An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced cell-based molography as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells.

Automated summary

Cell-based assays are frequently used for molecular interaction analysis, offering specific and real-time insights into interactions involving native membrane receptors in living cells. The introduction of cell-based molography has expanded the range of interactions that can be analyzed by biosensors, now including cytosolic protein-protein interactions. This approach overcomes the limitations of label-based assays, providing a novel method for studying specific molecular interactions in living cells.