De novo [PSI +] prion formation involves multiple pathways to form infectious oligomers

Abstract

Prion and other neurodegenerative diseases are associated with misfolded protein assemblies called amyloid. Research has begun to uncover common mechanisms underlying transmission of amyloids, yet how amyloids form in vivo is still unclear. Here, we take advantage of the yeast prion, [PSI +], to uncover the early steps of amyloid formation in vivo. [PSI +] is the prion form of the Sup35 protein. While [PSI +] formation is quite rare, the prion can be greatly induced by overexpression of the prion domain of the Sup35 protein. This de novo induction of [PSI +] shows the appearance of fluorescent cytoplasmic rings when the prion domain is fused with GFP. Our current work shows that de novo induction is more complex than previously thought. Using 4D live cell imaging, we observed that fluorescent structures are formed by four different pathways to yield [PSI +] cells. Biochemical analysis of de novo induced cultures indicates that newly formed SDS resistant oligomers change in size over time and lysates made from de novo induced cultures are able to convert [psi −] cells to [PSI +] cells. Taken together, our findings suggest that newly formed prion oligomers are infectious.

Actin impacts the late stages of prion formation and prion propagation

“…Sup35PrD-GFP was overexpressed to induce the formation of fluorescent aggregates, such as early foci and rings, while observing Cof1-RFP localization. Capturing early foci is difficult because of their transient nature (Sharma et al, 2017) yet our observations of the few early foci containing cells obtained did not show any clear examples of colocalization between Sup35PrD-GFP and Cof1-RFP ( Fig 1A). In ring containing cells, we did notice overlap between Sup35PrD rings and Cof1-RFP foci in 'flattened' whole cell images.…”

“…Alternatively, since we previously showed that 50% of cells containing dots are not viable (Sharma et al, 2017), the formation of dot structures in act1-120 may be more toxic than in wildtype cells.…”

“…For each wildtype and mutant BY4741 strain, at least three fresh transformants were grown in selective synthetic media supplemented with 50µM CuSO 4 for 40-46 hours. Strains were then observed under the fluorescent microscope for the presence of newly formed fluorescent aggregates as previously described (Sharma et al, 2017). Approximately 200 cells were plated on SD-Leu and 50 or 100-fold higher cell density was plated on SD-Leu-Ura to assay for those colonies that are [PSI + ].…”

“…The visualization of newly formed aggregates during the prion induction process is mediated through the fusion of a fluorescent marker to the Sup35p prion domain (Sup35PrD-GFP; Zhou et al, 2001). Overexpression of Sup35PrD-GFP leads to the appearance of small, transient "early foci" that assemble into cytosolic ring or dot aggregates (Arslan et al, 2015;Sharma et al, 2017). Micromanipulation of either ring or dot containing cells gives rise to future generations that contain [PSI + ] (Ganusova et al, 2006;Sharma et al, 2017), indicating that cells containing these newly formed aggregates are precursors to [PSI + ].…”

The three faces of Sup35

“…Once residing at the periphery, the majority of the diffuse Sup35NM quickly converges into the aggregate within 20 min. This quick sequestration coincides with the growth of the aggregate into dots, or ring-and line-like structures (Sharma et al, 2017;Zhou, Derkatch, & Liebman, 2001). Cells containing these structures are considered to be hallmarks of [PSI + ] because cells that contain aggregates give rise to [PSI + ] cells but diffuse cells do not (Ganusova et al, 2006;Sharma et al, 2017).…”