Microtubule-dependent intracellular trafficking of cellular prion protein

Abstract

To investigate intracellular trafficking of cellular prion protein (PrP^C), we performed a real-time imaging of fluorescent PrP^C (GFP-PrP^C) in living cells. Localization of GFP-PrP^C correlated with endogenous PrP^C, however, its localization was congregated in the cytosol after the treatment with a microtubule depolymerizer, nocodazole, suggesting that the microtubule-dependent transport of PrP^C. This microtubule-associated intracellular trafficking of PrP^C exhibited an anterograde movement towards the direction of plasma membranes at a speed of 140–180 nm/s, and a retrograde movement inwardly at a speed of 1.0–1.2 µm/s. The anterograde and retrograde movements of GFP-PrP^C were blocked by a kinesin family inhibitor (AMP-PNP) and a dynein family inhibitor (vanadate), respectively. Anti-kinesin antibody (α-kinesin) blocked its anterograde motility, whereas anti-dynein antibody (α-dynein) blocked its retrograde motility. These data showed that the kinesin family-driven anterograde and the dynein-driven retrograde movements of GFP-PrP^C. Mapping of the interacting domains of PrP^C identified amino acid residues indispensable for interactions with kinesin family: NH2-terminal mouse (Mo) residues 53–91 and dynein: NH2-terminal Mo residues 23–33, respectively. Our findings argue that the discrete N-terminal amino acid residues are indispensable for the anterograde and retrograde intracellular movements of PrP^C. Furthermore, by utilizing double-labeled fluorescent cellular PrP^C, we revealed that the NH2-terminal and COOH-terminal PrP^C fragments exhibit distinct distribution patterns in mouse neuroblastoma neuro2a (N2a) cells. The double-labeled fluorescent cellular PrP^C was successfully processed in the cell and the processing was clearly inhibited by metalloprotease inhibitors, such as EDTA, and calpain inhibitor, calpastain.