Development of a Red-Light Optogenetic Phase Separation System

Abstract

Cells employ compartmentalization, typically through lipid bilayers, to coordinate interdependent biochemical reactions. Recently, membrane-less compartments formed by biomolecular condensates have gained interest, as they are thought to play an important biological function in many developmental and disease-associated contexts. These condensates arise by way of liquid-liquid phase separation driven by multivalent interactions, often between intrinsically disordered protein regions (IDR). To study such dynamic processes, optogenetic tools have been used to control phase separation with high spatiotemporal precision. However, the field is currently limited, as all available systems rely on blue light for control, which has high phototoxicity and low tissue transparency. Consequently, the development of red-light opto-tools, safer for utilization in tissues, would be beneficial. Thus, this thesis outlines the successful development of a phase separation system driven by the red and far-red light responsive dimer Phytochrome B (PhyB) and phytochrome-interacting factor (Pif). Specifically, our system relied on the design of Corelets, a blue-light responsive phase separation system, supplanting the traditional blue-light induced interaction with that of PhyB/Pif. Upon illumination by red light, rapid cytosolic liquid droplet formation was observed, with only exposure from the inactivating far-red wavelength able to cause droplet dissociation over a similar timescale. Droplets could be repeatedly assembled and dissolved without exhaustion over several on-off cycles. Additionally, components of our system were combined with those of Corelets to generate a two-color controlled phase separation system in which droplet formation could be induced simultaneously by two different wavelengths of light in separate compartments of the cell. Taken together, our results represent an advancement in the world of optogenetic phase separation tools, offering a novel system that can be utilized to examine condensates in more intricate ways and potentially within clinical settings.