Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Article activity feed

1. 

   Version published to 10.7554/elife.69832 on eLife

   Oct 29, 2021
2. 

   eLife

   Jul 1, 2021

   Evaluation Summary:

   Although the role of membrane potential in Cell Permeable Peptides (CPP) translocation has been consistently described in artificial systems, this multi scale study, combining cell biology, genetics and in silico approaches, further extends this topic to a live cell context where it shows that internalization stops when the membrane polarization is decreased by the removal of potassium channels. It proposes an original mechanism of CPP translocation based on water pore formation, which should be of interest for biophysicists, cell biologists and for applications such as drug delivery.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the …

   Evaluation Summary:

   Although the role of membrane potential in Cell Permeable Peptides (CPP) translocation has been consistently described in artificial systems, this multi scale study, combining cell biology, genetics and in silico approaches, further extends this topic to a live cell context where it shows that internalization stops when the membrane polarization is decreased by the removal of potassium channels. It proposes an original mechanism of CPP translocation based on water pore formation, which should be of interest for biophysicists, cell biologists and for applications such as drug delivery.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

   Read the original source
3. 

   eLife

   Jul 1, 2021

   Reviewer #1 (Public Review):

   The authors used a wide range of systems and approaches to demonstrate the importance of the transmembrane potential for the internalization of Cell permeable peptides (CPPs). At a stronger (more negative) transmembrane potential, the three studied peptides (R9, TAT, and Penetratin) internalized easier, while their internalization stopped when the membrane polarization was decreased by the removal of potassium channels. The results are further supported by computer simulations and in vivo experiments providing consistent new insight into uptake of selected CPPs.

   Read the original source
4. 

   eLife

   Jul 1, 2021

   Reviewer #2 (Public Review):

   While the role of membrane potential in CPP translocation have been consistently described in artificial systems, this multi scale study, combining cell biology, genetic and in silico approaches, further extends this topic to a live cell context and proposes an original mechanism of CPP translocation based on water pore formation.

   Contrasting with numerous studies focusing on the role of carbohydrates and lipids in this process, here the authors address the role of proteins, using a CRISPR screen. Depending on the cell context, distinct potassium channels were shown to be required for translocation and based on genetic and pharmacological approaches, the authors propose that their action mostly rely on their ability to modulate membrane potential. Hyperpolarisation enhances CPP translocation while …

   Reviewer #2 (Public Review):

   While the role of membrane potential in CPP translocation have been consistently described in artificial systems, this multi scale study, combining cell biology, genetic and in silico approaches, further extends this topic to a live cell context and proposes an original mechanism of CPP translocation based on water pore formation.

   Contrasting with numerous studies focusing on the role of carbohydrates and lipids in this process, here the authors address the role of proteins, using a CRISPR screen. Depending on the cell context, distinct potassium channels were shown to be required for translocation and based on genetic and pharmacological approaches, the authors propose that their action mostly rely on their ability to modulate membrane potential. Hyperpolarisation enhances CPP translocation while depolarization has opposite effects. MD simulation revealed that hyperpolarization favors the transient formation of water pores in the membrane, induced by a massive and local hyperpolarization (megapolarisation) due to CPP accumulation at the membrane interface. Co-translocation of small soluble molecules with CPP observed in live cells is in agreement with the formation of pore proposed in silico model.

   Quantification of CPP uptake that takes into account the intracellular localization of the CPP (cytosolic versus vesicular) is a critical issue in cellular models. In this study, quantification relies either or the toxicity induced by the cytosolic accumulation of the Tat-RasGAP peptide or by the direct visualization of fluorescently-labelled CPPs. In the latter case, the choice made by the authors to define the 3 categories used for quantification has to be justified. More precisely, merging the low and strong cytosolic signal categories into a single one is questionable as these two categories might correspond to distinct functional fates due to the highly variable extent of the cytosolic staining (much more than between vesicular and low cytosolic). Indeed, the water pore mechanism invoked by the authors might better fit with an all or none mechanism for cytosolic delivery that would correspond to low and high cytosolic content respectively. How each of the two categories (low and high cytosolic categories) are affected by membrane potential might be highly relevant. In order to link functional (toxicity) and live imaging analysis assays, one possibility would be to analyze the remaining live cells following long incubation (16-24h) with FITC-TAT RasGAP to estimate which staining categories are actually killed (ideally with or without hyperpolarization).

   A second striking observation is the huge heterogeneity of the translocation efficiency within a same cell culture, which unless I missed some points, is not explained. Measuring the membrane potential of each cell (DiBAC4(3) labelling or one of the numerous genetically encoded membrane potential sensor) together with CPP translocation (TMR labelled if using DiBAC), would allow evaluating if the heterogeneity of CPP translocation within a same culture correlates with membrane potential variations.

   Although distinct types of potassium channels have been characterized in the screen, the authors only consider their action on the membrane potential, supported by the effects of drugs that specifically act on membrane potential. However, translocation efficiency does not strictly correlate with membrane potential (e. g. KCNJ2 expression in WT and KCNN4 KO Hela figure 3B). It would be interesting to evaluate if KCNQ5 expression would rescue or even increase internalization (additive effect) in SW6.4 and HeLa cells (WT and KCNN4 KO) and vice versa (KCNN4 expression in Raji). This would also avoid any potential interference of the CRISPR system on ectopic expression. Indeed, the kinetic of CPP uptake significantly differs between cell lines (Figure 1B, almost 100% negative cells for Raji at 20 minutes and only 20% for the 2 others), suggesting partially distinct mechanisms.

   This study brings new data not only in the CPP field but also in our vision of membrane permeability. The authors proposed an original mechanism of CPP translocation, based on water pore formation. To be fully conclusive, it would require the actual detection of megapolarisation events through electrophysiological recordings to corroborate the in silico model. Whether this mechanism only accounts for very high CPP concentrations or could also apply to lower concentrations often used in functional CPP-delivery strategies remains an open question.

   Read the original source
5. 

   Review Commons

   Mar 18, 2021

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   We thank the reviewers for their insightful comments and suggestions. Addressing them will improve our work. Please find below our point-by-points answers to the issues raised. We also provide a partially revised version of the manuscript with changes indicated in blue.

   ---

   Reviewer #1 (Evidence, reproducibility and clarity (Required

   **Summary**

   The authors propose a mechanism through which voltage dependent water pore formation is key to the internalization of Cell permeable peptides (CPPs). The claim is based on an in-silico study and on several experimental approaches. The authors compare 5 peptides (R9, TAT-48-57, Penetratin, MAP and Transportan and use …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   We thank the reviewers for their insightful comments and suggestions. Addressing them will improve our work. Please find below our point-by-points answers to the issues raised. We also provide a partially revised version of the manuscript with changes indicated in blue.

   ---

   Reviewer #1 (Evidence, reproducibility and clarity (Required

   **Summary**

   The authors propose a mechanism through which voltage dependent water pore formation is key to the internalization of Cell permeable peptides (CPPs). The claim is based on an in-silico study and on several experimental approaches. The authors compare 5 peptides (R9, TAT-48-57, Penetratin, MAP and Transportan and use 3 distinct cell lines (Raji, SKW6.4 and HeLa cells), plus neurons in primary cultures. The also present in vivo experiment (mouse skin and zebrafish embryo). All in all, it is an interesting study, but it raises several issues that need to be addressed. Moreover, the length and structure of the manuscript make it very difficult to read (see below under "Reviewer statement")

   **Reviewer statement**

   The instructions are to use the "Major comments" section to answer 6 precise questions. Unfortunately, this is not possible due to the structure of the document to review. The main manuscript (22 pages) comes with 4 primary figures and 19 supplemental ones. Most of these figures have an enormous number of panels and their legends occupy 17 pages. To this, are added 6 supplemental tables and 7 supplemental movies (with 2 pages of legends), 28 pages of Material and Methods, and 146 References (109 for the main manuscript and 37 for Supplemental information). To be frank, I was often tempted to send the manuscript back, asking for the authors to submit a document facilitating the task of the reviewers.

   Because of this complexity, my "Major comments" will come after a page by page, paragraph ({section sign}s) by paragraph and figure by figure "Detailed analysis" of the manuscript.

   **Detailed analysis**

   Q1. Page 4 {section sign} 3

   The test is based on the ability of TAT-RasGAP to kill the cells. Although controls exist, this is worrying since necrotic death might participate in the rupture of the membrane and artificially amplify internalization after a first physiological entry of the peptide. It is also a bit dangerous to add a FITC group to a short peptide without controlling that it has no effect on the interaction with the membrane (FITC-induced local hydrophobicity can provoke peptide tilting and membrane shearing). In the same vein, the very high peptide concentrations often used in the study (40µM for Raji and SKW6.4 cells and 80µM on HeLa cells) can be highly toxic.

   A1. We took advantage of the fact that TAT-RasGAP317-326 can kill cells to design a CRISPR/Cas9 screen based on cell survival for the identification of genes encoding proteins involved in CPP uptake. For this purpose, it was important therefore that the peptide was able to kill wild-type cells. Even if we consider the possibility that “necrotic death might participate in the rupture of the membrane and artificially amplify internalization after a first physiological entry of the peptide”, it remains that the cells that survived the screen did so because they were carrying mutations in genes that encoded potassium channels required for CPP uptake. And since the cells that survived the screen, by definition, were not dying, the issue raised by the reviewer is void in this case. The reviewer mentions that we included controls to validate the observations made with FITC-TAT-RasGAP317-326. Indeed, these controls were performed to address the potential problem raised by the reviewer. These controls, listed below, demonstrate that the genes identified through the CRISPR/Cas9 screen were also involved in the uptake of CPPs devoid of killing properties as well as CPPs that were not labelled with fluorophores.

   i) Three different cell lines, lacking specific potassium channels identified through the CRISPR/Cas9 screen, were unable to allow a non-labelled, non-toxic CPP (TAT-PNA) to enter cells (Supplementary Fig. 8a).

   ii) The Cre recombinase hooked to TAT, a construct that is not labelled with a fluorochrome and that is not toxic, did not enter Raji cells lacking the KCNQ5 potassium channels, also identified through a CRISPR/Cas9 screen (Supplementary Fig. 8b).

   iii) The internalization of a TAT-conjugated FITC-labelled cell-protective therapeutic compound was inhibited, sometimes fully, in three different cell lines, lacking specific potassium channels identified through the CRISPR/Cas9 screen (Supplementary Fig. 8c).

   Additionally, we are now reporting that the entry of FITC-labelled TAT, R9, and penetratin, all non-toxic CPPs, is impaired in Raji cells lacking the KCNQ5 potassium channel identified in the CRISPR/Cas9 screen. These new results will be incorporated in the revised version of our manuscript.

   As supportive evidence that a potential toxicity effect of TAT-RasGAP317-326 is not a confounding factor in experiments recording the initial uptake of the peptide is that internalization is measured after one hour of incubation with the cells (Figure 1), time at which the peptide only minimally impacts the survival of cells (PNAS December 15, 2020 117 31871-31881).

   Finally, please note that depolarizing cells, which is what happens in cells lacking the potassium channels identified through the CRISPR/Cas9 screen, not only blocked the uptake of TAT-RasGAP317-326, but also the uptake of a series of non-toxic CPPs (using short-time incubation protocols; Figure 2).

   Page 5 {section sign} 1

   Q2. Supp. Fig.1a shows no differences between the 3 cell types, even though they differ in their modes of peptide internalization, some favoring vesicular staining and others cytoplasmic diffusion.

   A2. The images shown in panel A of this figure depicts, for each cell line, examples of cells that do not take up the CPP, those that only display vesicular staining, and those that additionally take up the peptides in their cytosol. These images were picked to depict these uptake phenotypes and this is why they are similar in the three cells lines. Panel A does not provide any quantitative information on the prevalence of these different uptake modes in the three cell lines. This is shown in panel B of Supplementary Fig. 1. There is, therefore, no discrepancies between the two panels.

   Q3. Multiplying cell and peptide types contributes to the complexity of the manuscript without increasing its interest. If there is a conceptual breakthrough, as might be the case, it is obscured by the accumulation of useless images and data. A step into simplifying the manuscript would be (i), to concentrate on Raji cells (leaving out SKW6.4 and HeLa cells) and (ii) to only discuss the R9, TAT (including TAT-RasGAP) and Penetratin peptides.

