Vincent T.

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcu...

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.

Nucleotide signaling molecules are important messengers in key pathways that allow cellular responses to changing environments. Canonical secondary...