Drosophila TRAPPC8-Rab1 module regulates retrograde trafficking of Wingless and Evi/Wntless

  1. Satyam Sharma
  2. Juilee Sabnis
  3. Aendrila Adhikary
  4. Varun Chaudhary

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Abstract

Lipid-modified Wnt proteins are secreted from polarized epithelial cells through a multi-step pathway involving membrane delivery, internalization and resecretion. Internalization enables dissociation of Wnt from its cargo receptor Evi/Wntless in endosomes and promotes retromer-mediated recycling of Evi to the Golgi, however, the molecular basis of this retrograde Wnt trafficking remains poorly understood. Here, we identify an essential function of TRAPPC8, the TRAPPIII-specific subunit, in the trafficking of Drosophila Wingless (Wg), the Wnt1 homolog. Loss of TRAPPC8 impaired retrograde trafficking of both Wg and Evi, resulting in their intracellular accumulation in Wg-producing cells. Consistent with the role of TRAPPIII as a Rab1-specific GEF, inhibition of Rab1 phenocopied the trafficking defects caused by TRAPPC8 loss, whereas constitutively active Rab1 rescued these defects. Furthermore depletion of either TRAPPC8 or Rab1 increased the levels of Wg-unbound Evi, indicating that they act downstream of Evi-Wg dissociation. Together, these findings identify the TRAPPIII complex and its effector Rab1 as key regulators of retrograde Wg trafficking required for its efficient secretion.

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    Review Commons: RC-2026-03417

    We thank all three reviewers for their thoughtful and constructive evaluations of our manuscript. We are encouraged that the reviewers recognize the significance of identifying a role for the TRAPPIII-Rab1 module in Wingless (Wg) trafficking and secretion. We also appreciate the insightful comments that have helped us identify areas where our interpretations, experimental support, and presentation can be strengthened.

    Below, we provide a detailed point-by-point response to all comments and concerns raised by the reviewers.

    Reviewer #1

    __Evidence, reproducibility and clarity __

    Summary

    In this manuscript, the authors investigate the role of Transport Protein Particle (TRAPP) complexes in the secretion of Wingless (Wg) in Drosophila. Two TRAPP complexes are known to exist: TRAPPII and TRAPPIII. Through the systematic analysis of individual subunits, the authors identify the requirement of the TRAPPIII subunit TRAPPC8 for Wg trafficking.

    They demonstrate that the absence of TRAPPC8 disrupts the retrograde trafficking of both Wg and its carrier Evi, resulting in their intracellular accumulation within Wg- producing cells. Further analyses suggest that TRAPPC8 controls the post-apical internalisation and endosomal trafficking of Wg and Evi. Consistent with the established function of TRAPPIII as a Rab1-specific GEF, they demonstrate that inhibiting Rab1 produces similar effects as depleting TRAPPC8, while constitutively active Rab1 reverses the trafficking defects. Furthermore, depletion of either TRAPPC8 or Rab1 increases the levels of Wg-unbound Evi, suggesting that they act downstream of Evi-Wg dissociation. Taken together, these findings suggest that TRAPPIII and its effector Rab1 are essential regulators of retrograde Wg trafficking, which is necessary for efficient secretion.

    Overall, the work is carefully performed and the results are presented clearly. The controls are appropriate and the study expands the functional scope of Rab1-dependent trafficking beyond early secretory pathways. The identification of a previously unrecognised function of TRAPPC8 in Wg trafficking is a valuable contribution.

    Major Points

    It is not entirely clear whether the RNAi lines used in the initial screen were validated for knockdown efficiency. Notably, some core TRAPPIII subunits (e.g., TRAPPC3 and TRAPPC5) do not show a phenotype. This could indicate that the complex retains partial function upon their depletion, or alternatively that the RNAi lines are ineffective. While this point may not critically affect the main conclusion regarding TRAPPC8, it is important for drawing conclusions about the specificity of TRAPPIII versus TRAPPII involvement. For instance, TRAPPC10 (a TRAPPII-specific subunit) was analysed using a single RNAi line, yet no evidence of knockdown efficiency was provided. Validation of these RNAi reagents would strengthen the conclusions regarding complex specificity.

    Response: *We thank the reviewer for highlighting this shortcoming in the manuscript. We agree that testing the knockdown efficiency of the TRAPPII complex subunits will strengthen our conclusion on the specificity of TRAPPC8/TRAPPIII towards Wg trafficking. While testing the protein levels would be ideal for checking knockdown efficiency, specific antibodies to these subunits are not commercially available. Therefore, as an alternative approach, we will perform RT-PCR to assess the efficiency of RNAi-mediated depletion of the TRAPPII-specific subunits (TRAPPC9 and TRAPPC10). *

    Furthermore, we would also like to highlight that previous studies have reported variable phenotypic outcomes upon loss of TRAPP complex subunits, ranging from no apparent defects to early lethality (Sun and Sui 2023; Riedel et al. 2018). For example, complete deletion of the C9 gene and a frameshift mutation in the C10 gene were found to be homozygously viable, whereas C8 and C11 homozygous mutants showed early larval lethality (Riedel et al., 2018).

    *To better contextualize our findings, we will include a more detailed discussion comparing our results with the phenotypes reported for these published mutants, in addition to the RT-PCR analysis of RNAi efficiency. *

    Minor Points

    In Figure 2C, the clone appears restricted to the apical region, and I do not clearly observe GFP loss in the basolateral domain. Larger clones or additional sections would help clarify the spatial distribution and strengthen the interpretation.

    Response - *We thank the reviewer for pointing this out. This issue arises from the pseudostratified nature of the wing epithelium. For smaller clones, such as those observed in the TRAPPC8 mutant, cross-sections can occasionally pass through the tissue at an oblique angle, making it difficult to capture the entire clone within a single section. *

    We have repeated the experiment and now provide better images where the apical and basolateral distribution of the mutant clone is visible, and accumulation of exWg can be observed (see updated Figure 2A-C).

    * The authors should discuss why TRAPPC8 depletion results primarily in apical Wg enrichment, whereas Rab1 inhibition leads to Wg accumulation at both apical and basolateral membranes. This difference may provide insight into whether Rab1 has additional TRAPPIII-independent functions or whether TRAPPC8 affects a more spatially restricted trafficking step.*

    __ ____Response__: *We agree that, while inhibition of either TRAPPC8 or Rab1 led to a strong intracellular accumulation of Wg, subtle differences were observed in the localization of the accumulated Wg. In agreement with your comment and as suggested by previous studies, C8 may direct the Rab1-GEF activity of the core TRAPP complex towards a specific location, while Rab1DN will affect all downstream functions, possibly also including TRAPPIII-independent effects (Riedel et al. 2018; Gyurkovska et al. 2026). We will modify the text to incorporate these points more clearly. *

    __ ____Significance__

    This study broadens our understanding of Rab1-dependent membrane trafficking by identifying a previously unrecognized role for the TRAPPIII complex in retrograde transport during Wingless secretion. ____While the study convincingly establishes the involvement of the TRAPPIII-Rab1 module in Wg trafficking, it does not define the specific retrograde trafficking step that is regulated by this machinery. In addition, the functional relationship between TRAPPIII-Rab1 and established retrograde regulators, such as the retromer complex, is not addressed.

    * *____Response: We thank the reviewer for this comment. This issue was also raised by Reviewer 3. We agree that the precise retrograde trafficking step regulated by the TRAPPC8-Rab1 module remained unclear in the study. To address this, we will perform additional experiments to investigate the functional interactions between TRAPPC8-Rab1 and components of the retrograde trafficking machinery, including the retromer complex, and will incorporate these findings into the revised manuscript. These analyses should help clarify the specific trafficking step controlled by the TRAPPC8-Rab1 module.

    * Further molecular and mechanistic analyses will be required to position TRAPPIII within the broader retrograde trafficking network and to fully elucidate how Rab1 activity is coordinated with other pathways involved in Wnt secretion. These unresolved issues represent the main limitation of the current study.*

    __ ____Response__: Wg secretion in polarized epithelial cells involves multiple transport routes and regulatory mechanisms, including exosome-mediated transport, cytonemes, and association with carrier molecules. A systematic analysis of the contribution of TRAPPC8-Rab1 to each of these pathways would require substantial additional investigation and falls outside the scope of the present study.

    In this work, we focused on a key, relatively underexplored aspect of Wg transport: the retrograde pathway that mediates the separation and recycling of Wg and Evi/Wls. Our findings identify TRAPPC8-Rab1 as an important component of this process and provide a basis for future studies to define its broader mechanistic integration into intracellular trafficking pathways.

    Reviewer #2

    __Evidence, reproducibility and clarity __

    __Summary Sharma, Sabnis et al. show in this report that TRAPPC8 and Rab1 are important for Wingless (Wg) secretion in developing Drosophila wing discs.

    • Loss of TRAPPC8 (either knockdown or mutant clones) lead to higher levels of apical Wg in Wg-producing cells (total and extracellular staining).
    • The levels of the intracellular Wg transporter Evi/Wls are also increased, in particular in its 'unbound' form.
    • A similar phenotype is observed upon overexpression of Rab1 dominant negative.
    • Overexpression of constitutively active Rab1 has no effect in otherwise wild type discs but does rescue the effect of TRAPPC8 loss of function.

    Major concerns

    1. The author's key message is that TRAPPC8 is essential for retrograde transport of Wg and Evi. However, in apparent contradiction, TRAPPC8 loss does not appear to affect the basolateral distribution of exWg (or total Evi) (Fig2B', FigS3). Therefore, while Wg transport may be less efficient in the absence of TRAPPC8, the conclusion that TRAPPC8 is essential should be toned down. It is warranted to suggest that transport of unbound Evi is disrupted in the absence of TRAPPC8, as unbound Evi increases apically and is lost basally (Fig 4A-B).__

    Response:

    *Thank you for these comments, which prompted us to retest the extracellular Wg levels in these experiments. We repeated the experiments and reanalyzed the basolateral distribution of extracellular Wg in TRAPPC8-depleted cells. While we consistently observed an increase in apical extracellular Wg, the effects on basolateral Wg were more variable. In several samples, basolateral Wg appeared largely unaffected, whereas in others we observed some reduction. We speculate that this variability may arise from differences in RNAi efficiency, with stronger depletion of TRAPPC8 potentially required to reveal basolateral defects. Furthermore, these defects are completely rescued by the expression of Rab1CA, indicating Rab1-dependent effects of TRAPPC8 loss. *

    *We apologize for this oversight in our analysis in the original manuscript and would like to correct our conclusions regarding the basolateral Wg. We have included both results in the partially revised manuscript, indicating the variability observed in the basolateral Wg, albeit with consistent apical accumulation of extracellular Wg. *

    Furthermore, we have modified the statement (line 235, page 8) "These results suggest that TRAPPC8 functions after the dissociation of the Evi-Wg complex, likely promoting proper sorting of Evi and Wg within maturing endosomes" to "These results suggest that TRAPPC8 functions after the dissociation of the Evi-Wg complex, likely affecting proper retrograde trafficking of unbound-Evi."

    *Pending revision: **We will further update our discussion section to include these points after completing all additional experiments suggested by the reviewers. *

    2) The relative roles of Rab1 and TRAPPC8 are not equally considered. As the authors show, unlike TRAPPC8 loss of function, Rab1 DN causes a decrease in basal exWg. Doesn't this suggest that Rab1 is more important for Wg trafficking than TRAPPC8?

    __Response: __This issue is addressed in the previous comment.

    a) Rab1 DN causes also accumulation of total basal Wg. Does this mean that Wg can be trafficked basally but fails to be secreted at the basal surface?

    __Response: __Yes, we agree with this interpretation. In Rab1DN-expressing cells, total Wg accumulates throughout the cell, including at the basal side, while basolateral extracellular Wg is reduced. This suggests that Wg-containing vesicles can reach the basolateral region but are inefficiently secreted at the basal surface. One possibility is that Rab1 function is required for the apical-to-basolateral transcytosis and/or the final delivery of Wg-containing vesicles to sites of basolateral secretion.

    b) A similar assay (total basal Wg) is lacking in condition of TRAPPC8 loss (clones or RNAi); only exWg is shown.__

    __Response: __We will also include the basolateral sections of total Wg in TRAPPC8 depletion conditions (mutant clones and RNAi) in the revised manuscript.

    c) Also needed is an assessment of total and exWg at the basal surface in the Rab1 CA experiment. Based on the cross-section image shown in Fig6B, it seems that the distribution of Wg might be more confined to the apical plane of the cells compared to other conditions (Fig6F for example), but there is little difference in this regard between the anterior and posterior compartments.__

    __Response: __*We thank the reviewer for this important point, and we will reanalyze both total and extracellular Wg levels in Rab1CA samples, specifically the apical and basolateral distributions and provide images in the revised manuscript. *

    3) A significant claim of the paper is "TRAPPC8 regulates retrograde Wg trafficking post-apical internalization". a. The increase in apical extracellular Wg (FigS3), which seems to remain associated with the membrane of Wg-producing cells (i.e. no onward spread) would suggest that apical internalization may be affected, but this is not considered by the authors.

