Study Of Real Time Visulaization Of Prion Protein In Living Cells By Quantum Dots

Prion disease including Kuru syndrome, Gerstmann-Straussler-Scjeinker (GSS) and Creutzfeldt-Jakob disease (CJD) can be sporadic, inherited or acquired by transmission. The "infectious" factor prion is a protein only virus. The cellular prion protein (PrPC) is a harmless glycosylinositol phospholipids (GPI) anchored glycoprotein expressed in neuronal and other cells. The C-terminal domain of PrPc has three a-helices and a short section of antiparallelβ-sheet and it folds to a stable structure in vitro quickly. The N-terminal domain of PrPC, however, is His-rich and flexibly disordered in the full-length molecule, which can binds to Cu2+and Ni2+. The structural alteration of the PrPC from a normal a-helix rich form into a pathogenic and protease-resistant P-sheet rich isoform PrPSc (pathogenic scrapie prion protein) is tightly linked to transmissible spongiform encephalopathies (TSE). The actual conversion process of the prion, however, is not fully understood although there are evidences suggesting it may occur at the plasma membrane. Some reports showed that PrPC targets to the outer leaflet of the plasma membrane by the GPI anchor, or during the internalization pathways, as both clathrin-coated pits and caveolae-dependent structures have been found to be involved in the process. A direct and real-time visualization of PrPC transport in a living cell may help to understand how the entire process occurred and its relationship with prion associated diseases.The traditional way of visualizing and tracking of a protein is dependent on techniques of immunoprecipitation or fusing the target protein with a fluorescent protein. But to achieve single molecule sensitivity and tracking, quantum dots (QDs) have generated considerable interest in the biological community because their unique optical and electronic properties such as tunable emission from visible to infrared wavelengths by changing the size and composition, broader excitation spectra, strong brightness, photostability, and high resistance to photobleaching compare with that of the dye molecules. With these unique features, QDs are becoming suitable tools for both in vitro and in vivo imaging. Modified QDs may be conjugated with biomolecules through methods such as covalent attachment, electrostatic attraction, and thiol-exchange reaction. To conjugate QDs with prion, the virus made of single protein only, as a fluorescent detector with minimum influence to its suructure, we use Ni2+as a mediate, to conjugate QDs with the prion protein by the original His-rich region in its N-terminal.We confirmated QDs-Ni2+can be conjugated with PrP23-231 in vitro using agarose gel electrophoresis and ultrafiltration. Low concentration (1×10-8 M) and short time incubation (15 min) with QDs-Ni2+revealed PrP distinctly in western blot experiments. Then the atom force microscopy (AFM) and dynamic light scattering (DLS) show the conjugation of QDs with PrP directly from the particle size alteration. Then we tested the influence of QDs conjugation to the aggregation behaviors in pH condition by turbidity assay, and Circular Dichroism (CD) showed there is no alteration in PrP's secondary structure after QDs conjugation.The biotoxins of QDs are tested by MTT assay, shown while the QDs concentration is lower than 7.5×10-9 M, there is imperceptible influence to cells in 24h even the QDs were modified by less PEG. Using GFP fused PrP (SP-GFP-PrP) expressing in human neuron cells, we found QDs signal is co-localization with the GFP signal, with perfect specificity. We have demonstrated, for the first time, the actual process of PrPc transport from location on plasma membrane to translocation to the perinuclear region, in single living cell with a 12 min movie, and separated the process into four individual stages by the velocity changes. Furthermore, we showed that the critical role of lipid rafts in PrPc membrane localization and internalization.Finally, we established a cell line stably expressing PrP fused with RFP tag, for the further researchs on prion infection.