Identification and characterisation of novel molecular players in mechanotransduction

Cells sense and respond to the stiffness of the surrounding extracellular matrix (ECM) or sense and respond to forces elicited from adjacent cells and convert them into biological signals in a process known as mechanotransduction. In addition to propagating mechanical cues through remodelling of cytoskeleton stress fibres, cells rely on mechano-activated shuttling of proteins between the nucleus and the cytosol regulating gene expression that underpins multiple cellular behaviours such as proliferation, differentiation and migration. Already several proteins, including transcriptional regulators YAP/TAZ, have been identified to shuttle between the nucleus and cytosol depending on the presence of specific mechanical stimuli. However, the regulators and mechanisms of mechanotransduction are not fully understood. In this project, we established a screen by manipulating RhoA activity coupled with nuclear proteome specific proximity biotinylation and mass spectrometry analysis to search for mechanotransducers with nuclear function. This screen revealed 102 proteins significantly enriched in the nucleus after RhoA activation. A Gene Ontology analysis showed that most of the proteins are correlated with RNA regulation at both transcriptional and translational level such as RNA splicing and gene translation. Immunoblot analysis of selected protein candidates including the RNA splicing regulators PCBP1 and PTBP1 verified their nuclear enrichment was promoted by RhoA activity. The further investigation of PCBP1 and PTBP1 showed that the subcellular localisation of PCBP1 and PTBP1 are regulated by ECM stiffness and cell density. Furthermore, RT-PCR experiments indicated that ECM stiffness may regulate alternative splicing through PCBP1 and PTBP1 activity. We therefore suggest that PCBP1 and PTBP1 are potential mechanoregulators and play a role in ECM stiffness-mediated alternative splicing.