    __Response: __*We agree that the increased apical extracellular Wg observed in Fig. S3 (now Fig 2D-F and S3) could also suggest alterations in apical uptake dynamics. However, based on our internalization assay, we believe that the major defect is likely in the post-endocytic sorting/trafficking of Wg after apical internalization rather than a complete block in internalization itself. In these internalization assays, following the internalization pulse, the tissue was subjected to a brief acid wash to remove extracellular and surface-bound antibody. At the same time, the internalized antibody-antigen complexes remain protected. Therefore, the Wg signal detected after the acid wash represents internalized apical Wg, demonstrating that apical internalization does occur in the TRAPPC8 loss condition. *

    4) From the antibody chase experiment, it is not clear if the effect of TRAPPC8 loss affects endocytosis and/or trafficking in general or whether it is specific to Wg and Evi. A fluorescent Dextran control should be included as in Witte et al., 2020 (https://doi.org/10.1242/dev.186833 ). Also, can the authors be sure that they are visualising internalised Wg and not just internalised Wg antibody? Can the author show a field of view where Wg is not expressed?

    __ Response: __*Thank you for this important suggestion. We would first like to clarify that the Wg internalization assay, using the highly specific monoclonal anti-Wg antibody, is an established approach that has been used in multiple previous studies to monitor Wg internalization and trafficking (Hemalatha et al. 2016; Sharma and Chaudhary 2024). Furthermore, as shown in Figure S4C-C′ (now moved to Fig 3D), the internalized Wg signal is detected specifically in cells close to the DV boundary, while more distal receiving cells do not show comparable staining. This spatially restricted pattern strongly supports the specificity of the assay for Wg-producing cells and a limited number of nearby receiving cells that may internalize secreted Wg, rather than nonspecific internalization of the antibody alone. We will revise the text to clarify this point in the revised manuscript. *

    *To address whether TRAPPC8 loss affects general endocytosis, we will perform a fluorescent Dextran uptake assay, as suggested by the reviewer and similar to the approach described by (Witte et al. 2020). *

    5) The authors suggest that TRAPPC8 loss leads to increased Rab7 levels. This is taken as evidence for a role of acidification in driving Wg dissociation from Evi, with TRAPPC8 acting downstream to sort Wg from mature endosomes. ____Neither of these claims are supported because in Fig.S3G loss of TRAPPC8 results in an increase in Rab7 everywhere except at the DV boundary where the Wg-producing cells are. This should be acknowledged in text, and these data should appear in the main Fig4.

    Response: To address this concern, we have repeated the experiment and reanalyzed the RAb7 and lysotracker levels specifically in cells at the DV boundary. The data is now moved to the updated main Figure 5E-F and 5G-H. A significant increase in both lysotracker and Rab7 can be observed, suggesting that loss of TRAPPC8 affected late endosomal maturation.

    *a) *The increase in lysotracker also does not support the above claims as it could be due to an increase in unbound Evi that cannot be trafficked back to the ER (and hence targeted for degradation in lysosomes).

    __Response: __We respectfully disagree with this interpretation. If TRAPPC8 loss primarily caused increased lysosomal degradation of unbound Evi, we would expect a reduction in total Evi levels, as observed upon loss of retromer function, in which Evi is diverted to lysosomes and specifically depleted in Wg-producing cells (Port et al. 2008). In contrast, we observe an accumulation of both total and unbound Evi in TRAPPC8-depleted cells (Figures 4A-D and 5A-D), arguing against enhanced lysosomal degradation as the primary defect.

    b) In fig S3G, the purported Rab7 increase in the posterior compartment is not readily apparent (and not quantified), in contrast to the authors' description of the effect of TRAPPC8 depletion. This suggest that the model proposed by the authors needs to be revised (Fig6G) and the relevant paragraphs from Results and Discussion section must be significantly edited or removed.

    Response: We apologize for the lack of clarity in our previous images. As noted above, we now observe a significant increase in Rab7 and Lysotracker signals in Wg-producing cells, indicating an expansion of Rab7-positive acidic late endosomal compartments upon TRAPPC8 depletion. Furthermore, our interpretation is consistent with our previous findings showing that the Evi-Wg complex dissociates within maturing endosomes.

    __Minor concerns

    1. The way data in Fig 2, S3 and S4 is presented and referred to in text could be rearranged slightly to make it easier to follow: first talk about the work using clones (Fig2, FigS4 becomes FigS3), then mention similar results with the knockdown (FigS3 becomes FigS4). This arrangement would link naturally to the effect on Evi.__

    Response: Thanks for your suggestion, we have revised the supplementary figure order by changing FigS3 (now Fig S4) to FigS4 (now Fig S5) and FigS4 (now Fig S5) to FigS3 (now Fig S4), and the corresponding figure references in the main text have been updated accordingly. We have also moved some of the RNAi data from Fig S3 to main figures (Fig 2D-F and Fig 3D-F) to increase the clarity and make the results easier to follow.

    2) How do the authors explain that the knockdown of some core TRAPP subunits does not have a phenotype? Some sort of rationalisation (or experimental follow up) is desirable.

    *__Response: __The lack of a detectable phenotype following knockdown of some core TRAPP subunits may reflect several possibilities. Previous studies have suggested that certain core subunits, including TRAPPC2, TRAPPC2L, TRAPPC6A and TRAPPC6B, are not universally required for mammalian cell viability, indicating potential functional redundancy or their context-dependent requirements within the TRAPP core complex (Lipatova and Segev, 2019; Sun and Sui, 2023). It is also possible that residual protein levels after RNAi-mediated depletion are sufficient to support partial TRAPP function. Testing the RNAi-mediated protein depletion of these subunits is beyond the scope of the current study. *

    *We would like to highlight that the focus of the study is TRAPPC8, a TRAPPIII-specific subunit, which was validated with a genomic mutation. However, to strengthen our conclusions regarding the TRAPPIII-specific effect, we will validate the known efficiency of the TRAPPII complex subunits with RT-PCR, also addressing the comment from Reviewer 1. *

    3) The authors should give more detail about how they quantified normalised intensity profiles and clarify if the profiles correspond to just the representative image shown or the average of multiple discs (possible for the compartment experiments, but presumably impossible for the clone experiments as they would need to normalise by the size of the clone too).

    Response: *Thank you. We have updated the figure legend to clearly indicate the representative images corresponding to each plot profile. The plot profiles were generated from representative images only and do not represent averages from multiple discs. The intensity values were normalized by dividing each value by the mean intensity across the quantified region. We have also added this information in the Materials and Methods section (line 447, page 16). *

    __ ____Significance__

    This report is a valuable report for the Wg secretion subfield, and useful for the Wnt community. It makes some interesting observations and brings the importance of Rab1 back into the conversation after Ching et al., 2008. However, the insight remain limited and the mechanism/trafficking defects remain unclear. There is convincing evidence that TRAPPC8 and Rab1 affect Wg and Evi, but the claims that TRAPPC8 is essential and acts downstream of acidic endosomes is inadequate.

    __ ____Response:__ * Please refer to the responses for Reviewer 3. We have addressed these concerns in the next section.*

    Reviewer #2 (Significance (Required)):

    __This report is a valuable report for the Wg secretion subfield, and useful for the Wnt community. It makes some interesting observations and brings the importance of Rab1 back into the conversation after Ching et al., 2008. However, the insight remain limited and the mechanism/trafficking defects remain unclear. There is convincing evidence that TRAPPC8 and Rab1 affect Wg and Evi, but the claims that TRAPPC8 is essential and acts downstream of acidic endosomes) is inadequate. ____ __

    __Reviewer #3 __

    This manuscript uncovers a direct or indirect role of RAB1/TRAPPIII in regulating the intracellular fate of Wng in the columnar epithelial cells of the wing imaginal disc of the fruit fly. This observation is interesting, although it should come as no surprise that, given the fact that Wnts are secreted morphogens and considering the involvement of TRAPPIII in the early stages of the secretory pathway, the key TRAPPIII subunit, TRS85/TRAPPC8, is crucial for their normal trafficking. The main finding of this work is that cells deficient in TRAPPIII/RAB1 accumulate the morphogen and its receptor in the apical region of morphogen-producing cells, which is interpreted as a block in the retrograde trafficking of the morphogen.

    1) While it is unclear to me what the authors consider as retrograde trafficking of Wng (see below), the arguments fall short of being convincing, in part because the intracellular trafficking of Wng is rather intricate, but also because the physiological role of TRAPPIII is insufficiently understood. It is well established that Wng transits through endosomes to reach multivesicular bodies, where it is incorporated into the inwardly budding vesicles that are secreted as exosomes (Gross et al, cited). Do the authors consider this a retrograde pathway? The authors do not delineate further the location at which Wng accumulates, for example using co-localization studies.

    __Response: __*We would like to clarify that, in our manuscript, we use the term "retrograde trafficking" specifically to describe the trafficking route followed by apically internalized Wg/Evi complexes through the endosomal system before their re-secretion from Wg-producing cells. *

    *Wg trafficking after internalization is highly complex and can involve multiple intracellular routes. Importantly, besides trafficking of internalized Wg to multivesicular bodies (MVBs) for exosomal secretion, other pathways downstream of apical internalization have also been reported, including Rab4-dependent apical recycling, apical-to-basolateral transcytosis involving HSPGs, and secretion of Wg on lipoprotein particles. Testing the effect of TRAPPC8 in multiple trafficking routes is currently beyond the scope of this study. *

    *Our current study does not attempt to distinguish between these individual downstream secretory routes. Rather, our data support a requirement for TRAPPC8/Rab1 in trafficking steps occurring after apical internalization of Wg/Evi and before their redistribution and/or re-secretion. The detailed characterization of the precise endosomal compartments and carrier-specific secretion mechanisms affected by TRAPPC8/Rab1 will require significant additional lines of experiments, which are beyond the scope of the current work. *

    __2) They have also not provided a rationalization of their observations with current knowledge of retrograde trafficking between the endosomes and the Golgi. It would have been interesting to address the effects of Trs85 depletion in mutant backgrounds deficient in the master regulator of retrograde pathways, RAB6, its effector, the GARP complex, or RAB7 and the retromer; it would have been important to study TRAPPIII depletion under conditions in which endocytic internalization is blocked, or the biogenesis of multivesicular bodies is prevented. __

    __ ____Response: __We agree that understanding the functional relationship between TRAPPIII and other retrograde trafficking regulators would provide important mechanistic insight into Wg/Evi trafficking.

    *Among the known regulators of retrograde trafficking, we are particularly interested in testing the functional interaction between TRAPPC8 and the retromer complex, as retromer is one of the best-characterized regulators of Evi recycling in the Drosophila Wg pathway (Port et al., 2008; Franch-Marro et al., 2008; Belenkaya et al., 2008). Therefore, we will analyze the functional interactions between TRAPPC8 and retromer components and provide results in the revised manuscript. *

    However, the roles of other retrograde trafficking regulators, such as GARP and Rab6, in retrograde Wg trafficking in Drosophila are not yet well established and would first require independent characterization before meaningful epistasis analyses with TRAPPIII can be performed.

    *Regarding the suggestion to block endocytic internalization, our antibody internalization experiments indicate that early Wg internalization, including uptake of Wg in neighboring receiving (non-secreting) cells, is not detectably affected upon TRAPPC8 or Rab1 depletion. These observations suggest that TRAPPC8 and Rab1 are unlikely to play a general role in endocytic uptake. *

    We therefore focused our analyses on post-internalization trafficking events affecting Wg and Evi. Furthermore, blocking endocytosis globally would likely introduce strong secondary effects on both Evi-Wg complex internalization in secreting cells and uptake of extracellular Wg in receiving cells, making it difficult to distinguish direct effects on retrograde trafficking from broader defects in Wg trafficking dynamics.

    __Specific comments __

    3) There are no page or line numbers, which makes very cumbersome to comment on specific sections of the manuscript.

