Neue korrelative mikroskopische Methoden zur ultrastrukturellen Lokalisation von green fluorescent protein und Proteinkinase D2 in kultivierten humanen Pankreas-Karzinoidzellen

Abstract

The goal of this study is to combine the localisation of green fluorescent protein (GFP) and protein kinase D 2 (PKD2) with good ultrastructural preservation. This should be achieved by high-pressure freezing fixation of cultivated adherent pancreatic carcinoid cells (BON). In this study, PKD2 was overexpressed by transfection of BON cells using GFP as a marker. For the first part of the study we applied pmaxGFP- and PKD2-vector-DNA for the nucleofection, for a second set of experiments we used pEGFP-tagged PKD2-vector-DNA. The cells were grown as a monolayer on special sapphire discs that fit into aluminium platelets used for high-pressure freezing in a Compact 01. Afterwards the samples were freeze-substituted in acetone containing 5% of water and uranyl acetate. All samples were substituted without osmium and embedded in LR-Gold. Ultrathin sections were cut and collected on formvar-coated copper grids. The same grids were analysed in the fluorescent light microscope (LM) and in the transmission electron microscope (TEM). Ultrathin sections of pmaxGFP-DNA transfected cells clearly show a fluorescence signal. The same ultrathin section was then analyzed in the TEM. The correlation of LM and TEM provides an accurate ultrastructural localisation of the GFP signal. With the help of this method six anti-GFP antibodies were tested on the sections. On thin sections of pEGFP tagged PKD2 expressing cells, unfortunatelly, the GFP-fluorescence signal was too weak. Therefore we did GFP immunolabelling with a immunogold antibody and a Cy3 fluorescence antibody. The sections were analysed in the fluorescence microscope, and in the TEM. We detected areas with a high pEGFP tagged PKD2 expression rate. Afterwards we made a morphometry of the Golgi-apparatus of different pEGFP tagged PKD2 transfected cells. We found no differences between the Golgis. Three dimensional studies of the Golgi were performed by electron tomography in a 300 kV scanning transmission microscope.