   A3. We are sorry that the inclusion of several cell lines and several CPPs was seen as confusing by the reviewer. Our current vision is that our observations are strengthened if we show that the observed effects are seen in several cell lines and with a variety of CPPs. We would like therefore to not exclude supportive evidence presented in our work because if we do remove some of the data shown in the manuscript, we will definitely weaken some of our claims. We nevertheless remain open with this point that can be further discussed with the editors.

   Q4. TAT and R9 are poly-R peptides, which is not the case for Penetratin that has only 3 Rs. These 3 Rs are important (cannot be replaced by 3 Ks), but the two Ws absent in R9 and TAT are equally important as they cannot be replaced by Fs. This must be considered by the authors when they tend to generalize their model.

   A4. The point raised by the reviewer concerning the importance of W and R residues in CPPs is well taken. We have now developed this in the discussion with the addition of the paragraphs shown below.

   “An additional potential explanation to the internalization differences observed between arginine- and lysine-rich peptides is that even though both arginine and lysine are basic amino acids, they differ in their ability to form hydrogen bonds, the guanidinium group of arginine being able to form two hydrogen bonds1* while the lysyl group of lysine can only form one. Compared to lysine, arginine would therefore form more stable electrostatic interactions with the plasma membrane.*

   Cationic residues are not the only determinant in CPP direct translocation. The presence of tryptophan residues also plays important roles in the ability of CPPs to cross cellular membranes. This can be inferred from the observation that Penetratin, despite only bearing 3 arginine residues penetrates cells with similar or even greater propensities compared to R9 or TAT that contain 9 and 8 arginine residues, respectively (Supplementary Fig. 9g). The aromatic characteristics of tryptophan is not sufficient to explain how it favors direct translocation as replacing tryptophan residue with the aromatic amino acid phenylalanine decreases the translocation potency of the RW9 (RRWWRRWRR) CPP2. Rather, differences in the direct translocation promoting activities of tryptophan and phenylalanine residues may come from the higher lipid bilayer insertion capability of tryptophan compared to phenylalanine3-5. There is a certain degree of interchangeability between arginine and tryptophan residues as demonstrated by the fact that replacing up to 4 arginine residues with tryptophan amino acids in the R9 CPP preserves its ability to enter cells6. It appears that loss of positive charges that contribute to water pore formation can be compensated by acquisition of strengthened lipid interactions when arginine residues are replaced with tryptophan residues. This can explain why a limited number of arginine/tryptophan substitutions does not compromise CPP translocation through membranes.”

   Q5. Supp. Fig1c-d is not necessary (very little information in it) and Supp. Fig 1e is misleading as it takes a lot of imagination to see a difference between homogenous (top) and focal (bottom) diffusion.

   A5. Since we perform cytosolic quantitation to infer direct translocation, it appears important to us, for allowing others to potentially replicate our results, that we precisely report how methodologically we perform our experiments. For Supplementary Fig. 1e, we agree that the examples shown are not easily interpretable. We have now removed this panel, as well as the accompanying panel f, from the Supplementary Fig. 1.

   Q6. Supp. Fig.1g: How many cells are we looking at? Given the high variance, the result cannot be interpreted easily. A distribution according to fluorescence bits would be a better way to present the data.

   A6. Over 230 cells have been quantitated per condition, which includes all cells where CPP entry has occurred regardless of the intensity or the type of entry. We did not only focus on cells with strong cytosolic staining to avoid any bias with regards to detection limitations. High variance can also be explained by the fact that CPP cellular entry is not synchronized. We tested the way of showing the data as suggested by the reviewer but this did not improve the visualization of the results in our opinion. We will therefore keep the initial presentation. Note that regardless of the way the data are presented, the conclusion remains the same, namely that illumination in our hands is not the cause of CPP membrane translocation.

   Q7. Supp. Fig2i. This panel confirms that Raji cells differ from the two other cell types by showing clear temperature dependency. The explanation will come later with the energy barrier for low Vm-induced pore formation. This contradicts earlier reports showing that Penetratin translocation is not temperature-dependent, possibly because it was done on neurons naturally hyperpolarized. Or else because mechanisms are, at least in part, different from the one proposed here for R9 and TAT. This requires some clarification and supports the suggestion that, instead of multiplying models and peptides, it would be more efficient to compare TAT, R9 and Penetratin internalization by Raji cells and primary neurons.

   A7. Supplementary Fig. 1i (not Supplementary Fig. 2i as indicated by the reviewer) was reporting the overall CPP uptake, both through direct translocation and endocytosis as a function of temperature. As there is limited endocytosis in Raji cells, the data shown for this cell type mostly correspond to direct translocation. For Hela and SKW6.4, endocytosis is not marginal however and we will perform a new set of experiments to define the role of temperature (4, 20, 24, 28, 32°C) in CPP direct translocation (i.e. cytosolic acquisition) in HeLa cells and SKW6.4 (using the CPPs listed by the reviewer). We have partially performed this for HeLa cells already and this shows that direct translocation is indeed inhibited by low temperatures (more than 10-fold at 4°C compared to 37°C). Bear in mind that no endosomal escape occurs in our settings (see Supplementary Fig. 7c). This indicates that the decrease in cytoplasmic fluorescence induced by low temperature is not a consequence of diminished CPP endocytosis.

   Q8. Supp. Fig. 2a-f. Last sentence of the legend "Concentrations above 40µM led to too extensive cell death preventing analysis of peptide internalization". This confirms the warning against the use of concentrations varying between 40 µM and 80 µM and partially jeopardizes the validity of some experiments.

   A8. The reviewer has truncated this sentence that actually reads “Note: concentrations above 40 mM of TAT-RasGAP317-326 led to too extensive Raji and SKW6.4 cell death, preventing analysis of peptide internalization at these concentrations.” As different cell lines display various sensitivities to potential toxic effects induced by CPPs (Raji and SKW6.4 cells being more sensitive than HeLa cells for example), we have adapted the concentrations of CPPs used to monitor cellular uptake so that cell death was minimal or non-existent in order to prevent the potential confounding effects mentioned by the reviewer. Hence in contrast to what the reviewer is stating, we are taking care of the toxicity effect and perform our experiments in conditions were toxicity is minimal. The logic of the reviewer to state that we “jeopardize[d] the validity of some experiments” is therefore unclear to us as we did take care of not exposing our cells to toxic CPP concentrations.

   Page 6 {section sign} 2

   Q9. The authors advocate 2 modes of entry, opposing transport across the membrane and endocytosis. In contrast with R9, TAT and Penetratin, Transportan or MAP seem to be purely endocytosed but, if they reach the cytoplasm, they still have to cross a membrane (unless "a miracle happens"). For Penetratin and R9/TAT, the authors consider that water pore and inverted micelle formation are incompatible. This is a bit rapid as inverted micelles might induce water pores through W/lipids interactions requiring less R residues and, possibly, less energy. This provides the opportunity to signal that, in spite of their very high number, key references are missing or hidden in cited reviews, some of them written by colleagues who are not among the main contributors to the CPP field.

   A9. Transportan in our hands indeed appear to enter cells via endocytosis mostly. As reported by the reviewer, how Transportan reaches the cytosol remains unresolved.

   Our data support a model where CPPs enter cells via water pores that are not made by the CPPs themselves but that are created by the megapolarization state of the membrane. Our data therefore do not support toroidal or barrel-stave pore models because these pores would be built as a result of CPP assemblage.

   Inverted micelles have been hypothesized to mediate CPP translocation across membranes7 but to our knowledge, there is no in silico or cellular experimental evidence for this in the literature. To us, the data on which the involvement of inverted micelle in CPP translocation is based are also fully consistent with the water pore model. CPP translocation through water pores has been seen by several authors during in silico experiments but, to the best of our knowledge, simulations have not reported the formation of inverted micelles during CPP translocation across membranes.

   Finally, we would be grateful to this reviewer if the “key references” that are apparently missing from our manuscript are disclosed so that we could acknowledge them appropriately.

   Page 7 {section sign} 1

   Q10.Fig. 1b confirms that Raji cells provide a good model for loss and gain of function (lovely rescue experiment) and that the authors should drop the two other cell types that provide no decisive information.

   A10. Raji and HeLa cells display a stronger direct CPP uptake impairment phenotype when lacking a given potassium channel (KCNQ5 and KCNN4, respectively). In these cell lines, it appears that one potassium channel predominantly controls the plasma membrane potential. In contrast, in SKW6.4 cells, several potassium channels (e.g. KCNN4 and KCNK5) appear to be equally or redundantly involved in the control of the membrane potential. This probably explains the intermediate impact on the Vm and on CPP direct translocation when knocking out a given potassium channel in this cell line. When pharmacologically inducing cellular depolarization, a clear impairment in CPP translocation is however observed in this cell line. Thus, even though the Vm in SKW6.4 cells, is controlled predominantly by several potassium channels, it remains that an appropriate membrane potential is crucially required for these cells to take up CPP across their membrane. We agree with the reviewer that the stronger phenotypic effect observed in Raji and HeLa cells allows easy interpretation. On the other hand, it seems important to us that we provide data reporting intermediate situations so that readers can appreciate the variability that can be observed in different cell lines. Nevertheless, we would like to propose along the reviewer’s suggestion to move the SKW6.4 data from figure 1 to the supplemental data. Feedback from the editors would also be appreciated in this particular instance.

   Page 8 {section sign} 1

   Q11. A) Supp. Fig. 6b (no serum conditions) allows for the use of "normal" CPP concentrations and suggests that a fraction of the peptides may bind to serum components. No arrows in Supp. Fig.6b (but in 6c), and the R/pyrene butyrate interaction is not in 6c but in 6a. Still for Supp. Fig. 6c, the death of cells at 20µM (or less) even in the absence of K+ channels, confirms that we are borderline in term of peptide toxicity.

   B)There is a confusion between Supp. Fig. 6d and 6e and a legend problem (6e is not described). Cell death is assessed in % of PI-positive cells. Does this securely distinguish between death and holes allowing for PI entry without death?

   C) The CPP is incubated in the presence of Pyrene butyrate, making the KO cells less resistant. How does that demonstrate that the potassium channels are not involved in the killing if the peptide is already in? Unless the KO is done after internalization (but the cells should be already dead or dying?). This lacks clarity.

   A11. We apologize for the lack of clarity in the legend of Supplementary Fig. 6. This will be corrected in the revised version of the manuscript.

   A) Supp. Fig. 6b (no serum conditions) allows for the use of "normal" CPP concentrations and suggests that a fraction of the peptides may bind to serum components.

   A) The reviewer is correct that CPPs interact with serum components. This is indeed what is reported in this figure. The presence or absence of serum has therefore an important impact in experiments performed with CPPs and should be reported to allow proper interpretation of our data.

   No arrows in Supp. Fig.6b (but in 6c), and the R/pyrene butyrate interaction is not in 6c but in 6a.

   Thank you for noting this. This is now corrected.

   Still for Supp. Fig. 6c, the death of cells at 20µM (or less) even in the absence of K+ channels, confirms that we are borderline in term of peptide toxicity.

   It has to be understood that in Supplementary Fig. 6c, we use the TAT‑RasGAP317‑326 peptide that is inducing cell death when translocating into cells8. This cell death response is not provided by the CPP portion of TAT‑RasGAP317‑326 (i.e. TAT) but by its bioactive cargo (i.e. RasGAP317‑326). The read-out in this particular experiment is therefore cell death and this should not be confused with general CPP toxicity.

   B) There is a confusion between Supp. Fig. 6d and 6e and a legend problem (6e is not described).

   B) This has now been fixed.

   Cell death is assessed in % of PI-positive cells. Does this securely distinguish between death and holes allowing for PI entry without death?

