Single molecule Lipid Biosensors Mitigate Inhibition of Endogenous Effector Proteins

Genetically encoded lipid biosensors uniquely provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools have significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with effectors, potentially inhibiting signaling. Here, we show that although PI 3-kinase (PI3K)-mediated activation of Akt is not significantly reduced in a cell population transfected with a PH-Akt1 PIP 3 /PI(3,4)P 2 biosensor, single cells expressing PH-Akt at visible levels have reduced activation. Tagging endogenous AKT1 with neonGreen reveals its EGF-mediated translocation to the plasma membrane. Co-transfection with the PH-Akt1 or other PIP 3 biosensors eliminates this translocation, despite robust recruitment of the biosensors. Inhibition is even observed with PI(3,4)P 2 -selective biosensor. However, expressing lipid biosensors at low levels, comparable with those of endogenous AKT, produced no such inhibition. Helpfully, these single-molecule biosensors revealed improved dynamic range and kinetic fidelity compared with over-expressed biosensor. This approach represents a non-invasive way to probe spatiotemporal dynamics of PI3K signaling in living cells. eTOC summary Holmes and colleagues show that AKT activation is blocked by commonly used PIP 3 biosensors, but that this can be overcome by expressing biosensors at single molecule levels – which also improves kinetic fidelity and sensitivity.