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 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.

Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2’s interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2’s ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events.

TAT-RasGAP317–326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317–326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317–326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317–326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317–326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

30Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. CPPs 31 provide efficient methodology to transfer bioactive molecules in cells, in particular in 32 conditions when transcription or translation of cargo-encoding sequences is not 33 desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined 34 and controversial. This work identifies potassium channels as key regulators of cationic 35 CPP translocation. Using a CRISPR/Cas9-based screening, we discovered that 36 KCNQ5, KCNN4, and KCNK5 positively modulate CPP cellular direct translocation by 37 reducing transmembrane potential (Vm). Cationic CPPs further decrease the Vm to 38 megapolarization values (about -150 mV) leading to the formation of ~2 nm-wide water 39 pores used by CPPs to access the cell's cytoplasm. Pharmacological manipulation to 40 lower transmembrane potential boosted CPPs cellular uptake in zebrafish and mouse 41 models. Besides identifying the first genes that regulate CPP translocation, this work 42 characterizes key mechanistic steps used by CPPs to cross cellular membrane. This 43 opens the ground for pharmacological strategies augmenting the susceptibility of cells 44 to capture CPP-linked cargos in vitro and in vivo. 45 46Cell-penetrating peptides (CPPs) are non-toxic molecules of 5-30 amino acids that can 47 translocate into living cells. CPPs can be hooked to a variety of cargos (siRNAs, DNA, 48 polypeptides, liposomes, nanoparticles, etc.) to allow their transport into cells for 49 therapeutic or experimental purposes (1-10). The origin of CPPs is diverse. For 50 example, TAT48-57 is a 10 amino-acid fragment derived from the trans-activator of 51 transcription (TAT) HIV-1 protein (11, 12), penetratin is a 16 amino-acid peptide 52 derived from the Antennapedia Drosophila melanogaster protein (13), and MAP (model 53 amphipatic peptide) is a synthetic alanine/leucine/lysine-rich peptide (14). The vast 54 majority of CPPs are cationic (1, 3, 6, 7). 55How CPPs enter cells is debated and not fully characterized at the molecular level 56 (reviewed in (1-8)). Due to this knowledge gap, it is difficult to ameliorate CPP cellular 57 entry and this slows down development of CPP-based interventions. Two main modes 58 of CPP entry have been described: endocytosis and direct translocation(1-8). 59Endocytosed CPPs gain cytosolic access by escaping endosomes. Direct 60 translocation has been proposed to occur through transient pore formation or 61 membrane destabilization. Endocytosis and direct translocation are not mutually 62 exclusive. Several entry routes can be followed simultaneously by a given CPP in a 63 given cell line (9, 10). 64Here, we used CRISPR/Cas9-screenings to identify proteins required for the cellular 65 uptake of CPPs. This approach identified three potassium channels as mandatory for 66 the direct translocation of CPPs into various cell types. Further, we highlighted the 67 requirement of an appropriate membrane potential to generate a 2 nm-wide water 68 pores through which...