Development of a mechanical stress biosensor derived from myocardin related transcription factor A
Embargo Date
2027-09-24
OA Version
Citation
Abstract
Mechanotransduction is the ability of cells to sense and convert mechanical stimuli into intracellular changes. Mechanotransduction pathways regulate cell fate and disease pathways including fibrosis. Mechanical stimuli can come from the extracellular matrix (ECM), triggering signaling pathways that alter cell differentiation and function. In the case of fibrosis, aberrant ECM mechanics can lead to pro-fibrotic cellular phenotypes. In response to changes in the ECM, intracellular pathways trigger actin polymerization, allowing the transcription factor myocardin related transcription factor A (MRTFA) to translocate to the nucleus resulting in the activation of serum response factor (SRF) dependent gene expression. The goal of this study was to develop a biosensor based on the actin binding RPEL N-terminal domain of MRTFA. This biosensor fused the MRTFA-RPEL domain with enhanced green fluorescent protein (EGFP). To test the RPEL-EGFP biosensor, we treated cells on collagen-coated plates with varying mechanical properties and transforming growth factor β (TGFβ). A desired property of the biosensor is that it would not interfere with endogenous actin or transcriptional pathways. Using a luciferase-reporter gene expression assay, it was found that the RPEL-EGFP biosensor did not suppress SRF-MRTFA-dependent gene expression. In addition, staining with an ɑ-smooth muscle actin antibody (ɑ-SMA) demonstrated that the RPEL-EGFP biosensor did not interfere with actin dynamics. Lastly, a doxycycline inducible, lentiviral vector, RPEL-EGFP-pTRIPZ, was successfully generated. By developing this biosensor, mechanically sensitive pathways that influence the progression of fibrosis can be further investigated, leading to potential therapeutics.
Description
2024