A New Class of Targetable, Genetically Encoded Single-Color Biosensors for Multiplexed Monitoring of Phosphoinositides

Phosphoinositides are involved in numerous signaling processes, even though they represent only a small fraction of all phospholipids in cellular membranes. Phosphoinositide species can be interconverted reversibly, allowing them to serve as second messengers to activate signaling downstream or as membrane anchors by recruiting specific proteins. Many membrane proteins, especially ion channels, are regulated through direct interactions with specific phosphoinositide species. Several genetically encodable biosensors have been engineered to observe phosphoinositide signaling in living cells. These biosensors signal the binding of specific binding domains to phosphoinositides by either translocation of fluorescence or changes in FRET efficiency. However, monitoring further parameter or multiple phosphoinositide species simultaneously, especially in different cellular compartments, still poses a challenge. This hinders a full understanding of the role that phosphoinositides play in multi-step signaling pathways. In order to facilitate multiplexed imaging, we tested different strategies for developing single-color intensity based sensors and identified a generalizable unimolecular sensor design for phosphoinositides based on the sensor InPAkt. Targeting green and red fluorescent sensors for PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3 to different plasma membrane compartments, we were able to investigate phosphoinositide dynamics and additional parameters simultaneously during Gq and growth factor signaling.