# Protocol to identify mechanosensitive nuclear proteins using tunable actomyosin contractility and proximity biotinylation in mammalian cells

## TL;DR

This paper introduces a protocol to identify proteins in mammalian cells whose nuclear localization is influenced by mechanical forces.

## Contribution

A novel protocol combining tunable actomyosin contractility and proximity biotinylation to study mechanosensitive nuclear proteins.

## Key findings

- The protocol enables the identification of proteins with mechanosensitive nuclear localization.
- Proteins whose nuclear localization is controlled by actomyosin contractility can be quantified using LC-MS/MS.
- Candidate proteins can be validated by observing their subcellular localization in response to mechanical cues.

## Abstract

Mechanical forces influence a range of cellular behaviors; however, how these forces are sensed and converted into biochemical changes remains incompletely understood. A key aspect of mechanotransduction is the regulation of subcellular protein localization. Here, we present a protocol describing the engineering of cell lines with tunable actomyosin contractility combined with a proximity biotinylation strategy confined to the nucleus followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This approach allows the identification of proteins whose nuclear localization is controlled by changes of actomyosin contractility.

For complete details on the use and execution of this protocol, please refer to Tseng et al.1

•Protocol for the identification of proteins with mechanosensitive nuclear localization•Generation of a cell line with tunable actomyosin contractility•Quantification of nuclear proteome using proximity biotinylation and mass spectrometry•Candidate validation by monitoring subcellular localization upon direct mechanical cues

Protocol for the identification of proteins with mechanosensitive nuclear localization

Generation of a cell line with tunable actomyosin contractility

Quantification of nuclear proteome using proximity biotinylation and mass spectrometry

Candidate validation by monitoring subcellular localization upon direct mechanical cues

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Mechanical forces influence a range of cellular behaviors; however, how these forces are sensed and converted into biochemical changes remains incompletely understood. A key aspect of mechanotransduction is the regulation of subcellular protein localization. Here, we present a protocol describing the engineering of cell lines with tunable actomyosin contractility combined with a proximity biotinylation strategy confined to the nucleus followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This approach allows the identification of proteins whose nuclear localization is controlled by changes of actomyosin contractility.