Research On Mechanism Of Heat Induced Protein Aggregates Formation And Asymmetrical Inheritance Of Saccharomyces Cerevisiae

Saccharomyces cerevisiae is an aging organism as other ones in nature, and the “aging factors” formed and accumulated in the old cells including damaged, unfolded and aggregated proteins, which also appear in neurodegeneration disease cells (like Parkinson’s disease, Alzheimer’s disease and Huntington’s disease). Asymmetric cell division is key to cellular rejuvenation and budding yeast exploits this mode of cytokinesis to generate a young daughter cell from a mother cell that with each division grows progressively older. Thus, age physiognomies are reset in the progeny during division, a phenomenon that requires a mother-biased segregation of cytoplasmic”aging factors”. There are two models for how aggregated proteins are segregating in a mother cell-biased fashion: one holds that asymmetric inheritance is a purely passive outcome of the aggregates’ random but slow diffusion whereas the other model reasons that specific factors/organelles prevent free diffusion of aggregates into the daughter cell. The research on mechanism of aggregates/inclusion body formation and models of asymmetric inheritance provides the theoretical basis and potential therapy target for research on aging and neurodegeneration disease.In this study, we aimed to decipher the systems and pathway involved in inclusion body formation by an unbiased, genome-wide, approach utilizing the power of the yeast synthetic genetic array (SGA) technology and high-through-put/high-content microscopy (HCM). The Hsp104-GFP fusion marker is introduced into yeast single deletion mutants library including about4600nonessential gene deletion strains, construct genomic collection harboring Hsp104-GFP marker and a nonessential gene deletion. Utilizing high throughput automated platform, the incusion bodies are induced by heat shock at38℃for90min by pinning robot system. Using the high content microscope and software analysis,204mutants were identified as1fold higher increase in the fraction of cells displaying3or more than3aggregates than wild type.146mutants verified by manual confirmation are identified to be required for inclusion body formation, which covers71.6%. Functional enrichment analysis identified3functional categories required for inclusion body formation; vesicle-mediated transport, actin organization, and regulatory small GTPases. Vesicle-mediated transport includes vacular targeting, Golgi trafficking and Phosphatidyl-inositol metabolism.Sir2is a central regulator of yeast aging and its deficiency increases daughter cell inheritance of stress-and aging-induced misfold proteins deposited in aggregates and inclusion bodies, such that daughter cells inherit a higher load of damage and age prematurely. Here, we approached the questions of whether Sir2affects inheritance in line with the passive diffusion model or the opposing factor-dependent model for aggregate partitioning. By quantifying traits (mother-daughter cell geometry, bud neck diameter, generation time and aggregates abundance) predicted to affect aggregate inheritance in a passive manner, we found that a passive diffusion model cannot explain Sir2-dependent failures in mother-biased segregation of either the small aggregates formed by the misfolded Huntingtin, Htt103Q, disease protein or heat-induced Hsp104-associated aggregates. We used three-dimensional structured illumination microscopy (3D-SIM) to analysis co-localization of heat induced aggregates (Hsp104Y662A-GFP) and Huntington protein aggregates (Htt103Q-GFP), demonstrated that aggregates are lining up along actin cables and are in some instances wrapping around the cable. These data support the role of actin cable in aggregates segregation. The genetic interaction network of Sir2is constructed by using syntheticgenetic array (SGA), and specific essential genes required for mother-biased segregation of Hsp104-associated aggregates are identified, including those encoding components of the actin cytoskeleton, the actin-associated myosin V motor protein Myo2, and the actin organization protein calmodulin, Cmd1. Co-staining with Hsp104-GFP demonstrated that misfolded Htt103Q is sequestered into small aggregates, akin to stress foci formed upon heat stress, that fail to coalesce into inclusion bodies. Importantly, these Htt103Q foci, as well as the ATPase-defective Hsp104Y662A-associated structures previously shown to be stable stress foci, co-localized with Cmd1and Myo2-enriched structures and super-resolution3-D microscopy demonstrated that they are associated with actin cables. Moreover, we found that Hsp42is required for formation of heat-induced Hsp104Y662A foci but not Htt103Q foci suggesting that the routes employed for foci formation are not identical. In addition to genes involved in actin-dependent processes, SIR2-interactors required for asymmetrical inheritance of Htt103Q and heat-induced aggregates encode essential Sec genes involved in ER-to-Golgi trafficking/ER homeostasis.