The yeast Sup35NM domain propagates as a prion in mammalian cells - PubMed (original) (raw)

The yeast Sup35NM domain propagates as a prion in mammalian cells

Carmen Krammer et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Prions are infectious, self-propagating amyloid-like protein aggregates of mammals and fungi. We have studied aggregation propensities of a yeast prion domain in cell culture to gain insights into general mechanisms of prion replication in mammalian cells. Here, we report the artificial transmission of a yeast prion across a phylogenetic kingdom. HA epitope-tagged yeast Sup35p prion domain NM was stably expressed in murine neuroblastoma cells. Although cytosolically expressed NM-HA remained soluble, addition of fibrils of bacterially produced Sup35NM to the medium efficiently induced appearance of phenotypically and biochemically distinct NM-HA aggregates that were inherited by daughter cells. Importantly, NM-HA aggregates also were infectious to recipient mammalian cells expressing soluble NM-HA and, to a lesser extent, to yeast. The fact that the yeast Sup35NM domain can propagate as a prion in neuroblastoma cells strongly argues that cellular mechanisms support prion-like inheritance in the mammalian cytosol.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Induction of heritable NM-HA aggregates by exogenous NM fibrils. (A) Schematic diagram of the experiment. A bulk population of N2a cells stably expressing HA-tagged Sup35p yeast prion domain NM was generated by lentiviral transduction (N2a_NM-HA). Cells were exposed to recombinant NM fibrils and subsequently passaged (N2a_NM-HA+F). (B) Atomic force microscopy analysis of fibrillized recombinant NM (10 μM in PBS). (Scale bar: 1 μm.) (C) Sedimentation assay of NM-HA in control cells and 48 h after aggregate induction by exogenous recombinant NM fibrils (NM-HA+F). S indicates supernatant; P, pellet. (D and E) Confocal microscopy analysis of NM-HA with (D) and without (E) exposure to recombinant NM fibrils. (Scale bar: 10 μm.) (F) N2a_NM-HA cells were exposed to NM fibril preparations and subsequently passaged 1 to 10 times. Shown are sedimentation assays of untreated (N2a_NM-HA) or fibril-treated (N2a_NM-HA+F) cells. (G) Confocal microscopy analysis 10 passages after cell exposure to fibrils. (Scale bar: 10 μm.) Antibody (C–G): anti-HA.

Fig. 2.

Fig. 2.

Distinct aggregate phenotypes in progeny cells. (A and B) Higher-resolution images of N2a_NM-HA+F cells displaying distinct aggregate phenotypes. (C) Schematic diagram of the isolation of N2a_NM-HA subclones with distinct aggregate phenotypes. (D) Higher-resolution images of subclones demonstrate inheritance of distinct aggregate phenotypes by progeny cells. Note that clones 4G and 11C displayed no visible NM-HA aggregates. Antibody: anti-HA. (Scale bars: 10 μm.) (E) Relative expression levels of NM-HA by individual clones as detected by Western blot analysis. Antibody: anti-HA. GAPDH detection serves as a loading control.

Fig. 3.

Fig. 3.

NM-HA aggregates from individual clones exhibit distinct biochemical properties. (A) Relative resistance of NM-HA aggregates to thermal denaturation in the presence of SDS from individual cell clones using SDS/PAGE. Cell lysates were mixed with 1% SDS, and aliquots were incubated at different temperatures. To ensure comparable NM-HA levels, different amounts of lysates were loaded for individual clones. (B) Band intensities of SDS-soluble proteins were plotted against temperature and fitted to a sigmoidal function. Statistical analysis was performed by using the 2-way ANOVA with Bonferroni's multiple-comparison test (P < 0.0001).

Fig. 4.

Fig. 4.

NM-HA aggregates are infectious. (A) Schematic diagram of the experiment. The bulk population of N2a_NM-HA cells was exposed to cell lysates of N2a_NM-HA+F subclones 1C and 5D displaying NM-HA aggregates or to cell lysates of bulk N2a_NM-HA cells expressing soluble NM-HA. (B) Confocal microscopy analysis of wild-type N2a (control) and N2a_NM-HA bulk cells exposed to cell extracts 1 week after exposure. Individual cells within the population are shown. Note that the aggregate phenotype was not precisely phenocopied upon induction in the recipient cells. Antibody: anti-HA. (Scale bar: 10 μm.)

Fig. 5.

Fig. 5.

Clonal influence on NM-HA aggregate phenotype. (A) Schematic diagram of the experiment. Individual clones derived from the bulk population of N2a_NM-HA cells were exposed to recombinant NM fibrils. (B) The majority of cells in a given clonal population exhibit a distinct aggregate phenotype as assessed by confocal microscopy using anti-HA antibodies. (Scale bar: 10 μm.) (C) Relative NM-HA expression levels in N2a_NM-HA cell clones before exposure to recombinant NM fibrils. The blot was probed with anti-HA antibodies, and GAPDH was used to demonstrate comparable protein loading.

Fig. 6.

Fig. 6.

NM-HA amyloids extracted from N2a cells are infectious to yeast. Cell extracts from N2a_NM-HA clones exhibiting visible aggregates (1C, 2E, 3B, 5D), and bulk N2a_NM-HA cells as a negative control were used to induce [PSI+] in the yeast strain 74-D694. A total of 2,000 transformants per inoculum were tested. Recovered colonies were streaked on 1/2 YPD plates to identify possible [PSI+] isolates by color. Such colonies were found only after transformation of cell extracts from clones 1C and 5D (0.2% or 0.05% of ≈2,000 total transformants tested, respectively). Plates with 6 transformants of clone 1C, clone 5D, or bulk cells are shown. [_psi_−] indicates negative control; [PSI+], positive control.

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