Manipulation of Asymmetric Protein Aggregate Inheritance in Budding Yeast

Abstract

One hallmark of cellular ageing is the continuous accumulation of protein aggregates due to the gradual inability of cells to degrade misfolded proteins. Disruptions to the normal process of protein folding can occur for example due to genetic mutations, and external factors such as temperature change or environmental toxins. As cells age, the inability to clear misfolded proteins can lead to the formation of protein aggregate inclusions which interfere with cell homeostasis and can manifest as neurological diseases (e.g., Alzheimer’s, Parkinson’s and Huntington’s diseases) commonly seen in the ageing human population. In the budding yeast Saccharomyces cerevisiae, protein aggregate inclusions are typically retained in the mother cell during asymmetric cell division, contributing to its ageing, while rejuvenating the daughter cell at the start of its lifespan. Using fluorescence microscopy, this work quantitatively explores the efficiency of an artificial protein aggregate transport system (ATS) engineered to instead transport inclusions to the daughter cells of S. cerevisiae during asymmetric cell division. The ATS was mainly challenged with two toxic variants of the aggregate-forming N-terminal domain of human Huntingtin (Htt), which were fluorescently tagged to visualise and model inclusion formation in S. cerevisiae. This work also explores whether the ATS affects cell death, monitored using propidium iodide staining (fluorescence), and by the analysis of the growth curves of S. cerevisiae cells expressing the ATS in combination with toxic Huntingtin variants. In general, the integration of the ATS in S. cerevisiae cells bypasses the retention of Htt inclusions in the mother cell and was observed to effectively transport one inclusion to the daughter cell per asymmetric cell division. At times, inclusions were also observed in both mother and daughter cells midway through cell division which indicates the possibility of incomplete inclusion transport. In most cases, ATS integration also improved cell density at the stationary phase in both control and toxic aggregate producing S. cerevisiae cells. Interestingly, the integration of the ATS also showed a slight additive effect on cell death for yeast cells producing toxic aggregates. The most frequent type of cell death observed to occur is both mother and daughter concomitantly. This study provides preliminary insight into the phenotype of the ATS in S. cerevisiae and has the potential to be used to study the effects of enhanced aggregate clearance in mother cells. Additionally, the strategy employed in the design of the ATS may be repurposed for implementation in mammalian cells to reduce the toxicity of protein aggregates.