Tau-like Proteins Associated with Centrosomes in Cultured Cells

Abstract

The subcellular association of tau-like proteins with centrosomes in cultured cell lines and its effects in nucleating microtubule assembly were analyzed using biochemical and immunocytochemical approaches. Tau proteins, major components of microtubules, appear to be tightly associated with actin filaments in a variety of cell lines, while in pathological conditions of neurons, they are part of paired helical filaments found in Alzheimer's disease. Different studies suggest that, in addition to tau interactions with the components of the cytoskeletal network, tau polypeptides appear to be associated with highly structured cellular elements, in both interphase and mitotic cells. An in-depth analysis of tau subcellular distribution us- ing different polyclonal and monoclonal antibodies showed colocalization of tau-like components with centrosomes in interphase cells of the human Huh-7 hepatoma, in SW-13 adenocarcinoma, and in normal human fibroblasts. Tau associated with centrosomes in mitotic Huh-7 cells was also identified. However, antibodies against the tau binding repeats did not stain centrosomes. A set of different tau isoforms was also identified by Western blot analysis on isolated centrosomal preparations from Huh-7 cells, obtained by differential centrifugation through sucrose gradients. Microtubule nucleationin vitroover isolated centrosomes was inhibited by both the polyclonal antibody against native tau and an antibody to the N-terminal tau sequence, as revealed by immunofluorescence analysis and assembly kinetics experiments. The antibody TRS1.2 against the fragment containing the first binding repeat on tau did not affect nucleation. These studies allowed us to characterize tau association with the isolated centrosomal preparation and its involvement in microtubule assembly nucleated over centrosomes, thus suggesting possible structural and functional roles for these interactions.