Studio morfologico e funzionale dell’apparato di Golgi in relazione ad una struttura LTL-positiva nelle cellule di carcinoma prostatico DU145

The Golgi apparatus is a complex structure present in all eukaryotic cells. This organelle, which was first observed in 1989, still represents a fascinating enigma for much of its structural and functional peculiarity. Generally, the Golgi apparatus is known as the heart of the secretory pathway and glycosylation, which is one of the major post-traductional modifications. Most of the reactions of the glycosylation pathway occur in the Golgi apparatus, where glycosyltransferases and glycosidases modify glucidic chains by adding or removing monosaccharides. All the steps follow a precise sequential order from the cis-Golgi to the trans-Golgi network, depending on the exclusive presence of peculiar enzymes in each Golgi compartment. The sequential order of all these reactions is guaranteed by the morphological stability of the Golgi apparatus, which appears as many staked cisternae fused together forming a unique ribbon structure in vertebrates’ cells. The integrity of the Golgi ribbon is the result of a perfect harmonization between Golgi matrix proteins and the cytoskeleton. Defect in glycosylation has been observed in many pathologies, such as in neurodegenerative disease and malignant transformation of cancer cells. Particularly, the Golgi apparatus of cancer cells loses its typical ribbon shape and splits in smaller vesicles scattered in the cytosol. This thesis focuses on the defects of glycosylation in cancer cells, with a particular regard to fucosylation, since we observed a novel Golgi-derived α 1,2 fucosylated tubular structure exclusive of high proliferative cells. This structure, which was highlighted thanks to the α 1,2 fucose binding Lotus tetragonolobus lectin (LTL), seems to be responsible for a peculiar uptake system that lets many molecules, including LTL itself, enter the cell. To better understand 1) the relation between the morphological scattering of the cisternae and the functionality of the Golgi apparatus and 2) the link between the Golgi disarrangement and the origin of the LTL-positive tubular system, we analysed cells from the human prostatic tumor cell line DU145 by confocal microscopy using the lectins LTL and AAA (Aleuria aurantia Agglutinin), both specific for fucose, and different antibodies able to mark the organelle. Microscopic observations were parallelly performed with SDS-PAGE on DU145 extracts to analyze the localization of the Golgi proteins and glycans in the nuclear, cytoplasmic, membrane, and cytoskeleton fractions obtained using the Qproteome Cell Compartment Kit. Furthermore, in order to evaluate the relationship between the Golgi apparatus/tubular system and the actin cytoskeleton, DU145 were left to grow in presence of Cytochalasin D, a fungal toxin capable to depolymerize the microfilaments. Finally, we performed an uptake experiment to test the functionality of the tubular system, both in the presence and absence of cytochalasin.