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


Incaviglia, Ilaria; Frutiger, Andreas; Blickenstorfer, Yves; Treindl, Fridolin; Ammirati, Giulia; Lüchtefeld, Ines; Dreier, Birgit; Plückthun, Andreas; Vörös, Janos; Reichmuth, Andreas M (2021). An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells. ACS Sensors, 6(4):1572-1582.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Physical Sciences > Bioengineering
Physical Sciences > Instrumentation
Physical Sciences > Process Chemistry and Technology
Physical Sciences > Fluid Flow and Transfer Processes
Language:English
Date:23 April 2021
Deposited On:07 Dec 2021 07:54
Last Modified:01 Apr 2022 00:01
Publisher:American Chemical Society (ACS)
ISSN:2379-3694
OA Status:Green
Publisher DOI:https://doi.org/10.1021/acssensors.0c02480
PubMed ID:33759497

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