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.
Hachiya
Naomi S
NS
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.
Watanabe
Kota
K
Yamada
Makiko
M
Sakasegawa
Yuji
Y
Kaneko
Kiyotoshi
K
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