Project Summary – F32 Benjamin Stormo The Control of Membraneless Organelles Material Properties Allows Cells to Control Their Function. How cells pattern and compartmentalize their cytoplasm is a critical question in biology. Recent work has discovered that many cells rely on membraneless organelles. These organelles are not separated from the rest of the cell by a lipid bilayer. Instead membraneless organelles rely on a thermodynamic process called Liquid- Liquid Phase Separation (LLPS) in which a well-mixed solution can spontaneously demix into a concentrated droplet phase and a dilute bulk phase. These liquid-like droplets perform a number of important functions in the cell including RNA processing, altering reaction kinetics, and sequestering transcripts during stress. Droplets have a number of measurable biophysical properties including viscosity, diffusivity, and permeability. Interestingly, in cases where the properties of these droplets have been measured they vary substantially between different types of droplets. Despite these difference we have very little understanding about whether specific properties are important for the function of membraneless organelles, although it has been suggested that dysregulation of droplet properties can result in diseases such as Alzheimer and Huntington. Critically, because phase separation is a thermodynamic process it is influenced by both things within the cells control: i.e. concentration of the components, and things outside of the cells control: i.e. temperature. Our work seeks to understand how the material properties of membraneless organelles affect their function. To do this we will look for evidence that cells control the material properties of droplets following changes in temperature. Our lab has previously used the model filamentous fungus Ashbya gossypii to study liquid-like droplets. In Ashbya membraneless organelles are critical for controlling the cell cycle and polarized growth. Using these clear and relevant physiological readouts along with an in vitro reconstitution system we seek to understand how specific material properties of membraneless organelles are required for specific function in the cell. We will use a unique collection of wild Ashbya isolates collected from around the United States to address two specific aims: 1) How do the primary sequences of droplet components vary between isolates from different climates and how do these sequence changes affect the resulting droplets. 2) How do cells adapt to transient temperature changes to maintain droplet properties in vivo. To answer these questions we will use high resolution light microscopy. Through this work we will improve our understanding of how cells normally regulate the properties of membraneless organelles and how this regulation relates to cellular function. Understanding the role material properties of droplets play in normal function is crucial for understanding what happens when they are altered in disease con...