Investigation of PrPC metabolism and function in live cells : methods for studying individual cells and cell populations.
Prion protein (PrP)(C) expression levels and protein localization are known to be affected by factors such as metal ions and oxidative stress. By the development of a green fluorescent protein (GFP)-PrP(C) fusion protein, the movement of PrP can be followed in real time. Furthermore, alterations in cellular metabolism can be detected while cells are still viable. The internalization response of PrP to 20 microM manganese (Mn) in divalent metal ion-depleted media is used to demonstrate the movement of GFP-tagged proteins in live cells and real time. A live cell microtiter plate assay shows that PrP null cells are less capable of dealing with Mn-induced oxidative stress. In addition, this chapter outlines several complementary techniques for studying live cells and GFP fusion proteins.
Haigh
Cathryn L
CL
Department of Pathology and Mental Health Research Institute of Victoria, University of Melbourne, Melbourne, Australia.
Brown
David R
DR
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D018621:PrPC Proteins / Q000378:metabolism*; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D014162:Transfection
D018621:PrPC Proteins; D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins