VAMP8 function reveals tight linkage between endocytic recycling and endocytosis

  1. Ailing Liu
  2. Yueping Li
  3. Zheng Huang
  4. Wen Chen
  5. Peiliu Xu
  6. Xiangying Wei
  7. Guosheng Hu
  8. Shuangquan Liu
  9. Xiaoxia Liu
  10. Yaohui He
  11. Danling Wang
  12. Sandra L Schmid
  13. Zhiming Chen

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Abstract

Clathrin-mediated endocytosis (CME) is a multistage process that involves the initiation and stabilization of clathrin-coated pits (CCPs), which invaginate and finally detach from the plasma membrane to form clathrin-coated vesicles (CCVs). Given that SNARE proteins are essential for downstream vesicle targeting and fusion events, their recruitment into nascent CCVs has been suggested to play a role in regulating CME progression. However, which and how SNARE proteins regulate CME remains to be explored. Here, we showed that siRNA-mediated knockdown of the R-SNARE, VAMP8 impairs CCP initiation, stabilization and invagination and strongly inhibits CME. Mechanistically, recruitment of VAMP8 to CCVs is not required for CME regulation. Instead, VAMP8 regulates CME indirectly by mediating endocytic recycling. Depletion of VAMP8 skews endosomal recycling of CME cargo, exemplified here by transferrin receptor, toward lysosomal degradation, thereby inhibiting CCV formation. Overall, our study provides new insights into the crosstalk between endocytosis and VAMP8-mediated endocytic recycling of CME cargo and demonstrates the significance of cargo recruitment in CME regulation.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Liu et al. provided evidence of the interaction between endocytosis and VAMP8-mediated endocytic recycling of clathrin-mediated endocytosis (CME) cargo through a knockdown approach combined with total internal reflection fluorescence (TIRF) microscopy, western blotting, and functional assays in a mammalian cell line system. They demonstrated that VAMP8 impairs the initial stages of CME, such as the initiation, stabilization, and invagination of clathrin-coated pits (CCPs). VAMP8 indirectly regulates CME by facilitating endocytic recycling. The depletion of VAMP8 alters endosomal recycling, as shown here by the transferrin receptor, towards lysosomal degradation, thereby inhibiting clathrin-coated vesicle (CCV) formation. Overall, I found this study to be highly engaging because of its elucidation of the unexpected role of R-Snare in influencing the levels of cargo proteins within the context of clathrin-mediated endocytosis (CME). This MS will be helpful for researchers in endocytosis and protein trafficking fields. It appears to me that VAMP8 interacts with multiple targets within the endo-lysosomal pathway, collectively influencing the clathrin-mediated endocytosis (CME). Therefore, the contribution of lysosomes in this context should be evaluated. This matter should be addressed experimentally and discussed in the MS before considering publication.

    Major comments:

    1. Figure 4D demonstrates that the knockdown of VAMP8 leads to an increase in lysosome numbers and lysosomal perinuclear clustering, as evidenced by LAMP1 staining (Figure 5A). Additionally, the knockdown of VAMP8 results in the downregulation of most surface receptors, as illustrated in Figure 3A, which typically follows the lysosomal degradation pathway. The observed reduction in TfR cargo could be attributable to the decreased presence of the Tfn Receptor in siVAMP8-treated cells compared to that in control cells. How do the authors explain this phenomenon? Upon reviewing these observations, I suggest that the mechanism outlined in the manuscript-specifically, "Depletion of VAMP8 skews endosomal recycling of CME cargo, exemplified here by transferrin receptor, toward lysosomal degradation, thereby inhibiting CCV formation"-may serve as a secondary rather than a primary cause. This can be ruled out by the following experiments:
      • Assessment of lysosomal biogenesis markers through RT-PCR or Western blotting following VAMP8 knockdown.
      • Assessment of transferrin receptor stability under VAMP8 knockdown conditions using cycloheximide.
      • Previous studies have indicated that perinuclear clustering of lysosomes is correlated with increased degradative activity. Therefore, assessing the lysosomal perinuclear index in the images presented in Figure 5A (LAMP1) effectively determines the presence or absence of this phenomenon.
    2. Given that VAMP8 is implicated in lysosomal fusion events, I hypothesized that VAMP8 undergoes degradation via the lysosomal pathway. However, Figure 4F indicates that there was no restoration of VAMP8 following leupeptin treatment. Could you please provide an explanation for this discrepancy or is it trafficked to proteasomal degradation pathway?
    3. Figure 5A and 5C demonstrate that the restoration of TfnR in siVAMP8 under leupeptin conditions was similar to the levels observed in the sicontrol without leupeptin. However, no enhancement in TfnR uptake (Figure 5F) was detected in cells treated with siVAMP8 under leupeptin treatment conditions. How can these observations be reconciled with each other?

