Identification of cell context-dependent YAP-associated proteins reveals \u03b21 and \u03b24 integrin mediate YAP translocation independently of cell spreading

Ovijit Chaudhuri,Ryan S Stowers,Joanna Y Lee,Lei S Qi,Sungmin Nam,Antonia A Dominguez

doi:10.1038/s41598-019-53659-4

Ovijit Chaudhuri, Ryan S Stowers + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-53659-4

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Journal: Scientific Reports	Publication Date: Nov 20, 2019
Citations: 12	License type: open-access

Affiliation: Stanford University

Abstract

Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with β1 integrin under activating conditions, and β4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of β1 integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, β4 integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.

Highlights

Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture
YAP activation was not involved in mediating mechanotransduction in 3D culture, raising the question: what molecular interactions in 2D culture mediate mechanotransduction? Here, we identify proteins that differentially associate with YAP under activating and inactivating conditions, including β1 and β4 integrin, respectively
Cells plated on col-1coated PAM spread with increasing stiffness and 2 kPa stiffness resulted in YAP nuclear localization, consistent with previous studies (Fig. 1d,e and Supplementary Fig. 1)[13]

Summary

Introduction

Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Β4 integrin KO (ΔITGB4) cells showed no change in cell area and decreased nuclear YAP These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation. Enhanced stiffness alone in a hydrogel containing basement membrane (BM) ligands, absent of col-1, induces a highly invasive phenotype in MECs12 This occurred through a different mechanism, involving BM ligand binding to β4 integrin, followed by inhibition of hemidesmosome formation, altered β4 integrin signaling, and activation of Rac[1] and PI3K by increased stiffness[12]. These data provide compelling evidence that the ECM is a major regulator of BM invasion and ductal carcinoma progression. This reduction in YAP nuclear translocation coincided with both a decrease in cell area and a decrease in the ratio of nuclear/cytoplasmic YAP

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