Aspects of prion protein dynamics in cell culture models
The cell biology of Prion formation and transfer is not well understood. In order to further elucidate the dynamics of PrPc and PrPsc in a cellular context, fusions between Green Fluorescent Protein (GFP) and PrP were constructed and infected/uninfected cell line pairs expressing these constructs were created. Biochemical analysis indicated that the C-terminal PrPc portion of the fusion protein successfully converted to PrPsc. However, further studies demonstrated that proteolysis occurs between GFP and PrP and therefore the fusion protein cannot be employed as a direct reporter for PrPsc. A Time-Lapse microscopy system was set up and studies were undertaken with infected and uninfected cell lines expressing the fusion construct or cytoplasmic markers to observe events that may be related to transfer of infectivity. Although no exchange of fusion protein is observed, cytoplasmic material is released from both infected and uninfected cell lines. Fluorescence recovery after photobleaching (FRAP) was carried out to establish a system for further investigation of PrP dynamics in the plane of the membrane. Early experiments indicate the possibility of a difference in the diffusion of PrP between infected and uninfected contexts. It is not currently known how Prion glycoform profile is transmitted and maintained following a new infection. The glycoform profile of PrPscwas perturbed in order to investigate the causal role of PrPsc glycotypes in transmission and maintanence of Prion glycoform profile. The results indicate that perturbation of PrPsc glycoform profile in an infectious source does not lead to a correlated perturbation of glycoform profile in the newly established infection. Therefore the glycosylation of PrPsc in an infectious source is not a required source of information for establishing the glycoform profile of a Prion infection.