GRASP65 and GRASP55 proteins are engaged in structuring and organizing the Golgi apparatus (GA). GA has a central role in the biosynthetic pathways in urothelium. It participates in uroplakin processing and their organization into urothelial plaques, which are delivered to the apical plasma membrane of terminally differentiated urothelial cells, where they form the blood-urine permeability barrier. We investigated cellular distribution of GRASP65 and GRASP55 during urothelial regeneration after cyclophosphamide-induced injury in rats and during cell migration in SV-HUC-1 urothelial cell culture. Cyclophosphamide caused focal injuries of urothelial tissue, exposing the basal lamina, followed by temporarily hyperplastic urothelium and gradual increase of uroplakin expression. Western blot showed the presence of both GRASP65 and GRASP55. Immunohistochemical methods showed both GRASP proteins were distributed as dicreet spots diffused throughout the cytoplasm after injury, however, their distributions differed. Electron microscopy did not show any ultrastructural differences in the organelle. We documented the gradual reestablishment of fusiform vesicles and the blood-urine barrier. In mitotic cells both GRASP proteins were finely diffused throughout the cytoplasm, whereas they formed a continuous ribbon close to the nucleus in most other cells. The transmission electron microscopy of GA ultrastructure analysis showed the organelle in the shape of several cisternae which did not appear to be laterally connected to eachother. The results show that during urothelial regeneration GRASP proteins form a complex matrix, which is correlated to the changes of the apical plasmalemma of surface cells. Injury in cell culture induces cell migration to the site of injury. We also documented the localisation of GRASP proteins in SV-HUC-1 cells in interphase as well as during mitosis.
|
