Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
- University of Lausanne, Switzerland
- Università della Svizzera italiana, Scuola Universitaria Professionale della Svizzera Italiana, Switzerland
- Politecnico di Torino, Italy
- Université de Lausanne, Switzerland
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 (Vm). These findings provide the first unbiased genetic validation of the role of Vm 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 Vm 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 CPPs 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 membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.
Data availability
DNA sequencing data from the CRISPR/Cas9-based screens are available through the following link: https://www.ncbi.nlm.nih.gov/sra/SRP161445.
-
CRISPR/Cas9 screen to identify genes involved in uptake of cell-penetrating peptidesNCBI Gene Expression Omnibus, SRP161445.
Article and author information
Author details
Evgeniya Trofimenko
Funding
Swiss National Science Foundation (CRSII3_154420,IZCSZ0-174639)
- Christian Widmann
Swiss National Science Foundation (158116)
- Nadja Chevalier
Swiss National Science Foundation (320030_170062)
- Francesca Amati
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Patricia Bassereau, Institut Curie, France
Ethics
Animal experimentation: All experiments were performed according to the principles of laboratory animal care and Swiss legislation under ethical approval (Swiss Animal Protection Ordinance; permit number VD3374.a).
Version history
- Preprint posted: February 26, 2020 (view preprint)
- Received: April 27, 2021
- Accepted: October 28, 2021
- Accepted Manuscript published: October 29, 2021 (version 1)
- Version of Record published: December 2, 2021 (version 2)
Copyright
© 2021, Trofimenko et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,848
- views
-
- 617
- downloads
-
- 33
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
eLife 10:e69832.
https://doi.org/10.7554/eLife.69832
Further reading
-
- Cell Biology
- Stem Cells and Regenerative Medicine
Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47−/− mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47−/− spleens but significantly depleted in Thbs1−/− spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119−CD34+ progenitors and Ter119+CD34− committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1−/− spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.
-
- Cell Biology
Reports indicate that an interaction between TRPV4 and anoctamin 1 (ANO1) could be widely involved in water efflux of exocrine glands, suggesting that the interaction could play a role in perspiration. In secretory cells of sweat glands present in mouse foot pads, TRPV4 clearly colocalized with cytokeratin 8, ANO1, and aquaporin-5 (AQP5). Mouse sweat glands showed TRPV4-dependent cytosolic Ca2+ increases that were inhibited by menthol. Acetylcholine-stimulated sweating in foot pads was temperature-dependent in wild-type, but not in TRPV4-deficient mice and was inhibited by menthol both in wild-type and TRPM8KO mice. The basal sweating without acetylcholine stimulation was inhibited by an ANO1 inhibitor. Sweating could be important for maintaining friction forces in mouse foot pads, and this possibility is supported by the finding that wild-type mice climbed up a slippery slope more easily than TRPV4-deficient mice. Furthermore, TRPV4 expression was significantly higher in controls and normohidrotic skin from patients with acquired idiopathic generalized anhidrosis (AIGA) compared to anhidrotic skin from patients with AIGA. Collectively, TRPV4 is likely involved in temperature-dependent perspiration via interactions with ANO1, and TRPV4 itself or the TRPV4/ANO 1 complex would be targeted to develop agents that regulate perspiration.
