Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking.
Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we have re-examined here the kinetics of ON uptake, subcellular distribution and intracellular localisation in these cells, at concentrations relevant for the study of a receptor-dependent process. Kinetic parameters of ON endocytosis were comparable with those of the receptor-mediated endocytosis tracer, transferrin (uptake equilibrium, saturation with concentration, specific competition and rapid efflux) and were clearly distinct from those of fluid-phase endocytosis. By analytical subcellular fractionation, particulate ON showed a bimodal distribution after 2 h of uptake, with a low-density peak superimposed on the distribution of endosomes, and a high-density peak overlapping lysosomes. After an overnight chase, only the high-density peak remained, but it could be dissociated from lysosomes, based on its refractoriness to displacement upon chloroquine-induced swelling. After 2 h of uptake at 300 nM ON-Alexa, a punctate pattern was resolved, by confocal microscopy, from those of transferrin, of a fluid-phase tracer, and of vital staining of lysosomes by LysoTracker. At 3 microM ON-Alexa, its pattern largely overlapped with the fluid-phase tracer and LysoTracker. Taken together, these data suggest that ON may be internalised at low concentrations by receptor-mediated endocytosis into unique endosomes, then to dense structures that are distinct from lysosomes. The nature of these two compartments and their significance for ON effect deserve further investigation.
de Diesbach
Philippe
P
Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200 Brussels, Belgium.
N'Kuli
Francisca
F
Berens
Catherine
C
Sonveaux
Etienne
E
Monsigny
Michel
M
Roche
Annie-Claude
AC
Courtoy
Pierre J
PJ
D001692:Biological Transport; D005591:Chemical Fractionation; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D009841:Oligonucleotides / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000502:physiology*; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured / Q000378:metabolism
D007457:Iodine Radioisotopes; D009841:Oligonucleotides; D011956:Receptors, Cell Surface