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Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos
Author(s) -
Chugh Archana,
Eudes François
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06384.x
Subject(s) - embryo , pinocytosis , fluorescence microscope , biology , peptide , microbiology and biotechnology , endocytosis , fluorescein , biochemistry , intracellular , cell , cell penetrating peptide , fluorescence , physics , quantum mechanics
The uptake of five fluorescein labeled cell‐penetrating peptides (Tat, Tat 2 , mutated‐Tat, peptide vascular endothelial‐cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell‐penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell‐penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell‐penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2‐fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated‐Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active β‐glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat 2 ‐mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat 2 ‐mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat 2 –GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell‐penetrating peptide‐cargo uptake in wheat immature embryos. We also studied Tat 2 ‐plasmid DNA (carrying Act‐1GUS ) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat 2 –plasmid DNA complex showed 3.3‐fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine™ 2000 to the Tat 2 –plasmid DNA complex resulted in 1.5‐fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell‐penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.