Biology subjects

Trofimenko, E.

Publications and source records attributed to Trofimenko, E..

4 recordsLinked to original sources

bioRxiv2026

The cell cycle regulates cell-penetrating peptide cytosolic uptake via differential expression of KCNH1 potassium channel

Membrane potential fluctuations, driven by ion channel activity, are important regulators of processes such as action potential propagation in neurons and muscle cell contraction. These variations also occur in non-excitable cells, influencing various cellular processes, including, but not limited to, direct translocation of cell-penetrating peptides (CPPs) into the cytosol. CPPs are cationic peptide vectors that facilitate the delivery of cargos into cells. The efficiency of CPP direct translocation varies significantly within the same cell population, a phenomenon that is not understood mechanistically. In this study, we used an unbiased transcriptomic approach to identify genes involved in efficient CPP cytosolic uptake. This revealed a close correlation between the expression of cell cycle-related genes and the ability of cells to take up CPPs by direct translocation. Monitoring phases of the cell cycle with the PIP-FUCCI system showed that CPP direct translocation was most efficient in the G2/M and S phases and least efficient during the G1 phase. This was correlated with the cells plasma membrane potential: cells in G2/M and S phases being more polarized than cells in the G1 phase. The expression of the KCNH1 voltage-gated potassium channel was found to be highest in the phases of the cell cycle most permissive for CPP direct translocation. Pharmacological inhibition of KCNH potassium channels efficiently blocked CPP direct translocation in these cell cycle phases. These data indicate that KCNH1 controls the cell membrane potential in a cell cycle-dependent manner and consequently the ability of cells to take up CPPs by direct translocation. Our findings highlight the importance of the cell cycle in regulating CPP uptake. Variations in the plasma membrane potential along the cell cycle should be considered in the development of CPP-based therapeutics and their applications.

cell biology↗

bioRxiv2022

Repeat Detector: accurate, efficient, and flexible sizing of expanded CAG/CTG repeats from targeted DNA sequencing

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and pre-clinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntingtons disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntingtons disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and the development of novel therapies.

neuroscience↗

bioRxiv2021

Identification of a new, Rab14-dependent, endo-lysosomal pathway

Cells can endocytose material from the surrounding environment. Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Several endocytosis pathways have been described (e.g. clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway). Besides possible recycling routes to the plasma membrane and various organelles, these pathways all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs) used in research and clinic, we have discovered a second endocytic pathway that moves material to late endosomes/lysosomes and that is fully independent of Rab5 and Rab7 but requires the Rab14 protein. This newly identified pathway differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already and is not affected by a series of compounds that inhibit the Rab5-dependent pathway. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins. Rab14 is expressed by the last eukaryotic common ancestor. The Rab14-dependent pathway may therefore correspond to a primordial endosomal pathway taken by cationic cargos.

cell biology↗

bioRxiv2020

Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. 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 sizes, we assessed the diameter of the water pores in living cells and found that they readily accommodated the passage of 2 nm-wide molecules, 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.

cell biology↗