Fujioka Yuko, PhD
Competition Sponsor: Japan Agency for Medical Research and Development
Life science has entered a new phase to reconsider various phenomena, including autophagy, after discovery of liquid-liquid phase separation in cells. Autophagy is a major intracellular proteolysis pathway and plays an important role in proteostasis, which is essential for our healthy life expectancy. I found first in the world that the site of autophagosome formation, termed the pre-autophagosomal structure (PAS), is in fact a liquid droplet of Atg proteins formed by liquid-liquid phase separation (Fujioka et al., Nature 2020). However, the study was restricted to “early PAS” in budding yeast that is composed solely of the Atg1 complex. Autophagy progression is mediated by the PAS in a mature state, termed “mature PAS”, which contains various Atg proteins and lipid membranes. Moreover, the role of phase separation in the organization of autophagosome formation sites in mammals has remained elusive. This research proposal aims to unveil the molecular functions of the PAS and to answer the question of why the PAS must be a liquid droplet through in vitro reconstitution of mature PAS. Using these knowledges, the function and regulation of the autophagosome formation sites in mammals will also be analyzed. I also found that early PAS transforms into a solid state through liquid-solid phase transition in vitro and that PAS loses its function by solidification caused by loss of phospho-regulation in vivo. Through the analysis of the mechanism and structural basis of how PAS droplets lose the activity by liquid-solid phase transition and how phospho-regulation prevents that, I establish the basis for understanding liquid-liquid phase separation and liquid-solid phase transition regulating various aspects of proteostasis. These knowledges will contribute to understanding the mechanism of formation of pathogenic protein aggregates such as amyloids and provide a basis for developing preventive and/or curative medicines for neurodegenerative diseases through regulating phase separation and transition that affect both autophagy activity and state of pathogenic proteins.