Austausch zwischen Plasma- und Lamellärkörperchenmembran während Endo- und Exozytose

Abstract

Alveolar type II (ATII) cells produce a mixture of surface active substances, so-called surfactant, stored in lamellar bodies (LBs) and secreted into the alveolar space. Beside this, ATII cells recycle exocytosed surfactant. This requires an extensive membrane trafficking between exo- and endocytotic compartments. The aim of this study was to investigate the exchange of lipid components between plasma membrane and limiting LB membrane with life cell imaging techniques. Therefore, fusion proteins of lysosome-associated membrane glycoprotein 3 with green fluorescent protein (Lamp3-GFP) and farnesylated plasma membrane marker DsRed-Farn were developed and overexpressed in primary cultivated ATII cells using an adenoviral expression system. This allowed the specific labeling of the plasma and LB membrane in living ATII cells. Using confocal laser scanning microscopy I showed that secretagogues fascilitate the transport of plasma membrane components to the limiting LB membrane via a clathrin-independent endosomal/ lysosomal endocytotic pathway. The induction of clathrin-independent endocytosis by capsazepine and 2-ABP resulted in a comparable accumulation of DsRed-labeled plasma membrane components on the LB’s surface, with a simultaneous massive ultrastructural cell alteration. In both cases internalized plasma membrane components formed distinct domains at the LBs. Thus, the LBs are the common target organelle of internalized membrane components. In contrast, exocytotic fusion events visualized by darkfield microscopy were accompanied by diffusion of DsRed-Farn into the limiting LB membrane, resulting in a homogenous distribution of DsRed-Farn over the LB membrane. These results indicate that endo- and exocytotic processes cause a different integration of DsRed-Farn-labeled plasma membrane lipid fractions to the limiting LB membrane, reflecting different transport mechanisms (vesicular transport vs. diffusion) during endo- and exocytotic processes.