OPTOGENETIC RECONSTITUTION TO DETERMINE FUNCTIONS OF INTRACELLULAR PHASE SEPARATION

Abstract

It has long been known that cells organize key reactions into separate compartments. While this is traditionally thought to occur in membrane-enclosed organelles, recent work has demonstrated the ubiquity of membrane-less organelles that house proteins and nucleic acids. These organelles typically form via a process of liquid-liquid phase separation that involves the nucleation of biomolecules into droplets. While many studies illustrate the ubiquity of this process and these organelles in cells, less is known about how the process phase separation aids and organizes cellular reactions. Studying this question remains difficult as phase separation emerges from multiple weak interactions that occur between different biomolecules, making it hard to abolish it without offsetting effects and hard to induce with typical biological tools. However, the recently developed field of cellular optogenetics helps solve this issue as these tools are ideally suited to controlling emergent properties of cellular networks, like phase separation. In this thesis, I use and develop novel optogenetic tools to relate the formation and dissolution of phase-separated droplets to cellular functions. In chapter 2 of this thesis, I illustrate how phase-separated droplets “remember” spatial patterns and how that memory affects cellular movement as controlled by Receptor Tyrosine Kinases (RTKs). In chapter 3 of this thesis, I demonstrate that bringing two T Cell signaling molecules in phase-separated droplets leads to activation of downstream signaling pathways, while heterodimerizing these proteins does not. This activation stems from the ability of droplets to localize a three component positive feedback loop. These studies together highlight how both the biophysical and biochemical properties of phase separation contribute to their cellular functions. Collectively, this thesis utilizes optogenetic tools to determine how an emergent property of cellular networks – phase separation - affects cellular behavior.