synaptic hyperstimulation (Fig. 5G-J). We acknowledge, though, that Yao et al. (2009, Cell 138: 947– 960), showed increased uptake of Ca2+ into liposomes reconstituted with purified Flower protein. However, it cannot be ruled out that a protein contaminant could be responsible, as the controls were empty liposomes, not liposomes reconstituted with a mutated Flower protein purified the same way.
We also tested the E74Q mutant in its ability to rescue the reduced PI(4,5)P2 levels in coelomocytes (CCs), where we observed no positive effect. While we have not measured Ca2+ in CCs, we would assume that here a function of FLWR-1 affecting increased PI(4,5)P2 levels is not linked to a channel function. It was, nevertheless, compromised by E74Q (Fig. 8D).
Also, the aldicarb data presented in Figures 8B and 8D show notable inconsistencies that require clarification. While Figure 8B indicates that the 50% paralysis time for flwr-1 mutant worms occurs at 3.5-4 hours, Figure 8D shows that 50% paralysis takes approximately 2.5 hours for the same flwr-1 mutants. This discrepancy should be addressed. In addition, the manuscript mentions that the E74Q mutation impairs FLWR-1 folding, which could significantly affect its function. Can the authors show empirical data supporting this claim?
We performed the aldicarb assays in a consistent manner, but nonetheless note that some variability from day to day can affect such outcomes. Importantly, we always measured each control (wild type, flwr-1) along with each test strain (FLWR-1 point mutants), to ensure the relevant estimate of a point-mutant’s effect. These assays have been repeated, now including the FLWR-1 wild type rescue strain as a comparison. The data are now combined in Fig. 8B. Regarding the assumed instability of the E74Q mutant, as we, indeed, do not have any experimental data supporting this, we removed this sentence.
Reviewer #2 (Public review):
Summary:
The Flower protein is expressed in various cell types, including neurons. Previous studies in flies have proposed that Flower plays a role in neuronal endocytosis by functioning as a Ca2+ channel. However, its precise physiological roles and molecular mechanisms in neurons remain largely unclear. This study employs C. elegans as a model to explore the function and mechanism of FLWR-1, the C. elegans homolog of Flower. This study offers intriguing observations that could potentially challenge or expand our current understanding of the Flower protein. Nevertheless, further clarification or additional experiments are required to substantiate the study's conclusions.
Strengths:
A range of approaches was employed, including the use of a flwr-1 knockout strain, assessment of cholinergic synaptic activity via analyzing aldicarb (a cholinesterase inhibitor) sensitivity, imaging Ca2+ dynamics with GCaMP3, analyzing pHluorin fluorescence, examination of presynaptic ultrastructure by EM, and recording postsynaptic currents at the neuromuscular junction. The findings include notable observations on the effects of flwr-1 knockout, such as increased Ca2+ levels in motor neurons, changes in endosome numbers in motor neurons, altered aldicarb sensitivity, and potential involvement of a Ca2+-ATPase and PIP2 binding in FLWR-1's function.
Weaknesses:
(1) The observation that flwr-1 knockout increases Ca2+ levels in motor neurons is notable, especially as it contrasts with prior findings in flies. The authors propose that elevated Ca2+ levels in flwr-1 knockout motor neurons may stem from "deregulation of MCA-3" (a Ca2+ ATPase in the plasma membrane) due to FLWR-1 loss. However, this conclusion relies on limited and somewhat inconclusive data (Figure 7). Additional experiments could clarify FLWR-1's role in MCA-3 regulation. For instance, it would be informative to investigate whether mutations in other genes that cause elevated cytosolic Ca2+ produce similar effects, whether MCA-3 physically interacts with FLWR-1, and whether MCA-3 expression is reduced in the flwr-1 knockout.
We thank the reviewer for bringing up these critical points. As to other mutations that produce elevated cytosolic Ca2+: Possible mutations could be g.o.f. mutations of the ryanodine receptor UNC-68, the sarco-endoplasmatic Ca2+ ATPase, or mutants affecting VGCCs, like the L-type channel EGL-19 or the P/Q-type channel UNC-2. However, any such mutant would affect muscle contractions (as we have shown for r.o.f. mutations in unc-68, egl-19 and unc-2 in Nagel et al. 2005 Curr Biol 15: 2279-84) and thus would affect aldicarb assays (see aldicarb resistance induced by RNAi of these genes in Sieburth et al., 2005, Nature 436: 510). The same should be expected for g.o.f. mutations of any such gene. In neurons, we would expect increased or decreased Ca2+ levels in response to stimulation.
