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

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    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.)

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Abstract

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (V m ). These findings provide the first unbiased genetic validation of the role of V m in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the V m to very low values (–150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (–5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

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  1. 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 …

  2. 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.

  3. 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 …

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    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 …

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

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    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 …

  6. 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 …