    *__Response: __We apologize for this inconvenience and have now added page and line numbers. *

    __4) The introduction contains a factual mistake. TRAPPC11, 12, and 13 are not metazoan specific. They are present in fungi but have been lost in Saccharomyces cerevisiae. Pinar M, Arias-Palomo E, de Los Ríos V, Arst HN Jr, Peñalva MA. Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII. PLoS Genet. 2019 Dec 23;15(12):e1008557. doi: 10.1371/journal.pgen.1008557. This reference should have been cited. __

    __Response: __*We thank the reviewer for pointing this out and apologize for missing this reference. We have now updated the text and added the reference (see line 83 on page 3). *

    __5) Materials and methods are very incomplete, particularly in the section that deals with the antibodies, which are essential tools for understanding the experiments. __

    __Is the Wnt antibody a monoclonal antibody? __

    __Are there different antibodies specific for extracellular and intracellular Wnt? __

    __What is the molecular basis for this differential detection? __

    __The transgene expressing GFP under the engrailed driver is not described anywhere. __

    __ ____Response:__ We apologize for the lack of sufficient detail in the Materials and Methods section. We have now revised this section to include the missing methodological details and clarifications requested by the reviewer.* *

    For all Wg-related stainings, including total, extracellular, and internalization assays, we used the mouse monoclonal anti-Wg antibody obtained from DSHB. Antibody details and working dilutions are provided in the revised Materials and Methods section.

    The differential detection of extracellular versus total Wg does not arise from the use of different antibodies, but rather from differences in the staining protocol. Total Wg staining was performed after permeabilization of wing imaginal discs using 0.2% Triton X-100 in 1X PBS, allowing detection of both intracellular and extracellular Wg pools. In contrast, extracellular Wg staining was performed without tissue permeabilization, thereby restricting antibody access to extracellular or cell surface-associated Wg. Similarly, the Wg internalization assay was performed using established protocols already described in the manuscript. We have now updated the Materials and Methods section to include additional details for the extracellular Wg staining procedure (line 409, page 14).

    • *The GFP expression used in our study is driven by the Gal4-UAS system, where engrailed-Gal4 (en-Gal) drives UAS-GFP expression. We have provided the details for these two fly lines in the Drosophila stocks section in Materials and Methods. For our experiments, we generated a recombinant fly stock having both en-Gal and UAS-GFP on the second chromosome using Drosophila genetics, and this genotype, along with different combinations of genes, is listed in our Supplementary Information.
    • *__6) The labeling of the figures and the figure legends themselves are excessively simple and appear to be accessible for fly experts only. __

    __Response: __We thank the reviewer for this suggestion. We have revised the details for labeling the figure in the legends and expanded the figure legends to improve clarity and accessibility for a broader audience. In particular, we added more detailed descriptions of the specific images with reference to their corresponding quantified graphs (as also suggested by Reviewer 2). We also incorporated additional minor explanatory details (for example, GFP-negative clones mean the homozygous mutant) wherever necessary to help non-fly experts to better understand the figures.

    __7) The authors have not considered the possibility that ablating TRAPPC8 of TRAPPIII can have off-target effects in TRAPPII. It would have been very interesting to address the phenotype of down-regulating TRAPPII and of down-regulating one of the core subunits of TRAPPs. __

    __ ____ Response: __*If the reviewer is suggesting the off-target effects of TRAPPC8 RNAi on TRAPPII complex member, we would like point out that the observations from the RNAi were validated by the TRAPPC8 mutant. However, if the concern is whether TRAPPC8 loss functionally affected TRAPPII complex besides TRAPPIII, then it we have not directly tested this. However, several past studies have shown that TRAPPC8 is a TRAPPIII-specific subunit and not part of TRAPPII complex. Furthermore, and more importantly, we have rescued the RNAi phenotype with the expression of Rab1CA, indicating the effects TRAPPIII-specific are unlikely to be via the TRAPPII-Rab11 pathway. *

    __8) Figure 1, Panel 1i: What is the basis at this point that justifies "likely by altering intra-cellular trafficking"? __

    Response:* Since loss of TRAPPC8 resulted in increased levels of total Wg, we wanted to determine whether this increase could be due to transcriptional upregulation of wg. To address this, we examined the established wg-LacZ reporter and found that its expression was not altered upon loss of TRAPPC8 (Fig 1i).*

    Therefore, the increased Wg levels are unlikely to arise from increased wg transcription. In addition, previous studies have shown that loss of Evi/Wls leads to intracellular accumulation of Wg as a consequence of trafficking defects rather than transcriptional regulation. Based on these observations, we concluded that the accumulation of Wg upon TRAPPC8 depletion is more likely due to altered intracellular trafficking.

    __9) Several figures: where are the boundaries of the cells in orthogonal views? If GFP labels whole cells, why is there an area at the top of the cross-sections that hasn't got GFP staining? __

    __ ____Response:__* In all orthogonal views of the GFP-negative mutant clones, we have used dotted lines to indicate the clone boundaries. T*he wing disc epithelium consists of two distinct epithelial layers: a squamous epithelium (the peripodial membrane) and a pseudostratified columnar epithelium. Although GFP-negative clones are generated in both layers, our analysis focuses specifically on the columnar epithelium, where Wg is expressed. Therefore, the signal observed from the peripodial membrane can vary in the orthogonal views and does not affect our interpretation of Wg localization in the columnar cells.

    __10) I can follow the point that accumulation in the apical side means that retrograde trafficking is impaired. I miss the connection between the observation and the conclusion. __

    __Response: __We apologize for the lack of clarity in explaining the retrograde trafficking defects and would like to clarify this point for Wg.

    In our experiments, we performed both total Wg staining (predominantly intracellular) and extracellular Wg staining. The apical accumulation observed in total Wg staining upon TRAPPC8/Rab1 depletion, by itself, does not directly demonstrate impaired retrograde trafficking. However, the increase in extracellular Wg indicates that Wg can still reach the plasma membrane, suggesting that the anterograde delivery pathway remains functional.

    Our conclusion regarding defective retrograde trafficking is primarily based on the antibody internalization assays. In these experiments, internalized Wg and Evi accumulate intracellularly upon loss of TRAPPC8/Rab1, consistent with a defect in post-endocytic trafficking and recycling. Since both Wg and Evi normally undergo endocytic recycling through retrograde pathways, the accumulation of internalized Evi and Wg supports the interpretation that retrograde trafficking is impaired in TRAPPC8/Rab1-depleted cells.

    __11) VPS34 is an effector of RAB5 and therefore its down-regulation impairs the maturation of early endosomes because their membranes cannot acquire the key component phosphatidylinositol-3-phosphate, which is recognized by the ESCRT machinery to proceed with multivesicular body biogenesis. __

    __ ____Response: __*We agree with the reviewer that VPS34 acts as an effector of Rab5 and plays an important role in early endosome maturation. However, more recent studies have shown that VPS34 functions within two related but functionally distinct complexes, VPS34 complex I and VPS34 complex II. VPS34 complex II, which contains UVRAG, functions predominantly in the endolysosomal system and is associated with Rab5. In contrast, VPS34 complex I, which contains Atg14, functions in autophagy and has been shown to interact with Rab1. Importantly, Rab1 and Rab5 bind VPS34 in a mutually exclusive manner at overlapping interaction sites (Scott and Burke 2026; Cook et al. 2025; Špokaitė et al. 2026; Tremel et al. 2021). *

    • *Therefore, while the reviewer's interpretation regarding Rab5-dependent VPS34 function in endosomal maturation is fully valid, these studies also support the possibility that Rab1 can regulate VPS34-dependent trafficking pathways through a distinct VPS34 complex.