Regarding the physical interaction of MCA-3 and FLWR-1, we performed bimolecular fluorescence complementation, with two fragments of mVenus fused to the two proteins. This assay shows mVenus reconstitution (i.e., fluorescence) if the two proteins are found in close vicinity to each other. Testing MCA-3 and FLWR-1 in muscle indeed showed a robust signal, evenly distributed on the plasma membrane. As a control, FLWR-1 did not interact with another plasma membrane protein, the stomatin UNC-1 interacting with gap junction proteins (Chen et al., 2007, Curr Biol 17: 1334-9). FLWR-1 also interacted with the ER chaperone Nicalin (NRA2 in C. elegans), which helps assembling the TM domains of integral membrane proteins in association with the SEC translocon. However, this signal only occurred in the ER membrane, demonstrating the specificity of the BiFC assay. This data is presented in Fig. 9A, B. Additionally, we show that FLWR-1 expression has a function in stabilizing MCA-3 localization at synapses, which is also in line with the idea of a direct interaction (Fig. 9C, D).
(2) In silico analysis identified residues R27 and K31 as potential PIP2 binding sites in FLWR-1. The authors observed that FLWR-1(R27A/K31A) was less effective than wild-type FLWR-1 in rescuing the aldicarb sensitivity phenotype of the flwr-1 knockout, suggesting that FLWR-1 function may depend on PIP2 binding at these two residues. Given that mutations in various residues can impair protein function non-specifically, additional studies may be needed to confirm the significance of these residues for PIP2 binding and FLWR-1 function. In addition, the authors might consider explicitly discussing how this finding aligns or contrasts with the results of a previous study in flies, where alanine substitutions at K29 and R33 impaired a Flower-related function (Li et al., eLife 2020).
We further investigated the role of these two residues in an in vivo assay for PIP2 binding and membrane association of a reporter. We used the coelomocytes (CCs), in which a previous publication demonstrated that a GFP variant tagged with a PH domain would be recruited to the CC membrane (Bednarek et al., 2007, Traffic 8: 543-53). This assay was performed in wild type, flwr-1 mutants, and flwr-1 mutants rescued with wild type FLWR-1, the FLWR-1(E74Q) mutant, or the FLWR-1(K27A; R31A) double mutant. The data are shown in Fig. 8C, D. While the wild type FLWR-1 rescued PH-GFP levels at the CC membrane to the wild type control, the FLWR-1(K27A; R31A) double mutant did not rescue the reporter binding, indicating that, at least in CCs, reduced PIP2 levels are associated with non-functional FLWR-1. Mechanistically, this is not clear at present, though we noted a possible mechanism as found for synaptotagmin, that recruits the PIP2 kinase to the plasma membrane via a lysine and arginine containing motif (Bolz et al., 2023, Neuron 111: 3765-3774.e3767). We mention this now in the discussion. We also discussed our data with respect to the findings of Li et al., about the analogous residues K27, R31 (K29, R33) in the discussion section, i.e. lines 667-670, and the differences of our findings in electron microscopy compared to the Drosophila work (more rather than less bulk endosomes) were discussed in lines 713-720.
(3) A primary conclusion from the EM data was that FLWR-1 participates in the breakdown, rather than the formation, of bulk endosomes (lines 20-22). However, the reasoning behind this conclusion is somewhat unclear. Adding more explicit explanations in the Results section would help clarify and strengthen this interpretation.
We added a sentence trying to better explain our reasoning. Mainly, the argument is that accumulation of such endosomes of unusually large size is seen in mutants affecting formation of SVs from the endosome (in endophilin and synaptojanin mutants), while mutants affecting mainly endocytosis (dynamin) cause formation of many smaller endocytic structures that stay attached to the plasma membrane (Kittelmann et al., 2013, PNAS 110: E3007-3016). We changed our data analysis in that we collated the data for what we previously termed endosomes and large vesicles. According to the paper by Watanabe, 2013, eLife 2: e00723, endosomes are defined by their location in the synapse, and their size. However, this work used a much shorter stimulus and froze the preparations within a few dozens to hundreds of msec after the stimulus, while we used the protocol of Kittelmann 2013, which uses 30 sec stimulation and freezing after 5 sec. There, endosomes were defined as structures larger than SVs or DCVs, but no larger than 80 nm, with an electron dense lumen, and were very rarely observed. In contrast, large vesicles or ‘100 nm vesicles’, ranged from 50-200 nm diameter, with a clear lumen, were morphologically similar to the bulk endosomes as observed by Li et al., 2021. We thus reordered our data and jointly analyzed these structure as large vesicles / bulk endosomes. The outcome is still the same, i.e. photostimulated flwr-1 mutants showed more LVs than wild type synapses.
