Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites.
BACKGROUND
Spherulites are multi-lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B-subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non-toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures.


RESULTS
We demonstrate that STxB-spherulites (ST x B-functionalized spherulites) are internalized into HeLa cells in a receptor-dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane.


CONCLUSIONS
Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB-spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB-spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.
Université Bordeaux 1, Centre de Recherche Paul Pascal, av. du Docteur Schweitzer, 33600 Pessac, France. a.bouter@iecb.u-bordeaux.fr
Anthony
A
Bouter
Brigitte
B
Delord
Estelle
E
Dransart
Cécile
C
Poirier
Ludger
L
Johannes
Damien
D
van Effenterre
D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D021381:Protein Transport; D022641:Shiga Toxin 2 / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000235:genetics / Q000378:metabolism*; D014281:Trihexosylceramides / Q000235:genetics / Q000378:metabolism
D022641:Shiga Toxin 2; D014281:Trihexosylceramides; C450841:shiga toxin 2 B subunit; C018549:globotriaosylceramide