    __12) The Q70L mutation, widely used as a constitutive activator of RABs, is borrowed from studies in RAS and it might not lead to constitutive activation of RAB1 (Langemeyer, L., Nunes Bastos, R., Cai, Y., Itzen, A., Reinisch, K.M., and Barr, F.A. (2014). Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation. eLife 3, e01623). __

    Response: *We thank the reviewer for raising this important point. We admit that we have not performed an independent analysis of the GTP-locked status of the Rab1Q70L mutant in flies. Our rationale for using Rab1Q70L as a GTP-locked or functionally hyperactive Rab1 variant is based on its extensive prior use in the field (Tisdale et al. 1992; Levin et al. 2016; Russo et al. 2016; van Vliet et al. 2026). Importantly, a past study in Drosophila has shown functional hyperactivity of the Rab1Q70L compared with WT Rab1 (Sechi et al. 2017). ** *

    * *

    *References (response to reviewers): *

    Cook, Annan S. I., Minghao Chen, Thanh N. Nguyen, et al. 2025. "Structural Pathway for PI3-Kinase Regulation by VPS15 in Autophagy." Science (New York, N.Y.) 388 (6743): eadl3787.

    Gyurkovska, Valeriya, Rakhilya Murtazina, Sarah F. Zhao, Christopher B. Huppenbauer, Vadim Gaponenko, and Nava Segev. 2026. "Distinct TRAPP Complexes Activate Ypt/Rab GTPases in Secretion and Autophagy." The Journal of Cell Biology 225 (5). https://doi.org/10.1083/jcb.202507166.

    Hemalatha, Anupama, Chaitra Prabhakara, and Satyajit Mayor. 2016. "Endocytosis of Wingless via a Dynamin-Independent Pathway Is Necessary for Signaling in Drosophila Wing Discs." Proceedings of the National Academy of Sciences of the United States of America 113 (45): E6993-E7002.

    Levin, Rebecca S., Nicholas T. Hertz, Alma L. Burlingame, Kevan M. Shokat, and Shaeri Mukherjee. 2016. "Innate Immunity Kinase TAK1 Phosphorylates Rab1 on a Hotspot for Posttranslational Modifications by Host and Pathogen." Proceedings of the National Academy of Sciences of the United States of America 113 (33): E4776-83.

    Nakajima, Yu-Ichiro. 2021. "Analysis of Epithelial Architecture and Planar Spindle Orientation in the Drosophila Wing Disc." Methods in Molecular Biology (Clifton, N.J.) (New York, NY), Methods in molecular biology (Clifton, N.J.), vol. 2346: 51-62.

    Port, Fillip, Marco Kuster, Patrick Herr, et al. 2008. "Wingless Secretion Promotes and Requires Retromer-Dependent Cycling of Wntless." Nature Cell Biology 10 (2): 178-185.

    Riedel, Falko, Antonio Galindo, Nadine Muschalik, and Sean Munro. 2018. "The Two TRAPP Complexes of Metazoans Have Distinct Roles and Act on Different Rab GTPases." The Journal of Cell Biology 217 (2): 601-617.

    Russo, Ashley J., Alyssa J. Mathiowetz, Steven Hong, Matthew D. Welch, and Kenneth G. Campellone. 2016. "Rab1 Recruits WHAMM during Membrane Remodeling but Limits Actin Nucleation." Molecular Biology of the Cell 27 (6): 967-978.

    Scott, Mackenzie K., and John E. Burke. 2026. "Two Binding Sites Are Better than One." eLife 15 (e110917). https://doi.org/10.7554/eLife.110917.

    Sechi, Stefano, Anna Frappaolo, Roberta Fraschini, et al. 2017. "Rab1 Interacts with GOLPH3 and Controls Golgi Structure and Contractile Ring Constriction during Cytokinesis in Drosophila Melanogaster." Open Biology 7 (1): 160257.

    Sharma, Satyam, and Varun Chaudhary. 2024. "Dissociation of Drosophila Evi-Wg Complex Occurs Post Apical Internalization in the Maturing Acidic Endosomes." Traffic (Copenhagen, Denmark) 25 (9): e12955.

    Špokaitė, Saulė, Yohei Ohashi, Maxime Bourguet, Antoine N. Dessus, and Roger L. Williams. 2026. "A Novel RAB5 Binding Site in Human VPS34-CII That Is Likely the Primordial Site in Eukaryotic Evolution." In eLife. ELife, March 31. https://doi.org/10.7554/elife.110040.

    Sun, Shan, and Sen-Fang Sui. 2023. "Structural Insights into Assembly of TRAPPII and Its Activation of Rab11/Ypt32." Current Opinion in Structural Biology 80 (102596): 102596.

    Tisdale, E. J., J. R. Bourne, R. Khosravi-Far, C. J. Der, and W. E. Balch. 1992. "GTP-Binding Mutants of rab1 and rab2 Are Potent Inhibitors of Vesicular Transport from the Endoplasmic Reticulum to the Golgi Complex." The Journal of Cell Biology 119 (4): 749-761.

    Tremel, Shirley, Yohei Ohashi, Dustin R. Morado, et al. 2021. "Structural Basis for VPS34 Kinase Activation by Rab1 and Rab5 on Membranes." Nature Communications 12 (1): 1564.

    Vliet, Alexander R. van, Alison K. Gillingham, Tomos E. Morgan, et al. 2026. "A Rab1 Interactome Illuminates a Dual Role in Autophagy and Membrane Trafficking." The Journal of Cell Biology 225 (3): e202507084.