(4) The aldicarb assay results in Figure 3 are intriguing, indicating that reduced GABAergic neuron activity alone accounts for the flwr-1 mutant's hyposensitivity to aldicarb. Given that cholinergic motor neurons also showed increased activity in the flwr-1 mutant, one might expect the flwr-1 mutant to display hypersensitivity to aldicarb in the unc-47 knockout background. However, this was not observed. The authors might consider validating their conclusion with an alternative approach or, at the minimum, providing a plausible explanation for the unexpected result. Since aldicarb-induced paralysis can be influenced by factors beyond acetylcholine release from cholinergic motor neurons, interpreting aldicarb assay results with caution may be advisable. This is especially relevant here, as FLWR-1 function in muscle cells also impacts aldicarb sensitivity (Figure S3B). Previous electrophysiological studies have suggested that aldicarb sensitivity assays may sometimes yield misleading conclusions regarding protein roles in acetylcholine release.
We tested the unc-47; flwr-1 animals again at a lower concentration of aldicarb, to see if the high concentration may have leveled the differences between unc-47 animals and the double mutant. This experiment is shown in Fig. S3D, demonstrating that the double mutant is significantly less resistant to aldicarb. This verifies that FLWR-1 acts not only in GABAergic neurons, but also in cholinergic neurons (as we saw by electron microscopy and electrophysiology), and that the increased excitability of cholinergic cells leads to more acetylcholine being released. In the double mutant, where GABA release is defective, this conveys hypersensitivity to aldicarb.
(5) Previous studies have suggested that the Flower protein functions as a Ca2+ channel, with a conserved glutamate residue at the putative selectivity filter being essential for this role. However, mutating this conserved residue (E74Q) in C. elegans FLWR-1 altered aldicarb sensitivity in a direction opposite to what would be expected for a Ca2+ channel function. Moreover, the authors observed that E74 of FLWR1 is not located near a potential conduction pathway in the FLWR-1 tetramer, as predicted by Alphafold3. These findings raise the possibility that Flower may not function as a Ca2+ channel. While this is a potentially significant discovery, further experiments are needed to confirm and expand upon these results.
As above, we do not exclude that FLWR-1 may constitute a channel, however, based on our findings, AF3 structure predictions and data in the literature, we are considering alternative explanations for the observed effect on Ca2+ levels of Flower mutants in worms and flies. The observations of increase Ca2+ levels in stimulated flwr-1 mutant neurons could result from a reduced stimulation of the PMCA, and this was also observed with low stimulation in Drosophila (Yao et al., 2017). This idea is supported by the indications of a direct physical interaction, or proximity, of the two proteins. The reduced Ca2+ levels after hyperstimulation of Drosophila Flower mutants may have to do with increased levels of non-recycling PMCA in the plasma membrane, indicating that PMCA requires Flower for recycling. This could be underlying the rundown of evoked PSCs we find in worm flwr-1 mutants, and would also be in line with a function of FLWR-1 and MCA-3 in coelomocytes, cells that constantly endocytose, and in which both proteins are required for proper function (our data, Figs. 5A, B; 8D, E) and Bednarek et al., 2007 (Traffic 8: 543-553). CCs need to recycle / endocytose membranes and membrane proteins, and such proteins, likely including FLWR-1 and MCA-3, need to be returned to the PM effectively.
We thus refrained from testing a putative FLWR-1 channel function in Xenopus oocytes, in part also because we would not be able to acutely trigger possible FLWR-1 gating. A constitutive Ca2+ current, if it were present, would induce large Cl- conductance in oocytes, that would likely be problematic / killing the cells. The demonstration that FLWR-1(E74Q) does not rescue the PI(4,5)P2 levels in coelomocytes is also more in line with a non-channel function of FLWR-1.
(6) Phrases like "increased excitability" and "increased Ca2+ influx" are used throughout the manuscript. However, there is no direct evidence that motor neurons exhibit increased excitability or Ca2+ influx. The authors appear to interpret the elevated Ca2+ signal in motor neurons as indicative of both increased excitability and Ca2+ influx. However, this elevated Ca2+ signal in the flwr-1 mutant could occur independently of changes in excitability or Ca2+ influx, such as in cases of reduced MCA-3 activity. The authors may wish to consider alternative terminology that more accurately reflects their findings.
Thank you, we rephrased the imprecise wording. Ca2+ influx was meant with respect to the cytosol.
Reviewer #3 (Public review):
Summary:
Seidenthal et al. investigated the role of the Flower protein, FLWR-1, in C. elegans and confirmed its involvement in endocytosis within both synaptic and non-neuronal cells, possibly by contributing to the fission of bulk endosomes. They also uncovered that FLWR-1 has a novel inhibitory effect on neuronal excitability at GABAergic and cholinergic synapses in neuromuscular junctions.