   The answer to this question is yes. In this manuscript we used PI in two very different experimental set-ups.

   i) the conventional cell death detection assay where cells are incubated with 8 mg/ml PI prior to flow cytometry. In this set-up, dead cells with compromised membrane integrity have their nucleus brightly stained with PI.

   ii) the detection of small pores in the plasma membrane (water pore) where cells are incubated with ~30 mg/ml PI and the fluorescence of PI measured in the cytosol by confocal microscopy. In this set-up, PI enters into the cytosol through small plasma membrane pores but PI does not stain the DNA in the nucleus. This protocol has been previously described9 and we have further validated it in the present work (Figure 3 and Supplementary Fig. 12).

   PI does not fluoresce well unless it binds to DNA. In solution without cells, PI cannot be detected below 128 mg/ml (Supplementary Fig. 12e). At low PI concentrations (8 mg/ml), living cells (even when treated with compounds such as CPPs that create transitory pores) do not display cytosolic PI fluorescence. At high PI concentrations (32 mg/ml), the cytosol of CPP-treated cells becomes PI fluorescent. PI is positively charged and is attracted by the negative membrane potential of the cells. Its movement across the cell membrane is therefore unidirectional. This enables the PI molecules to accumulate/concentrate within the cytosol to values (> 64 mg/ml) allowing its detection (Supplementary Fig. 12a-c). PI and CPPs do no interact (Supplementary Figure 12d); hence they move independently from one another. If PI enters through the water pores induced by CPPs, the entry kinetics of PI and CPPs should be identical. Indeed, this is what we show now in a new figure (refer to our answer #31).

   C) The CPP is incubated in the presence of Pyrene butyrate, making the KO cells less resistant. How does that demonstrate that the potassium channels are not involved in the killing if the peptide is already in? Unless the KO is done after internalization (but the cells should be already dead or dying?). This lacks clarity.

   C) For the pyrene butyrate experiments the rationale was the following. The CRISPR/Cas9-identified potassium channels could either be involved in CPP internalization or they could be required for the killing activity of TAT-RasGAP317-326 when the peptide is already in the cytosol. To experimentally introduce TAT-RasGAP317-326 in the cytosol and to bypass any potential entry depending on potassium channels, we used pyrene butyrate that efficiently creates an artificial entry route for CPPs into cells. Our data show that when TAT-RasGAP317-326 is introduced in the cytosol through the use of pyrene butyrate, cells died whether they lack specific potassium channels or not. This led to our interpretation that potassium channels are not modulating the cell death activity of TAT-RasGAP317-326 once in the cytosol but that they are required for the entry of the CPP in the cytosol.

   Page 9 {section sign} 1

   Q12.The conclusion that the diffuse staining does not come from endosomal escape is based on the certainty that LLOME disrupts both endosomes and lysosomes. First, it should be verified with specific markers (rab5, rab7) that the fluorescent vesicles are endosomes. Second, the literature strongly suggests that LLOME primarily disrupts lysosomes and not endosomes. Finally, even if some endosomes are disrupted, the endosomal population is heterogenous and some CPPs may be in a subpopulation insensitive to LLOME. In addition, the importance of this issue is not well explained. In practice, access to the cytoplasm and nucleus requires crossing the plasma and/or the endosomal membrane and the latter, at least in early endosomes (thus the need of identifying the CPP-enriched vesicles), might not be very different from the plasma membrane.

   A12. The conclusion that diffuse staining does not come from endosomal escape is based on experiments where HeLa cells were incubated in the presence of CPP for 30 minutes to allow CPP entry into cells, then the cells were washed to prevent further uptake (Supplementary Fig. 7c). __We only monitored the cells that initially took up the CPP by endocytosis and not through direct translocation __(for the HeLa cell line, there is always a substantial fraction of such cells; see Supplementary Fig. 1b). We measured the cytosolic CPP fluorescence intensity in these cells by time-lapse confocal microscopy for 4 ½ hours. The procedure to do this is now explained in new Supplementary Fig. 7c. We then assessed the CPP fluorescence intensity within the cytosol. No increase in cytosolic fluorescence was detected in this condition, speaking against the possibility that cytosolic acquisition of CPPs by the cells resulted from vesicular escape (the identity of the vesicles being unimportant in this context). Our set-up has the potential to detect CPPs in the cytosol if these CPPs leak out from vesicles because we could measure increased CPP fluorescence in the cytosol in cells treated with LLOME. It did not matter in this positive control experiment what types of CPP-containing vesicles are disrupted by LLOME. What was important to show in this control condition was that the disruption of at least some CPP-containing vesicles permitted us to detect a cytosolic signal.

   Page 9 {section sign} 2

   Q13. Is Supp. Fig. 7e really necessary? First, as mentioned several times, if 20 µM is a borderline concentration in term of toxicity, raising the concentration up to 100 µM is problematic. Secondly, what matters is not "binding" in general, but binding to the proper membrane components. As mentioned by the authors themselves (Supp. Fig. 1e and movie), there are privileged sites of entry that may correspond to the recognition of specific molecular entities/structures.

   A13. The goal of the experiments presented in Supplementary Figure 7e was to determine whether the CRISPR/Cas9-identified potassium channels modulate CPP/membrane interaction. If those channels were to be required for the initial binding of the CPPs to the plasma membrane, this would have not hampered cells to take up the CPPs. Our data showed (Figure 7e) that Raji cells lacking the KCNQ5 potassium channel had a slightly decreased ability to bind TAT-RasGAP317-326 but importantly, these cells, at similar or even higher initial surface binding compared to wild-type cells (this was achieved by adequately varying the CPP concentrations), were still drastically impaired in taking up the peptide. Note that after one hour of incubation with TAT-RasGAP317-326 in the presence of serum there is only marginal amount of cell death (317-326, we have now performed an additional experiment with TAT that is not toxic to cells that confirms our data obtained with TAT-RasGAP317-326.

   Page 9 {section sign} 3 and Page 10 {section sign} 1

   Q14.The authors should have used a construct that does not kill the cells much earlier, just after the screening experiments based on resistance to necrosis induced by TAT-rasGAP. For Supp. Fig 8a and b: I am fully convinced by Raji cells and HeLa cells but not by the SKW6.4 cells.

   A14. As mentioned in our answer to point 10, we agree that SKW6.4 cells present intermediate phenotypes probably because, unlike Raji and HeLa cells, a combination of ion channels seems to regulate the plasma membrane potential. As indicated above, we can move the SKW6.4 data to the supplementary information to clarify the message presented in the main text. Again, feedback from the editors is welcome here.

   Page 10 {section sign} 2

   Q15. A) Supp. Fig 9 is quite convincing but adds the information that 2 µM are sufficient in neurons. This again makes the 20 to 80 µM concentrations used on transformed cells unsatisfactory.

   B) If one needs a cell line (more user friendly than primary cultures), there are several neural ones that can be differentiated (SHY, LHUMES, etc.) that may have an appropriate membrane potential (below -90mV). Indeed, it would then be important to verify if pore formation is still induced by TAT, R9 and Penetratin (separately) on "naturally" hyperpolarized cells.

   C) Figure 2a confirms that changes in Vm are not solid for HeLa and SKW6.4 cells. This casts a doubt on the validity of the results obtained with the latter 2 cell lines.

   A15. A) The experiments performed in Supplementary Fig. 9d with cortical rat neurons and HeLa cells were performed in the absence of serum accounting for the low concentrations used. We apologize for not emphasizing enough when experiments were performed in the presence or absence of serum, explaining the use of high CPP concentrations (40-80 mM) and low CPP concentrations (2-10 mM), respectively. We would like to emphasize however that we have adjusted the concentrations of CPPs in our study so as to get similar levels of CPP activity or CPP uptake between the different cell lines used. The concentrations used should not be compared as mere numbers, it is the CPP activity or uptake that should be considered.

   B) We thank the reviewer for his/her suggestion. To address this point, we will perform a new experiment to determine if in neurons TAT, R9, and Penetratin induce pores (using the PI uptake approach).

   C) Please see our answer to point 10.

   Page 11 {section sign} 2

   Q16. Why valinomycin was only tried on Raji cells?

   A16. In this study, valinomycin was used on Raji and HeLa cells (Figure 2 and 3). We did not use valinomycin on SKW6.4 cells, as the drug-induced hyperpolarization levels were insufficient in this cell line. As we got a nice hyperpolarization in HeLa wild-type and KCNN4 KO cells through ectopic expression of the KCNJ2 potassium channels (which restored the ability of the KO cells to take up the CPPs), we did not perform the CPP uptake experiment with valinomycin in HeLa cells (although we had tested that valinomycin is able to hyperpolarize HeLa cells).

   Page 12 {section sign} 2

   Q17.A)Looking at Fig. 2c, it seems that low Vm increases the uptake of all CPPs, except Transportan. Is there any reason why this Figure does not provide the number of vesicles per cell in the hyperpolarized conditions?

   B) In fact, if one goes to Supp. Fig. 9c, it appears that, among all peptides, only Penetratin is almost entirely cytoplasmic after 90' of incubation, whereas MAP and Transportan remain essentially vesicular. TAT and R9 are at mid-distance between these two extremes. This leads to send again the warning that all CPPs cannot be placed in a single category. The table that describes the sequences strongly suggests that, TAT and R9 uptake is due to the numerous Rs that cannot be replaced by Ks. In the case of Penetratin, that only has 3 Rs, the situation is thus different with the presence of 2 Ws previously shown to be mandatory for internalization, although absent in TAT ad R9.

   C) In Supp. Fig9, panel g is useless.

   D) A difference between peptides is also visible in Figure 2d where depolarization with KCl does not show the same efficiency on all peptides. The issue is whether these differences are significant and, if so, why? This discussion could be restricted to TAT, R9 and Penetratin.

   E) Supp. Fig. 10a also suggests that all peptides do not respond similarly to depolarization and that the effects differ between cell types and concentrations used. However, given the high concentrations used and the high variance between replicates, this figure might not be a priority in the reorganization of the manuscript.

   A17. A) As mentioned in the figure legend “Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal.” This would create a bias towards underestimation of vesicles numbers in cells displaying strong cytosolic signal.

   B) We agree with the reviewer that Transportan enters cells primarily through endocytosis. This is mentioned in the text as well as other differences that were observed with regards to the prevalence of endocytosis or direct translocation. These mentions are reported below.

   Page 12: “With the notable exception of Transportan, depolarization led to decreased cytosolic fluorescence of all CPPs, while hyperpolarization favored CPP translocation in the cytosol (Fig. 2c, Supplementary Fig. 9h and 10a). Transportan, unlike the other tested CPPs, enters cells predominantly through endocytosis (Supplementary Fig. 9e), which could explain the difference in response to Vm modulation.”

   Page 14: “Even though this extrapolation is likely to lack accuracy because of the well-known limitation of the MARTINI forcefield in describing the absolute kinetics of the molecular events, the values obtained are consistent with the kinetics of CPP direct translocation observed in living cells (Figure 1c and Supplementary Fig. 1b and 9e). With the exception of Transportan, the estimated CPP translocation occurred within minutes. This is consistent with our observation that Transportan enters cells predominantly through endocytosis and its internalization is therefore not affected by changes in Vm (Fig 2c-d and Supplemental Fig. 9e)”.

   Page 20: “On the other hand, when endocytosis is the predominant type of entry, CPP cytosolic uptake will be less affected by both hyperpolarization and depolarization, which is what is observed for Transportan internalization in HeLa cells (Fig. 2c and Supplementary Fig. 10a).”

   Concerning the roles of arginine and tryptophan residues, please refer to our answer #4.

   C) We do not think this panel (now panel h) is useless as it shows representative examples of the quantitation shown in Figure 2c. We can however remove it if requested by the editors.

   D) The reviewer is correct with the observation that KCl-induced depolarization does not lead to similar inhibition in uptake of the tested CPPs. As mentioned in the text, these differences can be explained by the prevalence of direct translocation in the cells. For example, transportan enters cells primarily through endocytosis, which as we show is not regulated/affected by the membrane potential (Figure 2c, lower graphs). Consequently, it is expected that KCl treatment will not impact on transportan cellular uptake.

   E) The reviewer is correct in mentioning that there is quantitative heterogeneity between the different CPP tested. We mentioned these differences in the manuscript. These mentions are those that are reported under B, plus those listed below.

