UC San Diego

Liquid-liquid phase separation (LLPS) contributes to signaling regulation. Cyclic GMP-AMP synthase (cGAS) in the cGAS-STING signaling pathway shows phase separation behavior but the exact function of cGAS condensates is not clear. ALIX, a vital protein in the ESCRT pathway that plays an important role in multivesicular body sorting and virus budding, has been shown to form fibril and gel-like condensates in vitro, yet its phase separation property has not been examined in mammalian cells. Here, I focused on investigating cGAS and ALIX condensates in living mammalian cells. We used fluorescent protein tagging and live-cell imaging to observe their localization and dynamics in HEK293 cells and found both cGAS and ALIX showed phase separation in cells. To visualize cyclic GMP-AMP (cGAMP) level, we co-expressed a FRET-based cGAMP BioSTING sensor with cGAS, and the result suggests that both the response of BioSTING and the number of the condensates is cGAS concentration dependent. To optimize the BioSTING sensor and improve its performance, I tested several cyan-yellow FRET pairs and developed a new sensor variant that showed an enhanced response. With regard to ALIX, both in vitro experiments and FRAP analysis suggested it forms gel-like condensates. Using truncation analyses, we found that the proline-richdomain (PRD) in ALIX contributes to phase separation, and the Bro1 domain may play an inhibitory role. Furthermore, co-expression of CHMP4B, a subunit of ESCRTIII, increased the number of ALIX puncta, suggesting CHMP4B may facilitate ALIX phase separation. Through this study, I confirmed cGAS phase separation property, developed an improved cGAMP biosensor, and set up the LLPS model to further study cGAS condensates. In addition, I demonstrated ALIX phase separation behavior in cells, investigated the contribution of several domains of ALIX to its LLPS, and identified the role of CHMP4B in facilitating ALIX LLPS. These studies laid a foundation for further studies of these important biomolecular condensates.