Strengths:
This study not only reinforces the conserved role of the Flower protein in endocytosis across species but also provides valuable ultrastructural data to support its function in the bulk endosome fission process. Additionally, the discovery of FLWR-1's role in modulating neuronal excitability broadens our understanding of its functions and opens new avenues for research into synaptic regulation.
Weaknesses:
The study does not address the ongoing debate about the Flower protein's proposed Ca2+ channel activity, leaving an important aspect of its function unexplored. Furthermore, the evidence supporting the mechanism by which FLWR-1 inhibits neuronal excitability is limited. The suggested involvement of MCA-3 as a mediator of this inhibition lacks conclusive evidence, and a more detailed exploration of this pathway would strengthen the findings.
We added new data showing the likely direct interaction of FLWR-1 with the PMCA, possibly upregulating / stimulating its function. This data is shown now in Fig. 9A, B. Also, we show now that FLWR-1 is required to stabilize MCA-3 expression / localization in the pre-synaptic plasma membrane (Fig. 9C, D). These findings are not supporting the putative function of FLWR-1 as an ion channel, but suggest that increased Ca2+ levels following neuron stimulation in flwr-1 mutants are due to an impairment of MCA-3 and thus reduced Ca2+ extrusion.
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors):
The authors might consider focusing on one or two key findings from this study and providing robust evidence to substantiate their conclusions.
We did substantiate the interactions of FLWR-1 and the PMCA, as well as assessing the function of FLWR-1 in the coelomocytes and the function of FLWR-1 in regulating PIP2 levels in the plasma membrane.
Reviewer #3 (Recommendations for the authors):
(1) Behavioral Analysis of Locomotion
In Figure 1, the authors are encouraged to examine whether flwr-1 mutants show altered locomotion behaviors, such as velocity, in a solid medium.
We performed such an analysis for wild type, comparing to flwr-1 mutants and flwr-1 mutants rescued with FLWR-1 expressed from the endogenous promoter. The data are shown in Fig. S1C. There was no difference. We note that we observed differences in swimming assays also only when we strongly stimulated the cholinergic neurons by optogenetic depolarization, but not during unstimulated, normal swimming.
(2) Validation of FLWR-1 Tagging
In Figure 2A, it is recommended that the authors confirm the functionality of the C-terminal-tagged FLWR-1.
We performed such rescue assays during swimming. The data is shown in Fig. S2S, E. While the GFP::FLWR-1 animals were slightly affected right after the photostimulation, they quickly caught up with the wild type controls, while flwr-1 mutants remained affected even after several minutes.
(3) Explanation of Differential Rescue in GABAergic Neurons and Muscle
The authors should provide a rationale for why restoring FLWR-1 in GABAergic neurons fully rescues the aldicarb resistance phenotype, while its restoration in muscle also partially rescues it.
We think that these effects are independent of each other, i.e. loss of FLWR-1 in muscles increases muscular excitability, which becomes apparent in the behavioral assay that depends on locomotion and muscle contraction. To assess this further, we performed combined GABAergic neuron and muscle rescue assays, as shown in Fig. S3B. The double rescue was not different from wild type, and performed better than the muscle rescue alone.
(4) Rescue Experiments for Swimming Defect in GABAergic Neurons
Consider adding rescue experiments to determine whether expressing FLWR-1 specifically in GABAergic neurons can restore the swimming defect phenotype.
We did not perform this assay as swimming is driven by cholinergic neurons, meaning that we would only indirectly probe GABAergic neuron function and a GABAergic FLWR-1 rescue would likely not improve swimming much. Also, given the importance of the correct E/I balance in the motor neurons, it would likely require achieving expression levels that are very precisely matching endogenous expression levels, which is not possible in a cell-specific manner.
(5) Further Data on GCaMP Assay for mca-3; flwr-1 Additive Effect
The additive effect of the mca-3 and flwr-1 mutations on GCaMP signals requires further data for substantiation. Additional GCaMP recordings or statistical analysis would provide stronger support for the proposed interaction between MCA-3 and FLWR-1 in calcium signaling.
Thank you. We increased the number of observations, and could thus improve the outcome of the assay in that it became more conclusive. Meaning, the double mutation was not exacerbating the effect of either single mutant, demonstrating that FLWR-1 and MCA-3 are acting in the same pathway. The data are in Fig. 7B, C.
(6) Inclusion of Wild-Type FLWR-1 Rescue in Figures 8B and 8D
Figures 8B and 8D would benefit from the inclusion of wild-type FLWR-1 as a rescue control.
We included the FLWR-1 wild type rescue as suggested and summarized the data in Fig. 8B.