   Page 19: “It is known for example that peptides made of 9 lysines (K9) poorly reaches the cytosol (Fig. 3f and Supplementary Fig. 9e) and that replacing arginine by lysine in Penetratin significantly diminishes its internalization10,11. According to our model, K9 should induce megapolarization and formation of water pores that should then allow their translocation into cells. However, it has been determined that, once embedded into membranes, lysine residues tend to lose protons12,13. This will thus dissipate the strong membrane potential required for the formation of water pores and leave the lysine-containing CPPs stuck within the phospholipids of the membrane. In contrast, arginine residues are not deprotonated in membranes and water pores can therefore be maintained allowing the arginine-rich CPPs to be taken up by cells.”

   Page 21: “Therefore, the uptake kinetics of lysine-rich peptide, such as MAP, appears artefactually similar as the uptake kinetics of arginine-rich peptides such as R9 (Supplementary Fig. 11b).”

   Page 21: “The differences between CPPs in terms of how efficiently direct translocation is modulated by the Vm (Fig. 2c-d and Supplementary Fig. 10a) could be explained by their relative dependence on direct translocation or endocytosis to penetrate cells. The more positively charged a CPP is, the more it will enter cells through direct translocation and consequently the more sensitive it will be to cell depolarization (Fig. 2c). On the other hand, when endocytosis is the predominant type of entry, CPP cytosolic uptake will be less affected by both hyperpolarization and depolarization, which is what is observed for Transportan internalization in HeLa cells (Fig. 2c and Supplementary Fig. 10a).”

   However, what remains is that depolarization always affects CPP uptake, at most concentrations tested. The heterogeneity reported in Supplementary Fig. 10a for a given experimental condition in a given cell type is in itself of interest as it suggests that there are varying factors within a cell population (e.g. cell cycle, metabolism, etc.) that may impact on the ability of cells to take up CPPs. As per reviewer’s suggestion we may remove this panel from the figure if instructed to do so by the editors.

   Page 12 {section sign} 3 and Page 13 {section sign} 1

   Q18. The pH story is either too long or too short.

   A18. One mechanism put forward to explain direct translocation relies on pH variation between the extracellular milieu and the cytosol14. It was therefore of interest in the context of the model we putting forward to see if pH is affecting the uptake of CPPs in our experimental model. Our data show that pH variations do not affect CPP direct translocation. This information should in our opinion be disclosed.

   Page 14 {section sign} 2

   Q19. At low Vm values, there is a decrease in free energy barrier. Does this modify temperature-dependency for internalization? Do cells really require energy when the Vm is very low, like is often the case for neurons?

   A19. We thank the reviewer for this interesting comment. We will now address this by visualizing under a confocal microscope CPP direct translocation in rat cortical neurons incubated at various temperature (4°C, 24°C, 37°C).

   Page 15 {section sign} 2

   Q20. Figure 2e is not explained, not even in the legend while the statement that CPPs induce a local hyperpolarization is central to the study.

   A20. As there is no Figure 2e, we believe that the reviewer is talking about Figure __3__e, the legend of which was present in the initial version of the manuscript.

   Page 16 {section sign} 1

   Q21. It is confusing that the same agent, here PI, is used to measure internalization (2 nm pore formation in response to hyperpolarization,) and cell death. I have seen the explanation below, but I do not find it fully satisfactory.

   A21. We have tried to explain this better under our answer to point 11B.

   Page 16 {section sign} 2

   Q22. Entry is not necessarily a size issue. Structure is an important parameter, including possible structure changes, for example in response to Vm modifications. Therefore, the statement that molecule with larger diameters are mostly prevented from internalization is not only vague ("mostly") but incorrect.

   A22. We agree with the reviewer’s comment in the sense that the secondary structure of a molecule will also play an important role in its internalization. For that reason, we have used a series of molecules of identical structure (dextrans) but that have different molecular weights. In these experiments we saw that dextran of higher molecular weight enter less efficiently than that of lower molecular weight (Figure 3). We will rephrase some of our sentences so to precise that the size and the shape (structure) of molecules will determine their ability to enter cells through water pores that are characterized by a certain diameter.

   Page 2: “Using dyes of varying sizes and shapes, we assessed the diameter of the water pores.”

   Page 4: “translocation and we characterize the diameter of the water pores used by CPPs.”

   Page 15: “cells were co-incubated with molecules of different sizes and structure and FITC-labelled CPPs at a peptide/lipid ratio of 0.012-0.018 (Supplementary Fig. 11c-d).”

   Page 16: “3 kDa, 10 kDa, and 40 kDa dextrans, 2.3 ±0.38 nm, 4.5 nm and 8.6 nm (diameter estimation provided by Thermofisher), respectively, were used to estimate the diameter of the water pores formed in the presence of CPP.”

   Page 16: “These results are in line with the in silico prediction of the water pore diameter obtained by analyzing the structure of the pore at the transition state.”

   Page 16: “The marginal cytosolic co-internalization of dextrans was inversely correlated with their diameter.”

   Page 35: “200 µg/ml dextran of different __molecular weight__* in the presence or in the absence of the indicated CPPs in normal […]*”.

   Page 17 {section sign} 4 and Page 18 {section sign} 1

   Q23. In Supp. Fig. 13b and c, since the GAP domain is mutated, death is not due to RasGAP activity. So what causes zebrafish death (hyperpolarization?) The results seem contradictory with those of Supp. Fig 13f where survival is 100% at 48 h.

   A23. Indeed, it appears that valinomycin in water leads to zebrafish embryo death, as can be seen in Supplementary Fig. 13c. However, the main difference between Supplementary Fig. 13c and S13f is that in Supplementary Fig. 13f zebrafish were not incubated in valinomycin-containing water, but were locally injected with a CPP in the presence or in the absence of valinomycin. This has now been clarified in the text. We saw that local injections with the hyperpolarizing agent are much less toxic and are well tolerated by the zebrafish embryos.

   Page 18 {section sign} 2

   Q24. The formation of inverted micelles is not incompatible with that of pores. CPP-induced hyperpolarization (Vm) is not measured directly, but deduced from experiments involving artificial membranes and in silico modeling. It would be useful to distinguish between what takes place on live cells (in vitro and in vivo) and what is speculated (based on modeling and artificial systems).

   A24.

   The formation of inverted micelles is not incompatible with that of pores.

   As mentioned above (point 9), we do also think that what has been presented as inverted micelles could have been in fact water pores.

   CPP-induced hyperpolarization (Vm) is not measured directly, but deduced from experiments involving artificial membranes and in silico modeling. It would be useful to distinguish between what takes place on live cells (in vitro and in vivo) and what is speculated (based on modeling and artificial systems).

   If we understand this point correctly, the reviewer is talking about the -150 mV hyperpolarization. This value is not a speculation but has been estimated from in silico experiments and also from experiments using live cells (not artificial membranes). In living cells, the hyperpolarization (megapolarization) has been estimated based on accumulation of intracellular PI over time in the presence or in the absence of CPP.

   Page 19 {section sign} 3

   Q25A. The model posits that the number of Rs influences the ability of the CPPs to hyperpolarize the membrane and, consequently, to induce pore formation. Since pore formation is key to the addressing to the cytoplasm, how can one explain that Penetratin which has only 3 Rs is transported to the cytoplasm more readily that TAT or R9? The authors should take this contradiction in consideration and should not leave aside, in the literature, what does not fit with their model.

   A25A. We fully agree that this should be discussed and not left aside. Please refer to point 4 for detailed discussion about the role of arginine and tryptophan in the ability of CPPs to translocate across membranes.

   Q25B. The fact that that Rs cannot be replaced by Ks, both in R9 and Penetratin is explained by differences in deprotonization. This is interesting but speculative. It might be that the interaction between Rs versus Ks with lipids and sugars are different and not only based on charge. After all their atomic structures, beyond charges, are different.

   A25B. We do not claim that protonation differences between R and K is the definitive answer for their ability to promote CPP translocation. It is one possible explanation that we find sound. As suggested by the reviewer, the ability of K and R to bind lipids and sugars can also play a role. We can mention in this context that the guanidinium group of arginine residues can form two hydrogen bonds1, which allow for more stable electrostatic interactions while the lysyl group of lysine residues can only form one hydrogen bond. We have included these additional possibilities in the revised version of our manuscript as indicated under point 4.

   Page 20 {section sign} 1 Q26. We still need to understand endosomal escape.

   A26. We agree with the reviewer that endosomal escape is still poorly understood. This is an interesting research topic that deserves its own separate study.

   **Major comments**

   • The key conclusions are convincing for a subset of CPPs and cell types
   • Yes, some claims should be qualified as speculative, but not preliminary
   • Many experiments should be removed. Neuronal primary cultures should be introduced to verify the main conclusions, at least for the 3 mains CPPs (TAT, R9, Penetratin). Answers must be given to the concentration issue. Vesicles should be characterized as well as the localization of the peptides in or around the vesicles. See above for less decisive but still important experiments that would benefit to the study.
   • Yes, the requested experiments correspond to a reasonable costs and amount of time (10 to 20,000 € and 3 to 5 months of work)
   • Yes, the methods are presented with great details. -Yes, the experiments are adequately replicated and statistical analysis is adequate

   **Minor comments (not so minor for some of them)**

   • See "Detailed analysis"
   • No, prior studies are not referenced appropriately (see above)
   • No, the text and figures are not clear and not accurate (see above)
   • (i) use Raji cells and primary neuronal cultures, plus in vivo model and forget the other cell types; (ii) forget MAP and Transportan and compare TAT/R9 and Penetratin; (iii) drastically reduce the number of figures, tables and movies (6 primary figures, 6 supplemental figures and 4 tables are reasonable numbers; movies are not absolutely necessary); (iv) limit to 6 (max) the number of panels per figure; (v) limit the number of references to less than 50 and cite the primary reports rather than reviews); (vi) reduce the size of the Material and Methods and the length of figure legends.

   Reviewer #1 (Significance (Required)):

   • The mode of CPP internalization is an unanswered question and the report, if revised, will represent a conceptual and technical advance.
   • Bits and pieces of the conclusions can be found in previous reports. But the Vm-dependent pore formation as well as the CPP-induced "megapolarization" (even if only shown for a subset of CPPs) would be an important contribution. The authors must resist the tentation to generalize to all CPPs what might only be true for a few of them.
   • I do not have the expertise for the in-silico work, but my field of expertise allows me to understand all other aspects of the manuscript.

   ---

   ---

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   In this manuscript, the authors investigated the effect of membrane potential on the internalization of CPPs into the cytosol of some cancer cell lines. Using a CRISPR/Cas9-based screening, they found that some potassium channels play an important role in the internalization of CPPs. The depolarization decreases the rate of internalization of CPPs and the hyperpolarization using valinomycin increases the rate. Using the coarse-grained MD simulations, the authors investigated the interaction of CPPs with a lipid bilayer in the presence of membrane potential. In the interaction of CPPs with the cells, propidium iodide (PI) enters the cytosol significantly. Based on this result, the authors concluded that pores with 2 nm diameter are formed in the plasma membrane.

   This reviewer raises one main issue concerning CPP endocytosis. The reviewer challenges our method to investigate CPP direct translocation and specifically how do we make sure that what we consider direct translocation is not a combination of CPP endocytosis (followed or not by endosomal escape) and CPP plasma membrane translocation. As explained below in details our methodology is able to accurately distinguish CPP uptake by direct translocation from CPP endocytosis and we further demonstrate that endosomal escape does not occur in our experimental settings.

   Q27. One of the defects in this manuscript is the method to determine the fraction of internalization of CPPs via direct translocation across plasma membrane. The authors estimated the fraction of the direct translocation of CPPs by the fluorescence intensity of the cytosolic region (devoid of endosomes) and the fraction of the internalization via endocytosis by the fluorescence intensity of vesicles. However, the CPPs can enter the cytoplasm via endocytosis, and thus the increase in the fluorescence intensity of the cytoplasm is due to two processes (via endocytosis and direct translocation). The authors should use inhibitors of clathrin-mediated endocytosis and macropinocytosis to determine the fraction of internalization of CPPs via direct translocation accurately. Low temperature (4 C) has been also used as the inhibitor of endocytosis (e.g., J. Biophysics, 414729, 2011; J. Biol. Chem., 284, 33957, 2009). Supplementary Figure 1i (the temperature dependence of internalization of TAT-RasGAP317-326) clearly shows that at 4 C the fraction of the internalization was very low, indicating that this peptide enters the cytosol mainly via endocytosis. The determination of the fraction of the internalization via endocytosis by the fluorescence intensity of vesicles in this manuscript is not accurate because it is difficult to examine all endosomes in cells and it is not easy to discriminate the fluorescence intensity due to the endosomes from that due to the cytosol.