    Minor comments:

    1. The manuscript does not provide details of the western blotting method and quantification criteria.
    2. Fig1A &B) - The siVAMP8 #1 blot indicates a reduction exceeding 90%, whereas the bar graph depicts a reduction of 70-80%. It is advisable to elucidate the quantification criteria in the Methods section to prevent potential confusion. Were the protein levels normalized to the loading control?
    3. Enhancing the readability of the graph could be achieved by labeling the Y-axis as either 'All CCP' or 'Bonafide CCP' of CME analysis graphs.
    4. The legends of panels 1M and N do not correlate with the corresponding figures. Need corrections.
    5. Fig 4D- Is the technique employed for electron immunogold staining utilizing a lysosome-specific antibody? How do the authors substantiate their assertion that the darkly stained structures are lysosomes and not other cellular compartments?
    6. Electron micrographs of siVAMP8 cells revealed the presence of dark-stained bodies near the plasma membrane. The implications of this observation should be explained in the discussion section.
    7. Fig5A- Provide the color code for the merged images.
    8. Fig5G- schematic needs to be improved to demonstrate the contribution of increased lysosomal content.

    Significance

    VAMP8 is an R-SNARE critical for late endosome/lysosome fusion and regulates exocytosis, especially in immune and secretory cells. It pairs with Q-SNAREs to mediate vesicle fusion, and its dysfunction alters immunity, inflammation, and secretory processes. This study revealed that the SNARE protein VAMP8 influences clathrin-mediated endocytosis (CME) by managing the recycling of endocytic cargo rather than being directly recruited to clathrin-coated vesicles. This study advances our understanding of cellular trafficking mechanisms and underscores the essential role of recycling pathways in maintaining membrane dynamics. This is an excellent piece of work, and the experiments were designed meticulously; however, the mechanism is not convincing enough at this point. This MS will surely benefit the general audience, specifically the membrane and protein trafficking and cell biology community.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors investigate the role of the SNARE protein VAMP8 in endocytic recycling and clathrin-mediated endocytosis (CME). Using siRNA knockdown, live-cell imaging, and recycling assays, they report that VAMP8 depletion impairs clathrin-coated pit (CCP) initiation, stabilisation, and invagination, thereby inhibiting CME. Furthermore, they suggest that VAMP8 knockdown promotes transferrin receptor (TfR) degradation and slows its recycling. Consistent with previous studies, knockdown of CALM expression inhibits CME, whereas overexpression of wild-type or L219S/M244K mutant CALM rescues CME.

    Major concerns:

    1. The authors claim their work "reshape our understanding" of CME by proposing that VAMP8 regulates CME through cargo recycling rather than by direct recruitment to clathrin-coated vesicles (CCVs). However, the concept that cargo recycling influences CME efficiency is not new. Prior work has established that cargo clustering stabilises CCPs and that cargo availability strongly impacts pit dynamics. Similarly, studies of CALM, Hrb, and SNAREs have implicated recycling and SNARE interactions in CME. The observation that reduced CME cargo expression (e.g. TfnR) in VAMP8-depleted cells impairs CME is therefore consistent with earlier findings, not a new paradigm. Moreover, the manuscript raises a conceptual paradox: if VAMP8 recruitment is dispensable for CME, why is VAMP8 recruited to CCPs, and why does its depletion produce such a striking phenotype?
    2. The authors note that VAMP8 knockdown reduces TfnR expression, which in turn reduces its surface levels (Figure 1N). Nevertheless, they report that VAMP8 knockdown also diminishes the endocytic efficiency of these TfRs already delivered to the plasma membrane (Figure 1M). Without rescue experiments - for example, re-expression of VAMP8 or TfnR - the specific roles of VAMP8 or cargo availability cannot be confirmed.
    3. The authors argue that overexpression of WT and L219S/M244K mutant CALM rescues CME, supporting the view that abolishing VAMP8 recruitment to CCVs does not impair CME. Yet previous studies have demonstrated that CALM is essential for CME through recruitment of multiple proteins, including the R-SNAREs VAMP8, VAMP3, and VAMP2. Miller et al. have shown a conserved interaction mechanism between CALM and these SNAREs. Thus, the finding that mutant CALM rescues CME does not sufficiently demonstrate that VAMP8 recruitment is unimportant. Furthermore, Sorkin's group showed that high levels of CALM overexpression inhibit transferrin and EGF receptor endocytosis and disrupt clathrin localisation in the trans-Golgi network (PMID: 10436022). In Figure S2, the authors clearly express CALM at levels far exceeding endogenous amounts. Such overexpression may itself perturb membrane trafficking, complicating interpretation of the rescue data.
    4. Most conclusions rely solely on TfR. Without examining additional receptors (e.g. EGFR, LDLR), the general claim regarding "cargo availability" remains unsubstantiated. The authors should quantify surface TfR levels following VAMP8 knockdown and/or leupeptin treatment. It also remains unclear why leupeptin treatment fails to induce TfR accumulation in lysosomes of control siRNA-treated cells.
    5. The manuscript presents several kymographs, but the appearance and disappearance of CCPs are difficult to discern. While this reviewer is not an expert in quantitative imaging analysis, it appears that in both siControl and siVAMP8 cells the tracks are either unusually persistent or very short-lived, with the only obvious differences being the brightness of the spots and tracks. Although some quantitative analyses are provided, the quality and representativeness of the imaging data remain unconvincing.
    6. Terms such as "productive" and "abortive" CCPs are used inconsistently and without clear definition in figure legends. In addition, the manuscript's claims of novelty, both in the Significance Statement and the main text, are overstated relative to prior literature.

    Significance

    General assessment: While the study shows that VAMP8 depletion negatively affects CME and TfR trafficking, the manuscript suffers from limited novelty, logical inconsistencies, and experimental shortcomings.

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    Referee #1

    Evidence, reproducibility and clarity

    Liu and colleagues show that the knockdown of VAMP8 impairs CME by downregulating various cargo receptors, including TfnR, by rerouting theses receptors to lysosomes for degradation instead of recycling them to the plasma membrane. The results also imply that the lack of sufficient receptors (CME-cargo and associated endocytic machinery) in turn impairs the initiation/stabilization of nascent CCPs and their subsequent invagination. As shown by specific mutations in CALM, VAMP8 is apparently not directly required for CME. The emloyed cmeAnalysis DASC assay appears to be state of the art. The data are overall convincing. Nevertheless, the authors should address/clarify the following points:

    Major comments:

    • A rescue experiment of the VAMP8 knockdown using VAMP8 and RFP-VAMP8 should be included to exclude off-target effects and demonstrate the functionality of the RFP-VAMP8 construct.
    • Please confirm that the RFP-VAMP8 expression levels in the CALM(WT) and CALM(SNARE) cells are comparable (compare first panels in Fig. 2A and 2B) and provide information about the RFP-VAMP8 expression levels compared to endogenous VAMP8. Figure 2D shows that RFP-VAMP8 is not enriched in CCPs in CALM(SNARE) cells. This raises the questions whether endocytic vesicles in the CALM(SNARE*) background indeed lack VAMP8 or still contain some residual VAMP8 levels. A complete lack of VAMP8 would imply that VAMP8 does not play a major role in determining the fate (fusion partner) of the endocytic vesicles (in the pathways analyzed by the authors). If possible, provide experimental data to solve this issue or discuss this point.

    Minor comments:

    • Fig. 1 N and M: The figure panels should be switched to fit to the legend, or vice versa.
    • In contrast to Table S1, which show a reduction of TfnR by a factor of 1.8, the Western blot analysis (Fig. 3C) shows a 4-fold reduction. Please explain the divergence.
    • It is surprising that the TfnR knockdown phenocopies the VAMP8 knockdown. Why does the knockdown of a single receptor affect endocytosis, measured by the eGFP-CLCa recruitment? Compared to other plasma membrane receptors, how abundant is TnfR? If available, please provide references demonstrating that the knockdown of other receptors has similar effects on endocytosis?
    • The authors should briefly discuss to which degree the knockdown of VAMP8 may also affect receptor exocytosis, thereby contributing to a reduction of cargo receptors at the plasma membrane and impaired CME.
    • VAMP8 has an established role in autophagosome - lysosome flux, favoring the fusion with the lysosomes. In the present study, VAMP8 knockdown seems to reroute receptors for lysosomal degradation in the absence of VAMP8. Please discuss.
    • For clarity, the authors may consider to restructure their abstract, directly starting with their finding that "Depletion of VAMP8 skews endosomal recycling of CME cargo, exemplified by ..........

    Significance

    Overall, this study provides significant insights into the role of VAMP8 in the recycling of receptors to the plasma membrane. The lack of VAMP8 results in rerouting of plasma membrane receptors to lysosomes and thereby indirectly reduces endocytosis. The results will be of broad interest in the field of membrane trafficking. The reviewers field of expertise is membrane trafficking, in particular molecular mechanisms of exocytosis.

  5. Version published to 10.1101/2025.08.31.673318 on bioRxiv