An unbiased screening approach for identifying proteins with a mechanosensitive nuclear localization

Mechanical forces profoundly influence a wide range of cellular behaviors. However, the mechanisms by which these forces are sensed and transduced into specific biological signals remain incompletely understood. One established principle of mechanotransduction is the mechanical regulation of subcellular protein localization, which enables control over the activity of key regulators such as transcription and splicing factors. In this protocol, we present a screening approach to identify proteins that exhibit mechanosensitive nuclear localization. We describe the engineering of cell lines with tuneable actomyosin contractility and the application of a spatially restricted in situ biotinylation strategy. This method, combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), enables the identification of proteins whose nuclear localization changes in response to mechanical cues. For a detailed application of this protocol, please refer to Tseng et al. (2025) [1]