| Budget Amount *help |
¥15,760,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2007: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2006: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2005: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Research Abstract |
Prions are infectious proteins; an abnormal form of the prion protein causes an auto-catalytic conversion of a normal (soluble) form of prion protein, which has the same primary structure as the abnormal one, to the abnormal insoluble form, and results in accumulation of infectious insoluble prion particles. In many cases, the particles are observed as amyloid fiber-like structures, which are called ‘prion-fibrils'. This concept originated from the studies of mammalian neurodegenerative diseases, but spread to include other proteinous genetic elements from yeast Saccharomyces cerevisiae. In particular, the prion-inducing fragment of yeast Sup35, a determinant of the yeast prion-like [PSI^+], is comprised of a glutamine/asparagine-rich N-terminal and medium domains of Sup35, and is a valuable model protein for studying the mechanism of prion-fiber formation.
Using the yeast prion as a model, we have previously developed a system to observe the growth of individual prion fibrils directly.
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As a first objective in this study, we have further expanded the imaging using total internal reflection fluorescent microscopy (TIRFM). By using the TIRFM system, we succeeded in monitoring live imaging of individual Sup35 fibrils elongation. Statistical analysis of rate of the fibril growth provides the kinetic information, and then a novel insight into the mechanism of fibril growth.
Second, we directly monitored the dynamics of the aggregates in living cells using an on-chip single-cell cultivation system as well as fluorescence correlation spectroscopy (FCS). Single-cell imaging revealed that the visible foci of yeast prion Sup35 fused with GFP are dispersed throughout the cytoplasm during cell growth, but retain the prion phenotype. FCS showed that [PSI^+] cells, irrespective of the presence of foci, contain diffuse oligomers, which are transmitted to their daughter cells. Single-cell observations of the oligomer-based transmission provide a link between previous in vivo and in vitro analyses of the prion and shed light on the relationship between the protein conformation and the phenotype. Less
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