Cell transduction pathways of transportans.
Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 mum diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportan-streptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportan-protein complexes is temperature dependent and strongly inhibited at 8-10 degrees C and blocked at 4 degrees C when only interaction with the plasma membrane takes place.
Padari
Kärt
K
Estonian Biocenter, 23 Riia Street, 51010 Tartu, Estonia.
Säälik
Pille
P
Hansen
Mats
M
Koppel
Kaida
K
Raid
Raivo
R
Langel
Ulo
U
Pooga
Margus
M
D002463:Cell Membrane Permeability; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004337:Drug Carriers / Q000493:pharmacokinetics; D004705:Endocytosis; D005456:Fluorescent Dyes; D019004:Galanin / Q000493:pharmacokinetics*; D006367:HeLa Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D019809:Streptavidin; D013696:Temperature; D014862:Wasp Venoms / Q000493:pharmacokinetics*
D004337:Drug Carriers; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D014862:Wasp Venoms; C110068:transportan; D019004:Galanin; D019809:Streptavidin