Implications of the Actin Cytoskeleton on the Multi-Step Process of [PSI+] Prion Formation.

Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin's precise role is unclear. Here, we investigate how actin influences the cell's ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell.

The yeast molecular chaperone, Hsp104, influences transthyretin aggregate formation.

Patients with the fatal disorder Transthyretin Amyloidosis (ATTR) experience polyneuropathy through the progressive destruction of peripheral nervous tissue. In these patients, the transthyretin (TTR) protein dissociates from its functional tetrameric structure, misfolds, and aggregates into extracellular amyloid deposits that are associated with disease progression. These aggregates form large fibrillar structures as well as shorter oligomeric aggregates that are suspected to be cytotoxic. Several studies have shown that these extracellular TTR aggregates enter the cell and accumulate intracellularly, which is associated with increased proteostasis response. However, there are limited experimental models to study how proteostasis influences internalized TTR aggregates. Here, we use a humanized yeast system to recapitulate intracellular TTR aggregating protein in vivo. The yeast molecular chaperone Hsp104 is a disaggregase that has been shown to fragment amyloidogenic aggregates associated with certain yeast prions and reduce protein aggregation associated with human neurogenerative diseases. In yeast, we found that TTR forms both SDS-resistant oligomers and SDS-sensitive large molecular weight complexes. In actively dividing cultures, Hsp104 has no impact on oligomeric or large aggregate populations, yet overexpression of Hsp104 is loosely associated with an increase in overall aggregate size. Interestingly, a potentiating mutation in the middle domain of Hsp104 consistently results in an increase in overall TTR aggregate size. These data suggest a novel approach to aggregate management, where the Hsp104 variant shifts aggregate populations away from toxic oligomeric species to more inert larger aggregates. In aged cultures Hsp104 overexpression has no impact on TTR aggregation profiles suggesting that these chaperone approaches to shift aggregate populations are not effective with age, possibly due to proteostasis decline.

Cytoduction and Plasmiduction in Yeast.

Cytoduction, and a related technique referred to as plasmiduction, have facilitated substantial advancements in the field of yeast prion biology by providing a streamlined method of transferring prions from one yeast strain to another. Prions are cytoplasmic elements consisting of aggregated misfolded proteins, and as such, they exhibit non-Mendelian patterns of inheritance. While prion transfer through mating and sporulation, or through protein transformation, is possible, these approaches yield non-isogenic strains or are technically complex, respectively. Cytoduction is a mating-based technique that takes advantage of a kar1 mutation with impaired nuclear fusion (karyogamy). It is a straightforward method for introducing a prion to any yeast strain (referred to as the recipient) by mating it with a donor strain containing the prion of interest. The only absolute requirement is that one of these two strains (donor or recipient) must carry the kar1-1 mutation to limit nuclear fusion. The resulting cytoductant contains the original nucleus of the recipient strain, but a cytoplasm reflecting a mix of all elements from the donor and the recipient. Modifications to the basic cytoduction strategy provide several options for successful cytoduction, including when working with slow growing or respiratory deficient strains. A significant advantage of the plasmiduction protocol presented is the ability to transfer a plasmid encoding a fluorescently tagged version of the prion protein, which allows for the direct verification of the prion state through visual protein aggregates. Graphic abstract: Transfer of Yeast Cytoplasmic Elements such as Prions using Cytoduction.

The actin cytoskeletal network plays a role in yeast prion transmission and contributes to prion stability.

Chaperone networks are required for the shearing and generation of transmissible propagons from pre-existing prion aggregates. However, other cellular networks needed for maintaining yeast prions are largely uncharacterized. Here, we establish a novel role for actin networks in prion maintenance. The [PIN+ ] prion, also known as [RNQ+ ], exists as stable variants dependent upon the chaperone machinery for the transmission of propagons to daughter cells during cell division and cytoplasmic transfer. Loss of the Hsp104 molecular chaperone leads to the growth of prion particles until they are too large to be transmitted. Here, we isolated a unique [PIN+ ] variant, which is unstable in actin mutants. This prion loss is observed over many generations, and coincides with the detection of both high molecular weight species of Rnq1 and large visible aggregates that are asymmetrically retained during cell division. Our data suggest that the irregular actin networks found in these mutants may influence propagon number by slowly permitting aggregate growth over time, resulting in the generation of nontransmissible large aggregates. Thus, we show the potential contribution of cytoskeletal networks in the transmission of prion propagons, which parallels models that have been proposed for cell-to-cell transmission of small amyloids in neurodegenerative protein aggregation diseases.

