Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.
We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.
Institute for Arteriosclerosis Research, University of Münster, Federal Republic of Germany.
W
W
Völker
P
P
Schön
P
P
Vischer
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002450:Cell Communication; D002465:Cell Movement; D002478:Cells, Cultured; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D006046:Gold; D007211:Indoles; D008854:Microscopy, Electron; D008985:Monensin / Q000494:pharmacology; D009942:Organometallic Compounds; D010446:Peptide Fragments / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D012834:Silver; D013552:Swine; D019699:Thrombospondins
D007211:Indoles; D009942:Organometallic Compounds; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D000643:Ammonium Chloride; D012834:Silver; C015445:copper phthalocyanine; D006046:Gold; D008985:Monensin