    Witte, Leonie, Karen Linnemannstöns, Kevin Schmidt, et al. 2020. "The Kinesin Motor Klp98A Mediates Apical to Basal Wg Transport." Development 147 (15). https://doi.org/10.1242/dev.186833.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript uncovers a direct or indirect role of RAB1/TRAPPIII in regulating the intracellular fate of Wng in the columnar epithelial cells of the wing imaginal disc of the fruit fly. This observation is interesting, although it should come as no surprise that, given the fact that Wnts are secreted morphogens and considering the involvement of TRAPPIII in the early stages of the secretory pathway, the key TRAPPIII subunit, TRS85/TRAPPC8, is crucial for their normal trafficking. The main finding of this work is that cells deficient in TRAPPIII/RAB1 accumulate the morphogen and its receptor in the apical region of morphogen-producing cells, which is interpreted as a block in the retrograde trafficking of the morphogen. While it is unclear to me what the authors consider as retrograde trafficking of Wng (see below), the arguments fall short of being convincing, in part because the intracellular trafficking of Wng is rather intricate, but also because the physiological role of TRAPPIII is insufficiently understood. It is well established that Wng transits through endosomes to reach multivesicular bodies, where it is incorporated into the inwardly budding vesicles that are secreted as exosomes (Gross et al, cited). Do the authors consider this a retrograde pathway? The authors do not delineate further the location at which Wng accumulates, for example using co-localization studies. They have also not provided a rationalization of their observations with current knowledge of retrograde trafficking between the endosomes and the Golgi. It would have been interesting to address the effects of Trs85 depletion in mutant backgrounds deficient in the master regulator of retrograde pathways, RAB6, its effector, the GARP complex, or RAB7 and the retromer; it would have been important to study TRAPPIII depletion under conditions in which endocytic internalization is blocked, or the biogenesis of multivesicular bodies is prevented.

    Specific comments

    There are no page or line numbers, which makes very cumbersome to comment on specific sections of the manuscript.

    The introduction contains a factual mistake. TRAPPC11, 12, and 13 are not metazoan specific. They are present in fungi but have been lost in Saccharomyces cerevisiae. Pinar M, Arias-Palomo E, de Los Ríos V, Arst HN Jr, Peñalva MA. Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII. PLoS Genet. 2019 Dec 23;15(12):e1008557. doi: 10.1371/journal.pgen.1008557. This reference should have been cited.

    Materials and methods are very incomplete, particularly in the section that deals with the antibodies, which are essential tools for understanding the experiments. Is the Wnt antibody a monoclonal antibody? Are there different antibodies specific for extracellular and intracellular Wnt? What is the molecular basis for this differential detection? The transgene expressing GFP under the engrailed driver is not described anywhere.

    The labeling of the figures and the figure legends themselves are excessively simple and appear to be accessible for fly experts only.

    The authors have not considered the possibility that ablating TRAPPC8 of TRAPPIII can have off-target effects in TRAPPII. It would have been very interesting to address the phenotype of down-regulating TRAPPII and of down-regulating one of the core subunits of TRAPPs.

    Figure 1, Panel 1i: What is the basis at this point that justifies "likely by altering intra-cellular trafficking"?

    Several figures: where are the boundaries of the cells in orthogonal views? If GFP labels whole cells, why is there an area at the top of the cross-sections that hasn't got GFP staining?

    I can follow the point that accumulation in the apical side means that retrograde trafficking is impaired. I miss the connection between the observation and the conclusion.

    VPS34 is an effector of RAB5 and therefore its down-regulation impairs the maturation of early endosomes because their membranes cannot acquire the key component phosphatidylinositol-3-phosphate, which is recognized by the ESCRT machinery to proceed with multivesicular body biogenesis.

    The Q70L mutation, widely used as a constitutive activator of RABs, is borrowed from studies in RAS and it might not lead to constitutive activation of RAB1 (Langemeyer, L., Nunes Bastos, R., Cai, Y., Itzen, A., Reinisch, K.M., and Barr, F.A. (2014). Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation. eLife 3, e01623).

    Referee cross-commenting

    I also feel that six months is a more realistic estimation

    Significance

    In summary an interesting observation that deserves a more detailed follow-up to unveil the actual role of TRAPPIII in the Wng pathway.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Sharma, Sabnis et al. show in this report that TRAPPC8 and Rab1 are important for Wingless (Wg) secretion in developing Drosophila wing discs.

    • Loss of TRAPPC8 (either knockdown or mutant clones) lead to higher levels of apical Wg in Wg-producing cells (total and extracellular staining).
    • The levels of the intracellular Wg transporter Evi/Wls are also increased, in particular in its 'unbound' form.
    • A similar phenotype is observed upon overexpression of Rab1 dominant negative.
    • Overexpression of constitutively active Rab1 has no effect in otherwise wild type discs but does rescue the effect of TRAPPC8 loss of function.

    Major concerns

    1. The authors key message is that TRAPPC8 is essential for retrograde transport of Wg and Evi. However, in apparent contradiction, TRAPPC8 loss does not appear to affect the basolateral distribution of exWg (or total Evi) (Fig2B', FigS3). Therefore, while Wg transport may be less efficient in the absence of TRAPPC8, the conclusion that TRAPPC8 is essential should be toned down. It is warranted to suggest that transport of unbound Evi is disrupted in the absence of TRAPPC8, as unbound Evi increases apically and is lost basally (Fig4A-B).

    2. The relative roles of Rab1 and TRAPPC8 are not equally considered. As the authors show, unlike TRAPPC8 loss of function, Rab1 DN causes a decrease in basal exWg. Doesn't this suggest that Rab1 is more important for Wg trafficking than TRAPPC8? a. Rab1 DN causes also accumulation of total basal Wg. Does this mean that Wg can be trafficked basally but fails to be secreted at the basal surface? b. A similar assay (total basal Wg) is lacking in condition of TRAPPC8 loss (clones or RNAi); only exWg is shown. c. Also needed is an assessment of total and exWg at the basal surface in the Rab1 CA experiment. Based on the cross-section image shown in Fig6B, it seems that the distribution of Wg might be more confined to the apical plane of the cells compared to other conditions (Fig6F for example), but there is little difference in this regard between the anterior and posterior compartments.

    3. A significant claim of the paper is "TRAPPC8 regulates retrograde Wg trafficking post-apical internalization". a. The increase in apical extracellular Wg (FigS3), which seems to remain associated with the membrane of Wg-producing cells (i.e. no onward spread) would suggest that apical internalization may be affected, but this is not considered by the authors. b. From the antibody chase experiment, it is not clear if the effect of TRAPPC8 loss affects endocytosis and/or trafficking in general or whether it is specific to Wg and Evi. A fluorescent Dextran control should be included as in Witte et al., 2020 (https://doi.org/10.1242/dev.186833 ). Also, can the authors be sure that they are visualising internalised Wg and not just internalised Wg antibody? Can the author show a field of view where Wg is not expressed?