   It is important to follow a time course of the fluorescence intensity of single cells from the beginning of the interaction of CPPs with the cells (at least from 5 min) in the presence and absence of inhibitors of the endocytosis (J. Biol. Chem., 278, 585, 2003) to elucidate the process of the internalization of CPPs in the cytosol.

   A27. The reviewer raises the possibility that the signal of fluorescent CPPs in endosomes somehow perturbs the acquisition of the signal in cytosol. This could occur in two ways: CPP endosomal escape and diffusion of the signal located in endosomes into adjacent cytosolic regions (halo effect). The second possibility can be readily dismissed because in situations where cells only take up fluorescent CPPs by endocytosis, the cytosol emits background fluorescence (autofluorescence). This can be seen in Supplementary Fig. 1a (“vesicular” condition) or in Supplementary Fig. 9h in the depolarized cells that cannot take up CPP by direct translocation. Also note that when we record the cytosolic signal we take great care of using regions of interest (ROI) that are distant from endosomes. In contrast to what the reviewer is saying (“it is not easy to discriminate the fluorescence intensity due to the endosomes from that due to the cytosol”), it is actually not difficult discriminating the cytosolic fluorescence from the endosome fluorescence. To illustrate this, we now provide examples of high magnification images of cells incubated with fluorescent CPPs (new Supplementary Fig. 1c, right[1]) to better explain/illustrate our methodology and to show that it is quite straightforward to find cytosolic areas devoid of endosomes. Such high magnification images are those that are used for our blinded quantitation. The other possibility is endosomal escape. We demonstrate in Supplementary Fig. 7c that in our experimental conditions, no endosomal escape is detected[2]. We may not have explained our methodology well enough in the earlier version. We will try and improve the description of our quantitation procedures better in the revised version. To this end, we have now added a scheme illustrating the experimental setup (now part of Supplementary Fig. 7c) that is used to assess endosomal escape.

   The reviewer also questions the way we quantitate the CPP signals in endosomes. In the present paper, our goal is to characterize the direct translocation process of CPPs in to cells. We do not wish here to investigate in details the endocytic pathway taken by CPPs. This has been done in a separate study that we are currently submitting for publication. In a nutshell, this work shows that the endocytic pathway taken by CPPs is different from the classical Rab5- and Rab7-dependent pathway and that the CPP endocytic pathway is not inhibited by compounds that affect the classical pathway. Thus, even if we had wanted to use the inhibitors mentioned by the reviewer, they would not have blocked CPP endocytosis.

   To sum up the issues raised under this point, we believe we have presented the reasons why there are no grounds to support the concerns raised by the reviewer.

   [1] Supplementary Fig. 1c (right) is mentioned in the “Cell death and CPP internalization measurements” section of the methods.

   [2] In this experiment, cells were incubated with CPPs for 30 minutes to allow CPP entry into cells. Then the cells were either washed (to prevent further uptake including uptake through direct translocation) or incubated in the continued presence of CPPs. In both conditions, cells where only endocytosis took place were followed by time-lapse confocal microscopy for 4 hours (i.e. these cells do not display any cytosolic CPP signal at the beginning of the recording). We then assessed the CPP fluorescence intensity within the cytosol (i.e. away from endosomes). From these experiments we saw that cytosolic fluorescence increased only in conditions where CPP was present in the media throughout the experiment. No increase of cytosolic fluorescence was detected in the condition where CPPs were washed out. In conclusion these results demonstrate that the cytosolic signal that we observed in our experiments is due to direct translocation and not endosomal escape. In these experiments we have used the LLOME lysosomotropic agent as a control to make sure that if endosomal escape had occurred (even if only from a subset of endosomes/lysosomes), we would have been able to detect it. Indeed, upon addition of LLOME we were able to record CPP release from endosomes to the cytosol. There is therefore no endosomal escape occurring in our experimental conditions. In conclusion, the observed cytosolic signal in our confocal experiments do not originate, even partly, from endosomal escape.

   Supplementary Figure 1i (the temperature dependence of internalization of TAT-RasGAP317-326) clearly shows that at 4 C the fraction of the internalization was very low, indicating that this peptide enters the cytosol mainly via endocytosis.

   The experiment shown in Supplementary Fig. 1i was analyzed by flow cytometry that cannot discriminate the cytosolic signal from the endosomal signal. We will therefore perform this experiment again but this time using confocal imaging to record the impact of temperature on CPP cytosolic acquisition. We have performed this for HeLa cells already and this shows that direct translocation is indeed inhibited by low temperatures (full blockage at 4°C). Bear in mind that no endosomal escape occurs in our settings (see Supplementary Fig. 7c). This indicates that the decrease in cytoplasmic fluorescence induced by low temperature is not a consequence of diminished CPP endocytosis.

   Q28. Recently, it has been well recognized that membrane potential greatly affects the structure, dynamics and function of plasma membranes (e.g., Science, 349, 873, 2015; PNAS, 107, 12281, 2010). The results of the effect of membrane potential on the internalization of CPPs (depolarization decreases the rate of internalization and hyperpolarization increases the rate), which is main results of this manuscript, can be interpreted by various ways. For example, the rate of endocytosis may be greatly controlled by membrane potential, which can explain the authors' results.

   A28. This reviewer may have missed the experiment presented in Figure 2c that clearly shows that CPP endocytosis is unaffected by depolarization or hyperpolarization of cells. We have also determined that transferrin uptake through endocytosis is not affected by potassium channel knockout (which also leads to depolarization). The possibility raised by the reviewer is therefore refuted by our experimental evidence.

   Q29. A) The authors used the similar concentrations of various CPPs for their experiments (10 to 40 microM), and did not examine the peptide concentration dependence of the internalization. It has been recognized that the CPP concentration affects the mode of internalization of CPPs (e.g., J. Biol. Chem., 284, 33957, 2009). The authors should examine the peptide concentration dependence of the mode of internalization (less than 10 micorM, e. g., 1 microM).

   B) In the case of depolarization, can higher concentrations of CPPs (e.g., 100 micorM) induce their internalization?

   A29. A) We agree that CPPs/cell ratio might prompt one mode of entry over the other. It has been reported by imaging that at lower CPP concentrations endocytosis is favored since only vesicles were observed15-19. Our data confirm this (new Supplementary Fig. 9f).

   B) In Supp. Fig. 7e we have incubated KCNQ5 KO Raji cells that are slightly more depolarized than WT cells in the presence of increasing CPP concentrations up to 100 m From the obtained results, we can see that at 100 mM, the uptake in depolarized cells is increased but does not reach the level of uptake seen in wild-type cells. Therefore, lack of hyperpolarization can be compensated to a mild extent by increased CPP availability.

   Q30. A) The effects of membrane potential on plasma membranes and lipid bilayers have been extensively investigated experimentally and thus are well understood, although currently the coarse-grained MD simulations cannot provide quantitative results which can be compared with experimental results. In this manuscript, using the coarse-grained MD simulations, the authors applied 2.2 V to a lipid bilayer to examine the translocation of CPPs. However, it is well known the experimental results that application of such large voltage to a lipid bilayer induces pore formation in the membrane or its rupture (Bioelectrochem. Bioenerg., 41, 135, 1996; Sci. Rep., 7, 12509, 2017), but at low membrane potential (B) What is the probability of the existence of R9 in the surface of the membrane? R9 cannot bind to the electrically neutral lipid bilayers (such as PC) under a physiological ion concentration (Biochemistry, 55, 4154, 2016). Even if in the case of R9 the membrane potential reaches at -150 mV, the other CPPs have lower surface charge density than that of R9, and hence, the decrement of membrane potential is lower. The authors should provide the data of other CPPs.

   C) It has been reported that the negative membrane potential increases the rate of entry of two kinds of CPPs into the lumen of giant unilamellar vesicles (GUVs) without leakage of water-soluble fluorescent probe (Stokes-Einstein radius; ~0.9 nm diameter), i.e., no pore formation in the GUV membrane (Biophys., 118, 57, 2020, J. Bacteriology, 2021, DOI: 10.1128/JB.00021-21). The authors should discuss the similarity and the difference between the results in these papers and the above results in this manuscript.

   A30. A) As correctly stated by this Reviewer, we reported simulations with high transmembrane potential values, which is a common procedure in in silico simulations used to accelerate the kinetics of the studied process. In this manuscript we have additionally developed and carefully validated a novel protocol to estimate the free energy landscape of water pore formation and CPP translocation under physiological transmembrane potential (further details about the methodological procedure, the convergence and the validation of the free energy estimation are reported in Supplementary Fig. 15-19 of the manuscript). This protocol allowed us to demonstrate the impact of megapolarization (‑150 mV) on the free energy barrier corresponding to the CPP translocation process. The results exemplify how the megapolarization process modifies the uptake probability of the R9 peptide, reducing locally the free energy barrier of the membrane translocation (Fig. 3c-d). Moreover, we have also demonstrated how a single CPP produces a local transmembrane potential of about -150 mV, in agreement with our hypothesis (Fig. 3e).

   Finally, the quantitative accuracy of the molecular simulations was found to be satisfactory because the water pore formation free energy in a symmetric DOPC membrane that we calculated is in excellent agreement with previous atomistic estimation (Table S5).

   B) It has been demonstrated that CPP/membrane interactions are mostly electrostatic between positively charged amino acids carried by the CPPs and various negatively charged cell membrane components, such as glycosaminoglycans20-31 and phosphate groups32. It is in line with our model that the more positively charged CPPs are the better they should translocate into cells. Therefore, we agree with the reviewer that the level of megapolarization may vary according to the charges carried by the CPPs. However, our data clearly indicate that a certain membrane potential hyperpolarization threshold must be achieved to induce water pore formation. As suggested by the reviewer we will now conduct additional modeling experiments with other CPPs.

   C) We have carefully read these papers and do not necessarily reach the same conclusions as the authors. In both papers, the translocation of CPPs in polarized GUVs is monitored through CPP acquisition on vesicles found within the GUVs (intraluminal vesicles; either smaller GUVs or LUVs). There is actually no evidence of the presence of luminal CPPs outside of the intraluminal vesicle membranes. We would therefore argue that these studies elegantly demonstrate that membrane potential increases CPP binding and insertion into the membrane of the mother GUVs but that the CPPs then move, by diffusion, from the lipidic boundary of the mother GUVs to the lipidic membranes of its intraluminal vesicles. This CPP diffusion would presumable occur when the intraluminal vesicles touch the outer membrane bilayer of the mother GUV. There is a marked lag between binding of the CPPs to the membrane of the mother GUV and appearance of CPPs on the intraluminal vesicles (Figure 3c of the Biophysical Journal paper). This lag is, according to us, more compatible with the explanation we are giving than with a translocation mechanism. If there were direct translocation of the CPP through the membrane of the mother GUV, such a large lag would not be expected to be seen (see next point). If there is no translocation of the CPPs across the GUV membrane, it could explain why the water soluble dye within the mother GUVs does not leak out.

   Q31. The authors consider that the translocation of CPPs induces depolarization, and as a result, the pore closes immediately. This kind of transient pore cannot explain the authors' result of the significant entry of PI into the cytosol during the interaction of CPPs with the cells. The authors should explain this point.

   A31. Our interpretation is that PI takes advantage of the water pore triggered by hyperpolarization to penetrate cells. PI is positively charged and is attracted by the negative membrane potential of the cells. Its movement across the cell membrane is therefore unidirectional. This enables the PI molecules to accumulate/concentrate within the cytosol (Supplementary Fig. 12). When PI is in the presence of a CPP, both molecules enter with similar kinetics (Supplementary Fig. 12a and the new quantitation provided in the partially revised version of the manuscript; Supplementary Fig. 12b). PI and CPPs do no interact (Supplementary Figure 12d); hence they move independently from one another.