DMSO-mediated curing of several yeast prion variants involves Hsp104 expression and protein solubilization, and is decreased in several autophagy related gene (atg) mutants.

Chaperones and autophagy are components of the protein quality control system that contribute to the management of proteins that are misfolded and aggregated. Here, we use yeast prions, which are self-perpetuating aggregating proteins, as a means to understand how these protein quality control systems influence aggregate loss. Chaperones, such as Hsp104, fragment prion aggregates to generate more prion seeds for propagation. While much is known about the role of chaperones, little is known about how other quality control systems contribute to prion propagation. We show that the aprotic solvent dimethyl sulfoxide (DMSO) cures a range of [PSI+] prion variants, which are related to several misfolded aggregated conformations of the Sup35 protein. Our studies show that DMSO-mediated curing is quicker and more efficient than guanidine hydrochloride, a prion curing agent that inactivates the Hsp104 chaperone. Instead, DMSO appears to induce Hsp104 expression. Using the yTRAP system, a recently developed transcriptional reporting system for tracking protein solubility, we found that DMSO also rapidly induces the accumulation of soluble Sup35 protein, suggesting a potential link between Hsp104 expression and disassembly of Sup35 from the prion aggregate. However, DMSO-mediated curing appears to also be associated with other quality control systems. While the induction of autophagy alone does not lead to curing, we found that DMSO-mediated curing is dramatically impaired in autophagy related (atg) gene mutants, suggesting that other factors influence this DMSO mechanism of curing. Our data suggest that DMSO-mediated curing is not simply dependent upon Hsp104 overexpression alone, but may further depend upon other aspects of proteostasis.

The three faces of Sup35.

Sup35p is an essential protein in yeast that functions in complex with Sup45p for efficient translation termination. Although some may argue that this function is the only important attribute of Sup35p, there are two additional known facets of Sup35p's biology that may provide equally important functions for yeast; both of which involve various strategies for coping with stress. Recently, the N-terminal and middle regions (NM) of Sup35p, which are not required for translation termination function, have been found to provide stress-sensing abilities and facilitate the phase separation of Sup35p into biomolecular condensates in response to transient stress. Interestingly, the same NM domain is also required for Sup35p to misfold and enter into aggregates associated with the [PSI+ ] prion. Here, we review these three different states or "faces" of Sup35p. For each, we compare the functionality and necessity of different Sup35p domains, including the role these domains play in facilitating interactions with important protein partners, and discuss the potential ramifications that each state affords yeast cells under varying environmental conditions.

Toxicity and infectivity: insights from de novo prion formation.

Prions are infectious misfolded proteins that assemble into oligomers and large aggregates, and are associated with neurodegeneration. It is believed that the oligomers contribute to cytotoxicity, although genetic and environmental factors have also been shown to have additional roles. The study of the yeast prion [PSI +] has provided valuable insights into how prions form and why they are toxic. Our recent work suggests that SDS-resistant oligomers arise and remodel early during the prion formation process, and lysates containing these newly formed oligomers are infectious. Previous work shows that toxicity is associated with prion formation and this toxicity is exacerbated by deletion of the VPS5 gene. Here, we show that newly made oligomer formation and infectivity of vps5∆ lysates are similar to wild-type strains. However using green fluorescent protein fusions, we observe that the assembly of fluorescent cytoplasmic aggregates during prion formation is different in vps5∆ strains. Instead of large immobile aggregates, vps5∆ strains have an additional population of small mobile foci. We speculate that changes in the cellular milieu in vps5∆ strains may reduce the cell's ability to efficiently recruit and sequester newly formed prion particles into central deposition sites, resulting in toxicity.

Spatial sequestration and oligomer remodeling during de novo [PSI+] formation.

Prions are misfolded, aggregated, infectious proteins found in a range of organisms from mammals to bacteria. In mammals, prion formation is difficult to study because misfolding and aggregation take place prior to symptom presentation. The study of the yeast prion [PSI+], which is the misfolded infectious form of Sup35p, provides a tractable system to monitor prion formation in real time. Recently, we showed that the de novo formation of prion aggregates begins with the appearance of highly mobile cytoplasmic foci, called early foci, which assemble into larger ring or dot structures. We also observed SDS-resistant oligomers during formation, and lysates containing newly formed oligomers can convert [psi-] cells to the [PSI+] state, suggesting that these oligomers have infectious potential. Here, we further characterize two aspects of prion formation: spatial sequestration of early foci and oligomerization of endogenous Sup35p. Our data provides important insights into the process of prion formation and explores the minimal oligomer requirement for infectivity.

A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

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

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.

Writing Assignments with a Metacognitive Component Enhance Learning in a Large Introductory Biology Course.

Writing assignments, including note taking and written recall, should enhance retention of knowledge, whereas analytical writing tasks with metacognitive aspects should enhance higher-order thinking. In this study, we assessed how certain writing-intensive "interventions," such as written exam corrections and peer-reviewed writing assignments using Calibrated Peer Review and including a metacognitive component, improve student learning. We designed and tested the possible benefits of these approaches using control and experimental variables across and between our three-section introductory biology course. Based on assessment, students who corrected exam questions showed significant improvement on postexam assessment compared with their nonparticipating peers. Differences were also observed between students participating in written and discussion-based exercises. Students with low ACT scores benefited equally from written and discussion-based exam corrections, whereas students with midrange to high ACT scores benefited more from written than discussion-based exam corrections. Students scored higher on topics learned via peer-reviewed writing assignments relative to learning in an active classroom discussion or traditional lecture. However, students with low ACT scores (17-23) did not show the same benefit from peer-reviewed written essays as the other students. These changes offer significant student learning benefits with minimal additional effort by the instructors.

Prion formation and polyglutamine aggregation are controlled by two classes of genes.

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN+]. When fused to GFP and overexpressed in [ps⁻] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI+] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI+]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI+] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI+] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.

Most, but not all, yeast strains in the deletion library contain the [PIN(+)] prion.

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non-Mendelian element, such as a prion, within this library could affect the outcome of many large-scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN(+)] prion. [PIN(+)] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1-GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN(+)]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN(+)], 139 deletions were able to efficiently maintain three different [PIN(+)] variants despite extensive growth and storage at 4 degrees C. One strain, cue2Delta, displayed an alteration in the RNQ1-GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild-type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN(+)], indicating that 5153 non-essential genes are not required for [PIN(+)] propagation.

An engineered nonsense URA3 allele provides a versatile system to detect the presence, absence and appearance of the [PSI+] prion in Saccharomyces cerevisiae.

Common methods to identify yeast cells containing the prion form of the Sup35 translation termination factor, [PSI+], involve a nonsense suppressor phenotype. Decreased function of Sup35p in [PSI+] cells leads to read-through of certain nonsense mutations in a few auxotrophic markers, e.g. ade1-14. This read-through results in growth on adenine-deficient media. While this powerful tool has dramatically facilitated the study of [PSI+], it is limited to a narrow range of laboratory strains and cannot easily be used to screen for cells that have lost the [PSI+] prion. Therefore we have engineered a nonsense mutation in the widely used URA3 gene, termed the ura3-14 allele. Introduction of the ura3-14 allele into an array of genetic backgrounds, carrying a loss-of-function URA3 mutation and [PSI+], allows for growth on media lacking uracil, indicative of decreased translational termination efficiency. This ura3-14 allele is able to distinguish various forms of the [PSI+] prion, called variants, and is able to detect the de novo appearance of [PSI+] in strains carrying the prion form of Rnq1p, [PIN+]. Furthermore, 5-fluoroorotic acid, which kills cells making functional Ura3p, provides a means to select for [psi-] derivatives in a population of [PSI+] cells marked with the ura3-14 allele, making this system much more versatile than previous methods.