    4. The authors suggest that TRAPPC8 loss leads to increased Rab7 levels. This is taken as evidence for a role of acidification in driving Wg dissociation from Evi, with TRAPPC8 acting downstream to sort Wg from mature endosomes. a. Neither of these claims are supported because in Fig.S3G loss of TRAPPC8 results in an increase in Rab7 everywhere except at the DV boundary where the Wg-producing cells are. This should be acknowledged in text, and these data should appear in the main Fig4. b. The increase in lysotracker also does not support the above claims as it could be due to an increase in unbound Evi that cannot be trafficked back to the ER (and hence targeted for degradation in lysosomes). c. In fig S3G, the purported Rab7 increase in the posterior compartment is not readily apparent (and not quantified), in contrast to the authors' description of the effect of TRAPPC8 depletion. This suggest that the model proposed by the authors needs to be revised (Fig6G) and the relevant paragraphs from Results and Discussion section must be significantly edited or removed.

    Minor concerns

    1. The way data in Fig 2, S3 and S4 is presented and referred to in text could be rearranged slightly to make it easier to follow: first talk about the work using clones (Fig2, FigS4 becomes FigS3), then mention similar results with the knockdown (FigS3 becomes FigS4). This arrangement would link naturally to the effect on Evi.

    2. How do the authors explain that the knockdown of some core TRAPP subunits does not have a phenotype? Some sort of rationalisation (or experimental follow up) is desirable.

    3. The authors should give more detail about how they quantified normalised intensity profiles and clarify if the profiles correspond to just the representative image shown or the average of multiple discs (possible for the compartment experiments, but presumably impossible for the clone experiments as they would need to normalise by the size of the clone too).

    Referee cross-commenting

    Reviewer 3 was thorough and makes some good points that I had not considered because of coming from a different research field (e.g. the fact that losing TRAPPC8 can have off-target effects in TRAPPII, or that the figures may not be clear to non-fly people). In my review I identified the lack of testing of other TRAPP subunits as a minor point, but having read R3's comments I would probably increase this to a major issue. Reviewer 1 and I agree on multiple points as well (e.g. the lack of testing of other TRAPP subunits, the difference between the TRAPPC8 and Rab1 phenotypes). I believe that 1 - 3 months to complete revisions is optimistic. 6 months is a more realistic, with the caveat that the results from some of the requested experiments could upend the conclusions of this study.

    Significance

    This report is a valuable report for the Wg secretion subfield, and useful for the Wnt community. It makes some interesting observations and brings the importance of Rab1 back into the conversation after Ching et al., 2008. However, the insight remain limited and the mechanism/trafficking defects remain unclear. There is convincing evidence that TRAPPC8 and Rab1 affect Wg and Evi, but the claims that TRAPPC8 is essential and acts downstream of acidic endosomes) is inadequate.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In this manuscript, the authors investigate the role of Transport Protein Particle (TRAPP) complexes in the secretion of Wingless (Wg) in Drosophila. Two TRAPP complexes are known to exist: TRAPPII and TRAPPIII. Through the systematic analysis of individual subunits, the authors identify the requirement of the TRAPPIII subunit TRAPPC8 for Wg trafficking. They demonstrate that the absence of TRAPPC8 disrupts the retrograde trafficking of both Wg and its carrier Evi, resulting in their intracellular accumulation within Wg-producing cells. Further analyses suggest that TRAPPC8 controls the post-apical internalisation and endosomal trafficking of Wg and Evi. Consistent with the established function of TRAPPIII as a Rab1-specific GEF, they demonstrate that inhibiting Rab1 produces similar effects as depleting TRAPPC8, while constitutively active Rab1 reverses the trafficking defects. Furthermore, depletion of either TRAPPC8 or Rab1 increases the levels of Wg-unbound Evi, suggesting that they act downstream of Evi-Wg dissociation. Taken together, these findings suggest that TRAPPIII and its effector Rab1 are essential regulators of retrograde Wg trafficking, which is necessary for efficient secretion. Overall, the work is carefully performed and the results are presented clearly. The controls are appropriate and the study expands the functional scope of Rab1-dependent trafficking beyond early secretory pathways. The identification of a previously unrecognised function of TRAPPC8 in Wg trafficking is a valuable contribution.

    Major Points

    It is not entirely clear whether the RNAi lines used in the initial screen were validated for knockdown efficiency. Notably, some core TRAPPIII subunits (e.g., TRAPPC3 and TRAPPC5) do not show a phenotype. This could indicate that the complex retains partial function upon their depletion, or alternatively that the RNAi lines are ineffective. While this point may not critically affect the main conclusion regarding TRAPPC8, it is important for drawing conclusions about the specificity of TRAPPIII versus TRAPPII involvement. For instance, TRAPPC10 (a TRAPPII-specific subunit) was analysed using a single RNAi line, yet no evidence of knockdown efficiency was provided. Validation of these RNAi reagents would strengthen the conclusions regarding complex specificity.

    Minor Points

    • In Figure 2C, the clone appears restricted to the apical region, and I do not clearly observe GFP loss in the basolateral domain. Larger clones or additional sections would help clarify the spatial distribution and strengthen the interpretation.
    • The authors should discuss why TRAPPC8 depletion results primarily in apical Wg enrichment, whereas Rab1 inhibition leads to Wg accumulation at both apical and basolateral membranes. This difference may provide insight into whether Rab1 has additional TRAPPIII-independent functions or whether TRAPPC8 affects a more spatially restricted trafficking step.

    Significance

    This study broadens our understanding of Rab1-dependent membrane trafficking by identifying a previously unrecognized role for the TRAPPIII complex in retrograde transport during Wingless secretion. While the study convincingly establishes the involvement of the TRAPPIII-Rab1 module in Wg trafficking, it does not define the specific retrograde trafficking step that is regulated by this machinery. In addition, the functional relationship between TRAPPIII-Rab1 and established retrograde regulators, such as the retromer complex, is not addressed. Further molecular and mechanistic analyses will be required to position TRAPPIII within the broader retrograde trafficking network and to fully elucidate how Rab1 activity is coordinated with other pathways involved in Wnt secretion. These unresolved issues represent the main limitation of the current study. This work will be of particular interest to researchers in the fields of cell signaling, membrane trafficking, and intercellular communication.

    My expertise lies in cell communication and the regulation of cellular proliferation.

  5. Version published to 10.64898/2026.01.25.701536 on bioRxiv