Developing Computational Tools to Investigate Stress Granules In-vivo

Abstract

The classical picture of an organelle is membrane-bound and stable, but in recent years we have been able to identify a number of membraneless organelles that may be dynamically assembled and disassembled in response to changes in the cellular environment. These condensates exhibit liquid-like behaviour and as such, can flow and merge. Despite this seemingly transient nature, these membraneless organelles perform a complex role in the cell, and in several instances, these roles may depend on the interactions between several species of membraneless organelle. Stress granules are such an example and arise from liquid-liquid phase separation triggered by oxidative, temperature or osmotic stress. Stress granules are composed of RNA and proteins that condense out of the cytoplasm. While stress granule composition and function has been a subject of intense work recently, many biophysical aspects remain poorly characterised.
In this work, we present two computational tools that are backed by state-of-the-art microscopy data. The first is a reaction-diffusion based model of granule formation and growth that accurately captures the count and size distribution of granules throughout the cell with time, providing us with insight into the dynamics of granule assembly.

Secondly, we introduce flicker spectroscopy as a method to measure the surface tension of granules in live cells directly and apply it to stress granules induced by different chemicals and genetic backgrounds. The measured surface tensions agree well with the current estimates used in the literature. Additionally, we quantify bending rigidity across the surface of the droplet, which to our knowledge has not yet been characterised for cellular droplets.

Finally, we present an open-source, user-friendly software package for performing flicker spectroscopy, to encourage the application of this method to other biological condensates.