Anterograde and retrograde intracellular trafficking of fluorescent cellular prion protein.
In order to investigate the microtubule-associated intracellular trafficking of the NH2-terminal cellular prion protein (PrPC) fragment [Biochem. Biophys. Res. Commun. 313 (2004) 818], we performed a real-time imaging of fluorescent PrPC (GFP-PrPC) in living cells. Such GFP-PrPC 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 microm/s. The anterograde and retrograde movements of GFP-PrPC were blocked by a kinesin family inhibitor (AMP-PNP) and a dynein family inhibitor (vanadate), respectively. Furthermore, anti-kinesin antibody (alpha-kinesin) blocked its anterograde motility, whereas anti-dynein antibody (alpha-dynein) blocked its retrograde motility. These data suggested the kinesin family-driven anterograde and the dynein-driven retrograde movements of GFP-PrPC. Mapping of the interacting domains of PrPC 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 PrPC.
Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry, and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 187-8502, Japan.
Naomi S
NS
Hachiya
Kota
K
Watanabe
Makiko
M
Yamada
Yuji
Y
Sakasegawa
Kiyotoshi
K
Kaneko
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D018621:PrPC Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D000596:Amino Acids; D008164:Luminescent Proteins; D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins