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Updated: Feb 11, 2026

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking
Published on: February 12, 2022
Cellular uptake mechanisms and endosomal trafficking of supercharged proteins
David B Thompson1, Roberto Villaseñor, Brent M Dorr
1Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Abstract:
Supercharged proteins (SCPs) can deliver functional macromolecules into the cytoplasm of mammalian cells more potently than unstructured cationic peptides. Thus far, neither the structural features of SCPs that determine their delivery effectiveness nor their intracellular fate postendocytosis, has been studied. Using a large set of supercharged GFP (scGFP) variants, we found that the level of cellular uptake is sigmoidally related to net charge and that scGFPs enter cells through multiple pathways, including clathrin-dependent endocytosis and macropinocytosis. SCPs activate Rho and ERK1/2 and also alter the endocytosis of transferrin and EGF. Finally, we discovered that the intracellular trafficking of endosomes containing scGFPs is altered in a manner that correlates with protein delivery potency. Collectively, our findings establish basic structure-activity relationships of SCPs and implicate the modulation of endosomal trafficking as a determinant of macromolecule delivery efficiency.
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