   Q32. In this manuscript, the authors used only cancer cell lines (Raji cell, SKW6.4 cell, and HeLa cell). The lipid compositions and the stability of the plasma membranes of these cells may be different from normal cells (e.g., 33; Cancer Res., 51, 3062, 1991). Is there a possibility that negatively charged lipids such as PS and PIP2 locate in the outer leaflet locally in these cells? At least, some discussions on this point is essential.

   A32. We agree with the reviewer that plasma membrane composition may vary between cancerous and not cancerous cells and that this may impact on the ability of CPPs to cross cellular membranes. We now mention this in the discussion: “While the nature of the CPPs likely dictate their uptake efficiency as discussed in the precedent paragraph, the composition of the plasma membrane could also modulate how CPPs translocate into cells. In the present work, we have recorded CPP direct translocation in transformed or cancerous cell lines as well as in primary cells. These cells display various abilities to take up CPPs by direct translocation and the present work indicates that this is modulated by their Vm. But as cancer cells display abnormal plasma membrane composition33, it will be of interest in the future to determine how important this is on their capacity to take up CPPs”.

   Q33. The authors found that PI enters the cytosol significantly when CPPs interact with these cells. Based on this result, the authors concluded that pores with 2 nm diameter are formed in the plasma membrane. However, they did not show the time courses of entry of PI and that of CPPs, and thus we cannot judge whether the pore formation in the plasma membrane is the cause of the entry of CPPs or the result of the entry of CPPs. We can reasonably consider that CPPs enters the cytosol via endocytosis and bind to the inner leaflet of the plasma membrane, inducing pore formation in the plasma membrane.

   A33. The kinetics we are now showing in point A31 indicate co-entry of CPPs and PI, an observation that is in line with our model. Also note that we have demonstrated that CPPs do not escape endosomes (please see our answers to questions 12 and 28). These data are therefore not compatible with the reviewer’s interpretation.

   Q34. It has been reported that the negative membrane potential increases the rate constant of antimicrobial peptide (AMP)-induced pore formation or local damage in the GUV membrane (J. Biol. Chem., 294, 10449, 2019; BBA-Biomembranes, 1862, 183381, 2020). These results are related to those in the present manuscript, because here the authors consider that CPPs induce pores in the plasma membrane in the presence of negative membrane potential.

   A34. We thank the reviewer for mentioning these interesting articles. As we understand them, they demonstrate that antimicrobial peptides (AMPs) bind membranes better as a function of increasing negative membrane potential and that this favors their ability to form pores in the membrane, compromising membrane integrity and inducing the release of cytosolic or luminal content. These AMPs do not behave exactly like CPPs because the latter do not compromise the integrity of the membranes.

   In conclusion, the results of the membrane potential dependence of the rate of the internalization of CPPs may be solid results, which is an important contribution. However, the other analyses and the interpretations are not conclusive at the current stage.

   We thank the reviewer for the positive assessment of our results concerning the membrane potential dependence on CPP uptake. Hopefully we have clarified the remaining points with our answers developed above and with the new data we are presenting.

   Reviewer #2 (Significance (Required)):

   (1) Using a CRISPR/Cas9-based screening, the authors found that some potassium channels play an important role in the internalization of CPP TAT-RasGAP317-326. This result advances the field of CPPs.

   (2) Several researches have suggested that the depolarization decreases the rate of internalization of CPPs into cell cytosol and the hyperpolarization increases the rate. It has been also reported that negative membrane potential increases the rate of entry of two kinds of CPPs into the lumen of GUVs of lipid bilayers. The authors provide a new genetic evidence that membrane potential plays an important role in the internalization of CPPs in the cytosol. However, modulation of membrane potential affects the structure, dynamics and function of plasma membranes greatly. At the current stage, it is difficult to judge which process of the internalization of CPPs is affected by the membrane potential.

   (3) The researchers of CPPs and AMPs are interested in their results after they improve the contents of the manuscript.

   (4) My field of expertise is membrane biophysics, especially the interaction of AMPs and CPPs with GUVs and cells.

   References

   1 Fromm, J. R., Hileman, R. E., Caldwell, E. E. O., Weiler, J. M. & Linhardt, R. J. Differences in the Interaction of Heparin with Arginine and Lysine and the Importance of these Basic Amino Acids in the Binding of Heparin to Acidic Fibroblast Growth Factor. Archives of Biochemistry and Biophysics 323, 279-287, doi:https://doi.org/10.1006/abbi.1995.9963 (1995).

   2 Derossi, D., Joliot, A. H., Chassaing, G. & Prochiantz, A. The third helix of the Antennapedia homeodomain translocates through biological membranes. The Journal of biological chemistry 269, 10444-10450 (1994).

   3 Jobin, M. L., Blanchet, M., Henry, S., Chaignepain, S., Manigand, C., Castano, S., Lecomte, S., Burlina, F., Sagan, S. & Alves, I. D. The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides. Biochim Biophys Acta 1848, 593-602, doi:10.1016/j.bbamem.2014.11.013 (2015).

   4 MacCallum, J. L., Bennett, W. F. D. & Tieleman, D. P. Distribution of amino acids in a lipid bilayer from computer simulations. Biophysical journal 94, 3393-3404, doi:10.1529/biophysj.107.112805 (2008).

   5 Christiaens, B., Symoens, S., Vanderheyden, S., Engelborghs, Y., Joliot, A., Prochiantz, A., Vandekerckhove, J., Rosseneu, M. & Vanloo, B. Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes. European Journal of Biochemistry 269, 2918-2926, doi:10.1046/j.1432-1033.2002.02963.x (2002).

   6 Walrant, A., Bauza, A., Girardet, C., Alves, I. D., Lecomte, S., Illien, F., Cardon, S., Chaianantakul, N., Pallerla, M., Burlina, F., Frontera, A. & Sagan, S. Ionpair-pi interactions favor cell penetration of arginine/tryptophan-rich cell-penetrating peptides. Biochim Biophys Acta Biomembr 1862, 183098, doi:10.1016/j.bbamem.2019.183098 (2020).

   7 Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. & Prochiantz, A. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem 271, 18188-18193, doi:10.1074/jbc.271.30.18188 (1996).

   8 Serulla, M., Ichim, G., Stojceski, F., Grasso, G., Afonin, S., Heulot, M., Schober, T., Roth, R., Godefroy, C., Milhiet, P. E., Das, K., Garcia-Saez, A. J., Danani, A. & Widmann, C. TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids. Proc Natl Acad Sci U S A, doi:10.1073/pnas.2014108117 (2020).

   9 Bowman, A. M., Nesin, O. M., Pakhomova, O. N. & Pakhomov, A. G. Analysis of plasma membrane integrity by fluorescent detection of Tl(+) uptake. J Membr Biol 236, 15-26, doi:10.1007/s00232-010-9269-y (2010).

   10 Mitchell, D. J., Kim, D. T., Steinman, L., Fathman, C. G. & Rothbard, J. B. Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56, 318-325 (2000).

   11 Amand, H. L., Rydberg, H. A., Fornander, L. H., Lincoln, P., Norden, B. & Esbjorner, E. K. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans. Biochim Biophys Acta 1818, 2669-2678, doi:10.1016/j.bbamem.2012.06.006 (2012).

   12 Armstrong, C. T., Mason, P. E., Anderson, J. L. & Dempsey, C. E. Arginine side chain interactions and the role of arginine as a gating charge carrier in voltage sensitive ion channels. Sci Rep 6, 21759, doi:10.1038/srep21759 (2016).

   13 Li, L., Vorobyov, I. & Allen, T. W. The different interactions of lysine and arginine side chains with lipid membranes. J Phys Chem B 117, 11906-11920, doi:10.1021/jp405418y (2013).

   14 Herce, H. D., Garcia, A. E. & Cardoso, M. C. Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules. J Am Chem Soc 136, 17459-17467, doi:10.1021/ja507790z (2014).

   15 Kosuge, M., Takeuchi, T., Nakase, I., Jones, A. T. & Futaki, S. Cellular Internalization and Distribution of Arginine-Rich Peptides as a Function of Extracellular Peptide Concentration, Serum, and Plasma Membrane Associated Proteoglycans. Bioconjugate Chemistry 19, 656-664, doi:10.1021/bc700289w (2008).

   16 Fretz, M. M., Penning, N. A., Al-Taei, S., Futaki, S., Takeuchi, T., Nakase, I., Storm, G. & Jones, A. T. Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. The Biochemical journal 403, 335-342, doi:10.1042/BJ20061808 (2007).

   17 Drin, G., Cottin, S., Blanc, E., Rees, A. R. & Temsamani, J. Studies on the internalization mechanism of cationic cell-penetrating peptides. J Biol Chem 278, 31192-31201, doi:10.1074/jbc.M303938200 (2003).

   18 Duchardt, F., Fotin‐Mleczek, M., Schwarz, H., Fischer, R. & Brock, R. A Comprehensive Model for the Cellular Uptake of Cationic Cell‐penetrating Peptides. Traffic 8, 848-866, doi:10.1111/j.1600-0854.2007.00572.x (2007).

   19 Ziegler, A., Nervi, P., Dürrenberger, M. & Seelig, J. The Cationic Cell-Penetrating Peptide CPPTAT Derived from the HIV-1 Protein TAT Is Rapidly Transported into Living Fibroblasts:  Optical, Biophysical, and Metabolic Evidence. Biochemistry 44, 138-148, doi:10.1021/bi0491604 (2005).

   20 Ziegler, A. Thermodynamic studies and binding mechanisms of cell-penetrating peptides with lipids and glycosaminoglycans. Advanced Drug Delivery Reviews 60, 580-597, doi:https://doi.org/10.1016/j.addr.2007.10.005 (2008).

   21 Rullo, A., Qian, J. & Nitz, M. Peptide–glycosaminoglycan cluster formation involving cell penetrating peptides. Biopolymers 95, 722-731, doi:10.1002/bip.21641 (2011).

   22 Bechara, C., Pallerla, M., Zaltsman, Y., Burlina, F., Alves, I. D., Lequin, O. & Sagan, S. Tryptophan within basic peptide sequences triggers glycosaminoglycan-dependent endocytosis. The FASEB Journal 27, 738-749, doi:10.1096/fj.12-216176 (2013).

   23 Gonçalves, E., Kitas, E. & Seelig, J. Binding of Oligoarginine to Membrane Lipids and Heparan Sulfate:  Structural and Thermodynamic Characterization of a Cell-Penetrating Peptide. Biochemistry 44, 2692-2702, doi:10.1021/bi048046i (2005).

   24 Rusnati, M., Tulipano, G., Spillmann, D., Tanghetti, E., Oreste, P., Zoppetti, G., Giacca, M. & Presta, M. Multiple Interactions of HIV-I Tat Protein with Size-defined Heparin Oligosaccharides. Journal of Biological Chemistry 274, 28198-28205, doi:10.1074/jbc.274.40.28198 (1999).

   25 Butterfield, K. C., Caplan, M. & Panitch, A. Identification and Sequence Composition Characterization of Chondroitin Sulfate-Binding Peptides through Peptide Array Screening. Biochemistry 49, 1549-1555, doi:10.1021/bi9021044 (2010).

   26 Åmand, H. L., Rydberg, H. A., Fornander, L. H., Lincoln, P., Nordén, B. & Esbjörner, E. K. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans. Biochimica et Biophysica Acta (BBA) - Biomembranes 1818, 2669-2678, doi:https://doi.org/10.1016/j.bbamem.2012.06.006 (2012).

   27 Ghibaudi, E., Boscolo, B., Inserra, G., Laurenti, E., Traversa, S., Barbero, L. & Ferrari, R. P. The interaction of the cell-penetrating peptide penetratin with heparin, heparansulfates and phospholipid vesicles investigated by ESR spectroscopy. Journal of Peptide Science 11, 401-409, doi:10.1002/psc.633 (2005).

   28 Fuchs, S. M. & Raines, R. T. Pathway for polyarginine entry into mammalian cells. Biochemistry 43, 2438-2444, doi:10.1021/bi035933x (2004).

