Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos.
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 beta-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.
Chugh
Archana
A
Lethbridge Research Centre, Agriculture and Agri-Food Canada, Alberta, Canada.
Eudes
François
F
D000595:Amino Acid Sequence; D000818:Animals; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004247:DNA / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D019004:Galanin / Q000235:genetics / Q000378:metabolism*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014908:Triticum / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*; D014862:Wasp Venoms / Q000235:genetics / Q000378:metabolism*
D015820:Cadherins; D015696:Gene Products, tat; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014862:Wasp Venoms; C110068:transportan; D019004:Galanin; D004247:DNA; D005966:Glucuronidase; D019793:Fluorescein