Vesicular uptake of macromolecules by human placental amniotic epithelial cells

Abstract

IntroductionStudies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy. MethodsPlacental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%–50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1–30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris® software. ResultsVesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions. ConclusionsIn human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism.