Quantification of volume and lipid filling of intracellular vesicles carrying the ABCA3 transporter

Publication date: Available online 6 September 2017 Source:Biochimica et Biophysica Acta (BBA) - Molecular Cell Research Author(s): Stefanie Höppner, Susanna Kinting, Adriano A. Torrano, Ulrike Schindlbeck, Christoph Bräuchle, Ralf Zarbock, Thomas Wittman, Matthias Griese The ABCA3 lipid transporter is located in the limiting membrane of lamellar bodies (LBs) in type-II-pneumocytes. Mutations within the ABCA3 gene may functionally impair the transporter, causing lung diseases in newborns, children and adults. Assays to quantify volume and lipid filling of the LBs on the level of the vesicular structures and thereby assess the function of ABCA3 are still lacking. In the present study human influenza haemagglutinin- (HA-) tagged wild type and mutant ABCA3 proteins were stably expressed in lung A549 cells. Fluorescently-labelled TopFluor phosphatidylcholine (TopF-PC) incorporated in surfactant-like liposomes was delivered to the cells and visualized by confocal microscopy. Subsequently, a comprehensive image analysis method was applied to quantify volume and fluorescence intensity of TopF-PC in ABCA3-HA-positive vesicles. TopF-PC accumulated within the vesicles in a time and concentration-dependent manner, whereas the volume remained unchanged, suggesting active transport into preformed ABCA3 containing vesicles. Furthermore, this finding was supported by a decrease of the fluorescence intensity within the vesicles when either the ATPase of the transporter was inhibi...
Source: Biochimica et Biophysica Acta (BBA) Molecular Cell Research - Category: Molecular Biology Source Type: research