   29 Ziegler, A. & Seelig, J. Contributions of Glycosaminoglycan Binding and Clustering to the Biological Uptake of the Nonamphipathic Cell-Penetrating Peptide WR9. Biochemistry 50, 4650-4664, doi:10.1021/bi1019429 (2011).

   30 Ziegler, A. & Seelig, J. Interaction of the Protein Transduction Domain of HIV-1 TAT with Heparan Sulfate: Binding Mechanism and Thermodynamic Parameters. Biophysical Journal 86, 254-263, doi:https://doi.org/10.1016/S0006-3495(04)74101-6 (2004).

   31 Hakansson, S. & Caffrey, M. Structural and Dynamic Properties of the HIV-1 Tat Transduction Domain in the Free and Heparin-Bound States. Biochemistry 42, 8999-9006, doi:10.1021/bi020715+ (2003).

   32 Kawamoto, S., Takasu, M., Miyakawa, T., Morikawa, R., Oda, T., Futaki, S. & Nagao, H. Inverted micelle formation of cell-penetrating peptide studied by coarse-grained simulation: importance of attractive force between cell-penetrating peptides and lipid head group. J Chem Phys 134, 095103, doi:10.1063/1.3555531 (2011).

   33 Szlasa, W., Zendran, I., Zalesinska, A., Tarek, M. & Kulbacka, J. Lipid composition of the cancer cell membrane. J Bioenerg Biomembr 52, 321-342, doi:10.1007/s10863-020-09846-4 (2020).

   Read the original source
6. 

   Review Commons

   Mar 18, 2021

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   In this manuscript, the authors investigated the effect of membrane potential on the internalization of CPPs into the cytosol of some cancer cell lines. Using a CRISPR/Cas9-based screening, they found that some potassium channels play an important role in the internalization of CPPs. The depolarization decreases the rate of internalization of CPPs and the hyperpolarization using valinomycin increases the rate. Using the coarse-grained MD simulations, the authors investigated the interaction of CPPs with a lipid bilayer in the presence of membrane potential. In the interaction of CPPs with the cells, propidium iodide (PI) enters the …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   In this manuscript, the authors investigated the effect of membrane potential on the internalization of CPPs into the cytosol of some cancer cell lines. Using a CRISPR/Cas9-based screening, they found that some potassium channels play an important role in the internalization of CPPs. The depolarization decreases the rate of internalization of CPPs and the hyperpolarization using valinomycin increases the rate. Using the coarse-grained MD simulations, the authors investigated the interaction of CPPs with a lipid bilayer in the presence of membrane potential. In the interaction of CPPs with the cells, propidium iodide (PI) enters the cytosol significantly. Based on this result, the authors concluded that pores with 2 nm diameter are formed in the plasma membrane.

   One of the defects in this manuscript is the method to determine the fraction of internalization of CPPs via direct translocation across plasma membrane. The authors estimated the fraction of the direct translocation of CPPs by the fluorescence intensity of the cytosolic region (devoid of endosomes) and the fraction of the internalization via endocytosis by the fluorescence intensity of vesicles. However, the CPPs can enter the cytoplasm via endocytosis, and thus the increase in the fluorescence intensity of the cytoplasm is due to two processes (via endocytosis and direct translocation). The authors should use inhibitors of clathrin-mediated endocytosis and macropinocytosis to determine the fraction of internalization of CPPs via direct translocation accurately. Low temperature (4 C) has been also used as the inhibitor of endocytosis (e.g., J. Biophysics, 414729, 2011; J. Biol. Chem., 284, 33957, 2009). Supplementary Figure 1i (the temperature dependence of internalization of TAT-RasGAP317-326) clearly shows that at 4 C the fraction of the internalization was very low, indicating that this peptide enters the cytosol mainly via endocytosis. The determination of the fraction of the internalization via endocytosis by the fluorescence intensity of vesicles in this manuscript is not accurate because it is difficult to examine all endosomes in cells and it is not easy to discriminate the fluorescence intensity due to the endosomes from that due to the cytosol.

   It is important to follow a time course of the fluorescence intensity of single cells from the beginning of the interaction of CPPs with the cells (at least from 5 min) in the presence and absence of inhibitors of the endocytosis (J. Biol. Chem., 278, 585, 2003) to elucidate the process of the internalization of CPPs in the cytosol

   Recently, it has been well recognized that membrane potential greatly affects the structure, dynamics and function of plasma membranes (e.g., Science, 349, 873, 2015; PNAS, 107, 12281, 2010). The results of the effect of membrane potential on the internalization of CPPs (depolarization decreases the rate of internalization and hyperpolarization increases the rate), which is main results of this manuscript, can be interpreted by various ways. For example, the rate of endocytosis may be greatly controlled by membrane potential, which can explain the authors' results.

   The authors used the similar concentrations of various CPPs for their experiments (10 to 40 microM), and did not examine the peptide concentration dependence of the internalization. It has been recognized that the CPP concentration affects the mode of internalization of CPPs (e.g., J. Biol. Chem., 284, 33957, 2009). The authors should examine the peptide concentration dependence of the mode of internalization (less than 10 micorM, e. g., 1 microM). In the case of depolarization, can higher concentrations of CPPs (e.g., 100 micorM) induce their internalization? The effects of membrane potential on plasma membranes and lipid bilayers have been extensively investigated experimentally and thus are well understood, although currently the coarse-grained MD simulations cannot provide quantitative results which can be compared with experimental results. In this manuscript, using the coarse-grained MD simulations, the authors applied 2.2 V to a lipid bilayer to examine the translocation of CPPs. However, it is well known the experimental results that application of such large voltage to a lipid bilayer induces pore formation in the membrane or its rupture (Bioelectrochem. Bioenerg., 41, 135, 1996; Sci. Rep., 7, 12509, 2017), but at low membrane potential (< ~200 mV) a lipid bilayer is stable although they have transient pre-pores (Biophys. J., 85, 2342, 2003; Biophys. J., 80, 1829, 2001). In the main results obtained in the experiments of cells in this manuscript, the values of the membrane potential are less than -100 mV. Therefore, this description of their results of MD simulations is misleading. According to their theory, only at much lower Vm values (-150 mV) induces a large decrease in free energy barrier of the translocation of CPPs across the lipid bilayer. Is this due to the pore formation in the membrane? The description of this point is poor, and thus it is diffiult to understand it. The authors consider that normal membrane potential is much higher than -150 mV, and thus the binding of positively charged CPPs in the membrane surface must increase the negative membrane potential to decrease the free energy barrier. Then, the authors suggest that the presence of R9 in contact with lipid membrane decreases the transmembrane potential to -150 mV according to the MD calculation. What is the probability of the existence of R9 in the surface of the membrane? R9 cannot bind to the electrically neutral lipid bilayers (such as PC) under a physiological ion concentration (Biochemistry, 55, 4154, 2016). Even if in the case of R9 the membrane potential reaches at -150 mV, the other CPPs have lower surface charge density than that of R9, and hence, the decrement of membrane potential is lower. The authors should provide the data of other CPPs. It has been reported that the negative membrane potential increases the rate of entry of two kinds of CPPs into the lumen of giant unilamellar vesicles (GUVs) without leakage of water-soluble fluorescent probe (Stokes-Einstein radius; ~0.9 nm diameter), i.e., no pore formation in the GUV membrane (Biophys., 118, 57, 2020, J. Bacteriology, 2021, DOI: 10.1128/JB.00021-21). The authors should discuss the similarity and the difference between the results in these papers and the above results in this manuscript.

   The authors consider that the translocation of CPPs induces depolarization, and as a result, the pore closes immediately. This kind of transient pore cannot explain the authors' result of the significant entry of PI into the cytosol during the interaction of CPPs with the cells. The authors should explain this point.

   In this manuscript, the authors used only cancer cell lines (Raji cell, SKW6.4 cell, and HeLa cell). The lipid compositions and the stability of the plasma membranes of these cells may be different from normal cells (e.g., J. Bioenergetics Biomem., 52, 321, 2020; Cancer Res., 51, 3062, 1991). Is there a possibility that negatively charged lipids such as PS and PIP2 locate in the outer leaflet locally in these cells? At least, some discussions on this point is essential.

   The authors found that PI enters the cytosol significantly when CPPs interact with these cells. Based on this result, the authors concluded that pores with 2 nm diameter are formed in the plasma membrane. However, they did not show the time courses of entry of PI and that of CPPs, and thus we cannot judge whether the pore formation in the plasma membrane is the cause of the entry of CPPs or the result of the entry of CPPs. We can reasonably consider that CPPs enters the cytosol via endocytosis and bind to the inner leaflet of the plasma membrane, inducing pore formation in the plasma membrane.

   It has been reported that the negative membrane potential increases the rate constant of antimicrobial peptide (AMP)-induced pore formation or local damage in the GUV membrane (J. Biol. Chem., 294, 10449, 2019; BBA-Biomembranes, 1862, 183381, 2020). These results are related to those in the present manuscript, because here the authors consider that CPPs induce pores in the plasma membrane in the presence of negative membrane potential.

   In conclusion, the results of the membrane potential dependence of the rate of the internalization of CPPs may be solid results, which is an important contribution. However, the other analyses and the interpretations are not conclusive at the current stage.

   Significance

   (1) Using a CRISPR/Cas9-based screening, the authors found that some potassium channels play an important role in the internalization of CPP TAT-RasGAP317-326. This result advances the field of CPPs.

   (2) Several researches have suggessted that the depolarization decreases the rate of internalization of CPPs into cell cytosol and the hyperpolarization increases the rate. It has been also reported that negative membrane potential increases the rate of entry of two kinds of CPPs into the lumen of GUVs of lipid bilayers. The authors provide a new genetic evidence that membrane potential plays an important role in the internalization of CPPs in the cytosol. However, modulation of membrane potential affects the structure, dynamics and function of plasma membranes greatly. At the current stage, it is difficult to judge which process of the internalization of CPPs is affected by the membrane potential.

   (3) The researchers of CPPs and AMPs are interested in their results after they improve the contents of the manuscript.

   (4) My field of expertise is membrane biophysics, especially the interaction of AMPs and CPPs with GUVs and cells.

   Read the original source
7. 

   Review Commons

   Mar 18, 2021

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary

   The authors propose a mechanism through which voltage dependent water pore formation is key to the internalization of Cell permeable peptides (CPPs). The claim is based on an in-silico study and on several experimental approaches. The authors compare 5 peptides (R9, TAT-48-57, Penetratin, MAP and Transportan and use 3 distinct cell lines (Raji, SKW6.4 and HeLa cells), plus neurons in primary cultures. The also present in vivo experiment (mouse skin and zebrafish embryo). All in all, it is an interesting study, but it raises several issues that need to be addressed. Moreover, the length and structure of the manuscript make it …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary

   The authors propose a mechanism through which voltage dependent water pore formation is key to the internalization of Cell permeable peptides (CPPs). The claim is based on an in-silico study and on several experimental approaches. The authors compare 5 peptides (R9, TAT-48-57, Penetratin, MAP and Transportan and use 3 distinct cell lines (Raji, SKW6.4 and HeLa cells), plus neurons in primary cultures. The also present in vivo experiment (mouse skin and zebrafish embryo). All in all, it is an interesting study, but it raises several issues that need to be addressed. Moreover, the length and structure of the manuscript make it very difficult to read (see below under "Reviewer statement")

   Reviewer statement

   The instructions are to use the "Major comments" section to answer 6 precise questions. Unfortunately, this is not possible due to the structure of the document to review. The main manuscript (22 pages) comes with 4 primary figures and 19 supplemental ones. Most of these figures have an enormous number of panels and their legends occupy 17 pages. To this, are added 6 supplemental tables and 7 supplemental movies (with 2 pages of legends), 28 pages of Material and Methods, and 146 References (109 for the main manuscript and 37 for Supplemental information). To be frank, I was often tempted to send the manuscript back, asking for the authors to submit a document facilitating the task of the reviewers.

   Because of this complexity, my "Major comments" will come after a page by page, paragraph ({section sign}) by paragraph and figure by figure "Detailed analysis" of the manuscript.

