The Features of the Genetically-Encoded Biosensor to PIP3 Influence on Insulin Signaling in MSCs

Abstract

Genetically encoded fluorescent biosensors (GEBs) are extensively utilized for investigating intracellular signaling processes. Nevertheless, the potential impact of these sensors on intracellular signaling pathways and overall cellular functionality is frequently overlooked, which may lead to inaccuracies in experimental results. In this study, the authors examined the effects of a widely used fluorescent biosensor, which binds to phosphatidylinositol 3,4,5-trisphosphate (PIP3), on the phosphoinositide 3-kinase (PI3K)-signaling pathway in an immortalized multipotent mesenchymal stromal cell (MSC) line. To achieve this, two immortalized MSC lines with distinct levels of biosensor expression were generated. The introduction of the biosensor led to a reduction in the adipogenic potential of these cell lines compared to the parental line. This effect was attributed to the inhibition of the PI3K signaling pathway, which is activated by insulin—a critical regulator of adipogenic differentiation. The biosensor to PIP3 interfered with the recruitment of downstream effectors, namely PDK and Akt. This was evidenced by diminished levels of phosphorylated Akt (p-Akt) following insulin stimulation in the transduced cell lines relative to the parental line, with the cell line exhibiting higher biosensor expression showing lower p-Akt levels than the line with lower expression. In contrast, the levels of phosphorylated ERK, a marker of another insulin-dependent signaling pathway, remained consistent across all cell lines, indicating that the biosensor selectively inhibited the PIP3-dependent pathway. This case emphasizes the necessity of a careful and cautious approach when employing GEBs in biological research. Prior to their use, it is imperative to verify that these biosensors do not disrupt intracellular signaling cascades or alter cellular behavior.