Using Optogenetic Systems to Map Out Liquid-Liquid Phase Separation in Living Cells
Over the course of evolution, living cells have developed new ways to organize their contents by categorizing certain biomolecules into various different organelles. Previous research has found that liquid-liquid phase separation gives rise to this assembly of structured compartments within the cell using weak multivalent interactions (such as IDRs/IDPs and nucleic acids); it is these interactions that allow for these stable forms of biomolecular organization to occur. However, this complex understanding of biomolecular phase separation in vivo has been limited in scientific research due to the lack of tools for stimulating, shaping, destabilizing, and mapping of these phase reactions in the cellular environment in vitro. This leaves us with a vague understanding of these patterned phase separations and other non-equilibrium features that are fundamental to phase separation in living, developing cells.
One way to close this gap of knowledge in biology is through the creation of an oligomerizing biomimetic system, called the “Corelet'' System. This two-module optogenetic system mimics the natural oligomerization of these weak multivalent reactions using a light-activated core and would provide a way to map out where these liquid-liquid phase separations can occur. Previous studies have shown that this is possible using blue-light activation, where they were able to successfully map out these phase diagrams. However, blue light systems can only be switched ON instantaneously but are limited to the kinetics of chromophore reversal to be switched OFF. In addition, blue light is toxic to living cells at moderate levels. Both of these facts about the blue light Corelet system limit its widespread application. I want to see if using a red-light activated Corelet system will overcome these challenges. In order to test this question, I am first going to use Python, a coding software, to learn skills that could allow me to simulate liquid-liquid phase separation I would expect to see in “real life”, until I can resume lab experiments in person. I am working to develop an algorithm in order to translate a given DNA sequence in Python, introduce mutations in sequences, and learn the principles of Cellular Automata in both 1D and 2D. This will help me translate how this red-light activated Corelet system should work in a lab setting, and ultimately help us better understand how different systems can work together to map out intracellular processes in living cells.