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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01094

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Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin

Alain Mangé^*, Carole Crozet^*, Sylvain Lehmann and Florence Béranger

Institut de Génétique Humaine, UPR CNRS1142, 141 Rue de la Cardonille 34396 Montpellier CEDEX 5, France

View larger version (35K): [in a new window]   Fig. 1. Accumulation of proteinase K resistant PrP in the nuclei of prion-infected cells independently of proteasome inhibition. (A) N2a cells were either untreated or treated with 150 µM ALLN for 12 hours and processed for PrP immunofluorescence detection with anti-PrP antibodies. (B) N2a and ScN2a cells were either untreated or treated for 12 hours with 150 µM ALLN. Nuclei were isolated and analyzed by western blots with anti-PrP antibodies. (C) Nuclei extracts or total cell lysates were treated with proteinase K (16 µg/mg protein) for 30 minutes at 37°C. Samples were run on the same gel, but the lane corresponding to the nuclei sample was exposed longer than the total cell lysate lane. (D) Absence of contamination of the nuclei preparations with endoplasmic reticulum fractions was tested with anti-calnexin antibodies. The control lane C represents a total cellular lysate from N2a cells. (E) Nuclei extracts were untreated (–) or treated (+) with N-glycosidase F (PGNase) (0.01 units/ml) for 16 hours at 37°C before analysis by western blot with anti-PrP antibodies.

View larger version (47K): [in a new window]   Fig. 2. Nuclear localization of PrPSc in prion-infected N2a cells by immunofluorescence. N2a and ScN2a cells were fixed, permeabilized, treated with 3M guanidine thiocyanate and processed for immunofluorescence labelling with antibodies against PrP (SAF61 antibody), followed by rhodamine-conjugated anti-mouse secondary antibodies. Cells were viewed with blue excitation/emission settings to detect Hoechst staining of the nuclei (e,f,g,h) and with red excitation/emission settings to detect PrP (a,b,c,d). (A) Non-infected N2a cells (a,e) or prion-infected cells (b-d,f-h) were observed by indirect immunofluorescence on a Leica microscope. (Ab) White arrows indicate cells that have a nuclear accumulation of PrPSc. (B) Staining of ScN2a cells was analyzed by confocal laser-scanning microscopy; arrow in panels i-k: localization of PrP in the nucleus is visible in this optical section of the cells (g-i). Bars, 8 µm.

View larger version (58K): [in a new window]   Fig. 3. Disruption of the microtubule network inhibits nuclear accumulation of PrP. (A) ScN2a cells were treated overnight with increasing concentrations of colchicine. Total and PK treated cell lysates and purified nuclei were analyzed by western blot with anti PrP antibodies. (B) Isolated nuclei from ScN2a cells (a-f) or whole cells (g-l), were either non-treated (control) or treated overnight with 10 µg/ml colchicine and analyzed by immunofluorescence as described in Fig. 2. Bar, 8 µm

View larger version (35K): [in a new window]   Fig. 4. Nuclear PrP is associated with chromatin and aggregates when released from DNA. (A) Nuclease treatment of nuclei. Triton X-100-permeabilized nuclei from ScN2a cells were incubated at 37°C for 20 or 40 minutes with DNase I. After centrifugation, the supernatant (S) and the pellet (P) fractions were analyzed by immunoblotting with anti-PrP antibodies. (B) Effect of NaCl. Triton X-100-permeabilized nuclei from ScN2a cells. were incubated with the indicated concentration of NaCl and kept on ice for 15 minutes. The released or insoluble proteins following low speed centrifugation were analysed by immunoblotting with anti-PrP antibodies. (C) Effect of EDTA. Triton X-100-permeabilized nuclei from ScN2a cells were supplemented with 10 mM EDTA. After incubation at 25°C for 20 minutes, the samples were processed as in panel B.

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