   Detailed analysis

   Page 4 {section sign} 3 The test is based on the ability of TAT-RasGAP to kill the cells. Although controls exist, this is worrying since necrotic death might participate in the rupture of the membrane and artificially amplify internalization after a first physiological entry of the peptide. It is also a bit dangerous to add a FITC group to a short peptide without controlling that it has no effect on the interaction with the membrane (FITC-induced local hydrophobicity can provoke peptide tilting and membrane shearing). In the same vein, the very high peptide concentrations often used in the study (40µM for Raji and SKW6.4 cells and 80µM on HeLa cells) can be highly toxic.

   Page 5 {section sign} 1 Supp. Fig.1a shows no differences between the 3 cell types, even though they differ in their modes of peptide internalization, some favoring vesicular staining and others cytoplasmic diffusion. Multiplying cell and peptide types contributes to the complexity of the manuscript without increasing its interest. If there is a conceptual breakthrough, as might be the case, it is obscured by the accumulation of useless images and data. A step into simplifying the manuscript would be (i), to concentrate on Raji cells (leaving out SKW6.4 and HeLa cells) and (ii) to only discuss the R9, TAT (including TAT-RasGAP) and Penetratin peptides. TAT and R9 are poly-R peptides, which is not the case for Penetratin that has only 3 Rs. These 3 Rs are important (cannot be replaced by 3 Ks), but the two Ws absent in R9 and TAT are equally important as they cannot be replaced by Fs. This must be considered by the authors when they tend to generalize their model. Supp. Fig1c-d is not necessary (very little information in it) and Supp. Fig 1e is misleading as it takes a lot of imagination to see a difference between homogenous (top) and focal (bottom) diffusion. Supp. Fig.1g: How many cells are we looking at? Given the high variance, the result cannot be interpreted easily. A distribution according to fluorescence bits would be a better way to present the data.

   Supp. Fig2i. This panel confirms that Raji cells differ from the two other cell types by showing clear temperature dependency. The explanation will come later with the energy barrier for low Vm-induced pore formation. This contradicts earlier reports showing that Penetratin translocation is not temperature-dependent, possibly because it was done on neurons naturally hyperpolarized. Or else because mechanisms are, at least in part, different from the one proposed here for R9 and TAT. This requires some clarification and supports the suggestion that, instead of multiplying models and peptides, it would be more efficient to compare TAT, R9 and Penetratin internalization by Raji cells and primary neurons. Supp. Fig. 2a-f. Last sentence of the legend "Concentrations above 40µM led to too extensive cell death preventing analysis of peptide internalization". This confirms the warning against the use of concentrations varying between 40µM and 80µ and partially jeopardizes the validity of some experiments.

   Page 6 {section sign} 2 The authors advocate 2 modes of entry, opposing transport across the membrane and endocytosis. In contrast with R9, TAT and Penetratin, Transportan or MAP seem to be purely endocytosed but, if they reach the cytoplasm, they still have to cross a membrane (unless "a miracle happens"). For Penetratin and R9/TAT, the authors consider that water pore and inverted micelle formation are incompatible. This is a bit rapid as inverted micelles might induce water pores through W/lipids interactions requiring less R residues and, possibly, less energy. This provides the opportunity to signal that, in spite of their very high number, key references are missing or hidden in cited reviews, some of them written by colleagues who are not among the main contributors to the CPP field.

   Page 7 {section sign} 1 Fig. 1b confirms that Raji cells provide a good model for loss and gain of function (lovely rescue experiment) and that the authors should drop the two other cell types that provide no decisive information.

   Page 8 {section sign} 1 Supp. Fig. 6b (no serum conditions) allows for the use of "normal" CPP concentrations and suggests that a fraction of the peptides may bind to serum components. No arrows in Supp. Fig.6b (but in 6c), and the R/pyrene butyrate interaction is not in 6c but in 6a. Still for Supp. Fig. 6c, the death of cells at 20µM (or less) even in the absence of K+ channels, confirms that we are borderline in term of peptide toxicity. There is a confusion between Supp. Fig. 6d and 6e and a legend problem (6e is not described). Cell death is assessed in % of PI-positive cells. Does this securely distinguish between death and holes allowing for PI entry without death? The CPP is incubated in the presence of Pyrene butyrate, making the KO cells less resistant. How does that demonstrate that the potassium channels are not involved in the killing if the peptide is already in? Unless the KO is done after internalization (but the cells should be already dead or dying?). This lacks clarity.

   Page 9 {section sign} 1 The conclusion that the diffuse staining does not come from endosomal escape is based on the certainty that LLOME disrupts both endosomes and lysosomes. First, it should be verified with specific markers (rab5, rab7) that the fluorescent vesicles are endosomes. Second, the literature strongly suggests that LLOME primarily disrupts lysosomes and not endosomes. Finally, even if some endosomes are disrupted, the endosomal population is heterogenous and some CPPs may be in a subpopulation insensitive to LLOME. In addition, the importance of this issue is not well explained. In practice, access to the cytoplasm and nucleus requires crossing the plasma and/or the endosomal membrane and the latter, at least in early endosomes (thus the need of identifying the CPP-enriched vesicles), might not be very different from the plasma membrane. Page 9 {section sign} 2 Is Supp. Fig. 7e really necessary? First, as mentioned several times, if 20µM is a borderline concentration in term of toxicity, raising the concentration up to 100µM is problematic. Secondly, what matters is not "binding" in general, but binding to the proper membrane components. As mentioned by the authors themselves (Supp. Fig. 1e and movie), there are privileged sites of entry that may correspond to the recognition of specific molecular entities/structures.

   Page 9 {section sign} 3 and Page 10 {section sign} 1 The authors should have used a construct that does not kill the cells much earlier, just after the screening experiments based on resistance to necrosis induced by TAT-rasGAP. For Supp. Fig 8a and b: I am fully convinced by Raji cells and HeLa cells but not by the SKW6.4 cells..

   Page 10 {section sign} 2 Supp. Fig 9 is quite convincing but adds the information that 2µM are sufficient in neurons. This again makes the 20 to 80µM concentrations used on transformed cells unsatisfactory. If one needs a cell line (more user friendly than primary cultures), there are several neural ones that can be differentiated (SHY, LHUMES, etc.) that may have an appropriate membrane potential (below -90mV). Indeed, it would then be important to verify if pore formation is still induced by TAT, R9 and Penetratin (separately) on "naturally" hyperpolarized cells. Figure 2a confirms that changes in Vm are not solid for HeLa and SKW6.4 cells. This casts a doubt on the validity of the results obtained with the latter 2 cell lines.

   Page 11 {section sign} 2 Why valinomycin was only tried on Raji cells?

   Page 12 {section sign} 2 Looking at Fig. 2c, it seems that low Vm increases the uptake of all CPPs, except Transportan. Is there any reason why this Figure does not provide the number of vesicles per cell in the hyperpolarized conditions? In fact, if one goes to Supp. Fig. 9c, it appears that, among all peptides, only Penetratin is almost entirely cytoplasmic after 90' of incubation, whereas MAP and Transportan remain essentially vesicular. TAT and R9 are at mid-distance between these two extremes. This leads to send again the warning that all CPPs cannot be placed in a single category. The table that describes the sequences strongly suggests that, TAT and R9 uptake is due to the numerous Rs that cannot be replaced by Ks. In the case of Penetratin, that only has 3 Rs, the situation is thus different with the presence of 2 Ws previously shown to be mandatory for internalization, although absent in TAT ad R9. In Supp. Fig9, panel g is useless. A difference between peptides is also visible in Figure 2d where depolarization with KCl does not show the same efficiency on all peptides. The issue is whether these differences are significant and, if so, why? This discussion could be restricted to TAT, R9 and Penetratin. Supp. Fig. 10a also suggests that all peptides do not respond similarly to depolarization and that the effects differ between cell types and concentrations used. However, given the high concentrations used and the high variance between replicates, this figure might not be a priority in the reorganization of the manuscript.

   Page 12 {section sign} 3 and Page 13 {section sign} 1 The pH story is either too long or too short.

   Page 14 {section sign} 2 At low Vm values, there is a decrease in free energy barrier. Does this modify temperature-dependency for internalization? Do cells really require energy when the Vm is very low, like is often the case for neurons?

   Page 15 {section sign} 2 Figure 2e is not explained, not even in the legend while the statement that CPPs induce a local hyperpolarization is central to the study.

   Page 16 {section sign} 1 It is confusing that the same agent, here PI, is used to measure internalization (2 nm pore formation in response to hyperpolarization,) and cell death. I have seen the explanation below, but I do not find it fully satisfactory.

   Page 16 {section sign} 2 Entry is not necessarily a size issue. Structure is an important parameter, including possible structure changes, for example in response to Vm modifications. Therefore, the statement that molecule with larger diameters are mostly prevented from internalization is not only vague ("mostly") but incorrect.

   Page 17 {section sign} 4 and Page 18 {section sign} 1 In Supp. Fig. 13b and c, since the GAP domain is mutated, death is not due to RasGAP activity. So what causes zebrafish death (hyperpolarization?) The results seem contradictory with those of Supp. Fig 13f where survival is 100% at 48 h.

   Page 18 {section sign} 2 The formation of inverted micelles is not incompatible with that of pores. CPP-induced hyperpolarization (Vm) is not measured directly, but deduced from experiments involving artificial membranes and in silico modeling. It would be useful to distinguish between what takes place on live cells (in vitro and in vivo) and what is speculated (based on modeling and artificial systems).

   Page 19 {section sign} 3 The model posits that the number of Rs influences the ability of the CPPs to hyperpolarize the membrane and, consequently, to induce pore formation. Since pore formation is key to the addressing to the cytoplasm, how can one explain that Penetratin which has only 3 Rs is transported to the cytoplasm more readily that TAT or R9? The authors should take this contradiction in consideration and should not leave aside, in the literature, what does not fit with their model. The fact that that Rs cannot be replaced by Ks, both in R9 and Penetratin is explained by differences in deprotonization. This is interesting but speculative. It might be that the interaction between Rs versus Ks with lipids and sugars are different and not only based on charge. After all their atomic structures, beyond charges, are different.

   Page 20 {section sign} 1 We still need to understand endosomal escape.

   Major comments

   • The key conclusions are convincing for a subset of CPPs and cell types
   • Yes, some claims should be qualified as speculative, but not preliminary
   • Many experiments should be removed. Neuronal primary cultures should be introduced to verify the main conclusions, at least for the 3 mains CPPs (TAT, R9, Penetratin). Answers must be given to the concentration issue. Vesicles should be characterized as well as the localization of the peptides in or around the vesicles. See above for less decisive but still important experiments that would benefit to the study.
   • Yes, the requested experiments correspond to a reasonable costs and amount of time (10 to 20,000 € and 3 to 5 months of work)
   • Yes, the methods are presented with great details. -Yes, the experiments are adequately replicated and statistical analysis is adequate

   Minor comments (not so minor for some of them)

   • See "Detailed analysis"
   • No, prior studies are not referenced appropriately (see above)
   • No, the text and figures are not clear and not accurate (see above)
   • (i) use Raji cells and primary neuronal cultures, plus in vivo model and forget the other cell types; (ii) forget MAP and Transportan and compare TAT/R9 and Penetratin; (iii) drastically reduce the number of figures, tables and movies (6 primary figures, 6 supplemental figures and 4 tables are reasonable numbers; movies are not absolutely necessary); (iv) limit to 6 (max) the number of panels per figure; (v) limit the number of references to less than 50 and cite the primary reports rather than reviews); (vi) reduce the size of the Material and Methods and the length of figure legends.

   Significance

   • The mode of CPP internalization is an unanswered question and the report, if revised, will represent a conceptual and technical advance.
   • Bits and pieces of the conclusions can be found in previous reports. But the Vm-dependent pore formation as well as the CPP-induced "megapolarization" (even if only shown for a subset of CPPs) would be an important contribution. The authors must resist the tentation to generalize to all CPPs what might only be true for a few of them.
   • I do not have the expertise for the in-silico work, but my field of expertise allows me to understand all other aspects of the manuscript.

   Read the original source
8. 

   Version published to 10.1101/2020.02.25.963017v2 on bioRxiv

   Jan 19, 2021
9. 

   Version published to 10.1101/2020.02.25.963017v1 on bioRxiv

   Feb 26, 2020