【For INTERNATIONAL COLLABORATIVE RESEARCH 2018】

SUMMARY of

DATE: Day 15 Month May Year 20192018 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title

Mechanism of amyloid fibril formation of -synuclein

Applicant

Name

UVERSKY, Vladimir

Affiliation

University of South Florida, College of Medicine, USA

Present Title

Associate Professor

Research Collaborator (Host PI)

GOTO, Yuji

Summary
Background: Although the cellular interior is crowded with various biological macromolecules, thedistribution of these macromolecules is highly inhomogeneous. Eukaryotic cells contain numerous
proteinaceous membrane-less organelles (PMLOs), which are condensed liquid droplets formed asa result of the reversible and highly controlled liquid-liquid phase transitions [Curr. Opin. Struct.
Biol. 44, 18-30 (2017)]. On the other hand, amyloid fibrils involved in various diseases are formed
by a nucleation-growth mechanism, similar to the crystallization of solutes from solution. Solubility
and supersaturation are two of the most important factors determining crystallization of solutes.
Moreover, crystallization competes with glass formation in which solutes collapse into amorphousaggregates. Recent studies on the formation of amyloid fibrils and amorphous aggregates indicatethat the partition between distinct types of aggregates can be explained by a kinetic andthermodynamic competition between them [Curr. Opin. Struct. Biol. 36, 32-39 (2016)].
Specific Aims: The role of supersaturation in forming PMLOs is far from unclear. It has been
observed that PMLOs have the propensity to mature, changing their properties from liquid-like tosolid-like [Nature Reviews Mol. Cell Biol. 18, 285-298 (2017)]. Initially, the components in thecondensed phase exhibit only transient interactions and lack appreciable order. Thus, the molecules
freely rearrange (and exchange with the surrounding solution) and the molecular dynamics can bedescribed as that of a liquid. Over time, the phase becomes more solid-like amyloid fibrils.
Methods: With -synuclein, an intrinsically disorder protein associated with Parkinson’s disease,
we study the liquid-to-solid transitions focusing on the role of supersaturation and phase transition.
We will use a C. elegance system expressing -synuclein with yellow fluorescence protein.
Results and Discussion: The experiments showed the ultrasonication-accelerated fibril formationof -synuclein. The formation of rigid fibrils was confirmed by TEM imaging of cell lysate. It ispossible that -synuclein first formed amorphous aggregates and then amorphous aggregates wereconverted to amyloid fibrils. However, at this stage, it was not possible to distinguish amorphousaggregates and amyloid fibrils directly inside cells. If we could use sophisticated methodologies
distinguishing the clustered amorphous aggregates and rigid and elongated amyloid fibrils, we may
be able to observe a kind of phase transition from droplet-like amorphous aggregates to amyloid
fibrils as observed for FUS protein (Cell 162, 1066-1077 (2015). One possibility is to use a highresolution fluorescence imaging system monitoring the process of aggregation.