Expression of transferrin receptors in endothelial cells transfected by electroporation.
Transferrin is the primary iron-binding protein in the plasma. Transferrin receptors (TfR) were detected in brain and liver endothelial cells (EC); however, little information exists about their intracellular routes. To detect the EC structures involved in TfR biosynthetic and endocytotic pathways, cultured aortic EC were transfected with the plasmid pSR alpha containing a construct encoding the human TfR, to which horseradish peroxidase (HRP) was anchored as reporter molecule. Since EC are difficult to be transfected, we tried different techniques, and two forms of the plasmid (circular and linearized), of which the electroporation method and the linearized plasmid were the most efficient in producing stable cell lines. Transfected cells were selected with geneticin, and the expression of TfR-HRP tested by cytochemistry. The stable transformants preserved the general characteristics of EC. At the ultrastructural level, TfR-HRP was associated with the nuclear envelope, rough endoplasmic reticulum, Golgi complex and adjacent secretory vesicles, cytoplasmic vesicles of various sizes (50-130 nm diameter), endosomes, plasma membrane, plasmalemmal pits, and a fraction of plasmalemmal vesicles. The intensity of the reaction product varied, suggesting a different concentration of TfR, in specific organelles. For example, (i) a gradient of HRP-reaction product was found within the Golgi cisternae, (ii) the plasmalemmal pits were more intensely stained than the adjacent plasma membrane, and (iii) the vesicle membrane was decorated stronger than the endosomal membrane (to which it fuses). A striking feature was the coexistence within the same EC of two vesicle populations (or subtypes): some containing TfR-HRP, whereas others lack the receptor. Quantitative data indicated a stronger expression of TfR in confluent cells (approximately 8-fold higher) than in EC at 2 days after plating; a significant decrease (approximately 9-fold) of TfR was found in postconfluent transfectants. Together, the data demonstrate that (i) after electroporation of EC, the stable lines maintain the characteristics of native cells; (ii) the newly synthesized TfR is located in variable concentration within the organelles involved in endocytosis and exocytosis, and (iii) the expression of TfR-HRP is particularly high in confluent cells.
Heltianu
C
C
Institute of Cellular Biology and Pathology Nicolae Simionescu, Bucharest/Romania.
Serban
G
G
Alexandru
V
V
Gafencu
A
A
Simionescu
N
N
Simionescu
M
M
D000818:Animals; D001011:Aorta / Q000166:cytology; D002130:Calcium Phosphates / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D018274:Electroporation; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002130:Calcium Phosphates; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous