Role of prion-like proteins in cell fate and memory

All cells establish an intracellular organization that fits their functions. They also have to respond quickly to external signals or change their architecture during cell migration for example. Recently, a novel level of dynamic cellular organization has emerged, the aggregation of proteins into granules. Aggregation supports the formation of membrane-less organelles involved in diverse functions including the protection of cellular factors upon unfavourable conditions (stress granules), the segregation of cell fate determinants (P-granules), the formation of prions and the storage of memory. Aggregation is driven by protein domains that confer specific properties to these assemblies. Some behave as liquids, other as gels and even solid structures. In most cases, these domains engage the protein in a self-templating conformational change, resulting in a specific unmixing of the granules from the rest of the cytoplasm. An important feature is that when aggregation is nucleated, it has an en masse effect, allowing cells to integrate environmental and intra-cellular cues to make switch-like decisions.
Protein aggregation was long associated with protein misfolding and pathological or even pathogenic structures, such as Creutzfledt-Jakob disease (prion) and neurodegenerative diseases like Alzheimer or Parkinson diseases. However, we now have evidences that a number of physiological cellular processes are controlled by aggregation in all kingdoms of life, suggesting that aggregation is a very fundamental molecular mechanism. Since there are currently no cures for prion or neurodegenerative diseases and since we are in an increasingly ageing society, we need to understand the molecular mechanisms of protein aggregation, what distinguishes the different granules and what transforms physiological assemblies in pathological aggregation.
We have recently discovered a new type of aggregation in the budding yeast Saccharomyces cerevisiae. Yeast cells can contain proteins that are in a prion form. The infectious property of prions lends them the ability to disseminate to daughter cells during cell division and to be clonally stable. Many yeast proteins contain prion-like domains and in fact most if not all organisms have prion-like domain containing proteins, including humans. We discovered that some of these proteins could adopt another state that we termed mnemon. Mnemons, like prions, adopt a new shape. In the case of the protein Whi3, this mnemon state promotes the formation and the maintenance of a cellular memory. However, only the cells that have learned contain the mnemon. In other words, unlike a prion, a mnemon is confined by the cell that established this state. As all other aggregation mechanisms, Whi3 conversion to the mnemon form is regulated by molecular chaperones. Interestingly, Whi3 also forms terminal inclusions during cellular ageing that resemble inclusion bodies found in many human pathologies. Additionally, we recently found that other new mnemons seem to work alongside Whi3 to promote memory formation. Thus, we have discovered a unique system in which we can compare and identify the molecular mechanisms of protein aggregation. We will exploit this system to understand the functions of these new mnemons and if mnemons aggregate as a network or work independently. Since replicative ageing in yeast is easily accessed, we will determine if all mnemons aggregate during ageing and identify the molecular causes for these pathological-like states. We propose to translate our research to zebrafish to understand the biology of mnemons in vertebrates.
Our research takes the advantage of using a genetically powerful organism to understand the physiology as well as the pathology of prion-like domain containing proteins. We aim to translate our results to other organisms in the future to help understanding the biology behind prion and neurodegenerative diseases.

Protein aggregation has emerged as a dynamic regulation of cellular functions. Until recently, the field was mainly driven by the idea that aggregates represent pathological conditions involved in neurodegenerative or prion diseases for example. However, a wealth of new data including ours has shown that protein aggregation is relevant for physiological functions such as metabolism rewiring, memory of single cells adaptations and neuronal memory. Thus, protein aggregation is a fundamental biological process that needs to be understood.
We have discovered a novel molecular mechanism for the asymmetric segregation of a cell fate determinant during cell division. Budding yeast cells exposed to mating pheromone arrest in the next G1 phase of the cell cycle to search for a mating partner. In the absence of a mating partner, cells can learn to ignore pheromone and re-enter the cell cycle. However, daughter cells of these adapted cells are born naïve and respond to pheromone. We found that the mRNA binding protein Whi3 forms protein aggregates through a conformational change during pheromone response that releases the break it normally imposes on the G1-S phase transition. Comparably to prions, Whi3 fold change is self-templating. However, confinement of these self-templating aggregates to the mother cell defines them as mnemons.
We have discovered new mnemons contributing to the memorization of deceptive mating attempts, including Akl1 (Ark kinase), Cbk1 (LATS1 kinase), Ssd1 (mRNA binding protein) and Ste18 (subunit of heterotrimeric G protein). Furthermore, we discovered that Whi3 also aggregates erratically during ageing. By using a combination of genetics and 3D time lapse microscopy, we aim to 1) understand the functions and regulations of these new mnemons; 2) ask if they aggregate and function as a network; 3) test if all mnemons aggregate during ageing and identify the causes of age-induced aggregation; 4) start translating this research to a vertebrate model.

Impact Summary - Fabrice Caudron - Role of Prion-like Proteins in Cell Fate and Memory

Academic Impact
The first academic impact of our work will be the discovery of new mnemons, how they interact with each other and their behaviour during ageing. A major following impact will be the translation of our research in yeast to a vertebrate model, zebrafish. Prion-like proteins are present in all organisms tested so far, yet their biology is only beginning to emerge. Moreover, many of these proteins are linked to age associated diseases such as neurodegenerative diseases. Our work will be of interest to a large community of researchers and is in the priority area of healthy ageing across the lifecourse.

Researcher Career Development
A post-doctoral research associate and a 40% technician will learn and apply all the modern techniques used in budding yeast research, including molecular cloning now often done with gene synthesis, CRISPR/Cas9 directed and seamless mutagenesis as well a state-of-the-art long-term fluorescence microscopy combined with custom microfluidic chips. They will be well prepared for an excellent future career.

Impact on Drug Development
We use our strength in the understanding of cellular aging and protein aggregation in yeast to test the molecular mechanisms underlying the effect of very potent anti-aggregations compounds discovered by our industrial collaborator SunRegen HealthCare AG. We hope to use the ease and rapidity of research in yeast to guide the understanding of the mode of action of drugs that are aimed at being on the market in the next decade. Using budding yeast for this research falls into the BBSRC priority of the replacement, refinement and reduction in research using animals.

Outreach to the public
We will seek to increase public understanding of our research through dissemination of our results and direct interaction with children.