Manuel Samantha K A, Bilenka Erika, Gupta Monica, Moy Vincent T, Landgraf Ralf
Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Coral Gables, FL, USA.
Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Coral Gables, FL, USA.
Sci Rep. 2025 Aug 21;15(1):30805. doi: 10.1038/s41598-025-15745-8.
Phosphoinositides (PIXPs) contribute to diverse biological functions that involve rapid cellular signaling as much as complex metabolic equilibria with slowly adjusting steady state levels. Existing biosensors focus on rapid response capability in cell signaling. Tools for the study of PI4P, especially over longer metabolism relevant time periods, are far more limited. PI4P itself serves as an adaptor but also precursor for two functionally distinct PIP2 species. However, the largest PI4P pool supports Golgi functionality and drives counter-current ER-Golgi transport of cholesterol. Based on the existing paradigm of a dimerization dependent RFP (ddRFP) sensor for PI(4,5)P2, we compared an analogous sensor for PI4P to a novel split GFP based sensor. The ddRFP(PI4P) sensor provides rapid response dynamics at the expense of sensitivity at biologically tolerated low expression levels. As the first implementation of a split GFP metabolite sensor, sGFP(PI4P) is slow responding but combines easily accessible readout options by FACS or in cell lysate with high sensitivity and spatial information, even at low, stably expressed sensor levels. We demonstrate the utility of the sGFP(PI4P) sensor using an inhibitor of cholesterol transport as well as two alternative inhibitors of PI4P synthesis, integrated into proliferation assays.
磷酸肌醇（PIXPs）参与多种生物学功能，这些功能既涉及快速的细胞信号传导，也涉及具有缓慢调节稳态水平的复杂代谢平衡。现有的生物传感器侧重于细胞信号传导中的快速响应能力。用于研究PI4P的工具，尤其是在更长的与代谢相关的时间段内，要有限得多。PI4P本身既是一种衔接蛋白，也是两种功能不同的PIP2种类的前体。然而，最大的PI4P池支持高尔基体功能，并驱动胆固醇的逆流内质网-高尔基体转运。基于现有的用于PI(4,5)P2的二聚化依赖性RFP（ddRFP）传感器范式，我们将一种类似的PI4P传感器与一种基于新型分裂GFP的传感器进行了比较。ddRFP(PI4P)传感器以在生物学耐受的低表达水平下的灵敏度为代价提供快速响应动力学。作为分裂GFP代谢物传感器的首次应用，sGFP(PI4P)响应缓慢，但即使在低水平、稳定表达的传感器水平下，也能通过FACS或细胞裂解液将易于获取的读出选项与高灵敏度和空间信息相结合。我们通过将胆固醇转运抑制剂以及两种PI4P合成的替代抑制剂整合到增殖试验中，展示了sGFP(PI4P)传感器的效用。
