Holmes Victoria, Ricci Morgan M C, Weckerly Claire C, Worcester Michael, Hammond Gerald R V
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
bioRxiv. 2025 Jan 8:2024.09.11.612480. doi: 10.1101/2024.09.11.612480.
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/PI(3,4)P 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 biosensors eliminates this translocation, despite robust recruitment of the biosensors. Inhibition is even observed with PI(3,4)P-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.
基因编码的脂质生物传感器独特地为活细胞中的脂质动力学提供实时、空间分辨的动力学数据。尽管有明显的优势，但这些工具也有显著的缺点；最明显的是，与生物传感器结合的脂质分子无法与效应器相互作用，可能会抑制信号传导。在这里，我们表明，虽然在用PH-Akt1 PIP/PI(3,4)P生物传感器转染的细胞群体中，PI 3-激酶（PI3K）介导的Akt激活没有显著降低，但表达可见水平的PH-Akt的单细胞激活有所降低。用neonGreen标记内源性AKT1可揭示其由表皮生长因子（EGF）介导的向质膜的转位。尽管生物传感器有强大的募集作用，但与PH-Akt1或其他PIP生物传感器共转染会消除这种转位。即使使用PI(3,4)P选择性生物传感器也观察到了抑制作用。然而，以与内源性AKT相当的低水平表达脂质生物传感器不会产生这种抑制作用。有益的是，与过表达的生物传感器相比，这些单分子生物传感器显示出更好的动态范围和动力学保真度。这种方法代表了一种探测活细胞中PI3K信号时空动态的非侵入性方式。
