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Identification of cell context-dependent YAP-associated proteins reveals β1 and β4 integrin mediate YAP translocation independently of cell spreading.
Scientific Reports ( IF 3.8 ) Pub Date : 2019-11-20 , DOI: 10.1038/s41598-019-53659-4
Joanna Y Lee 1, 2 , Antonia A Dominguez 3, 4, 5 , Sungmin Nam 1 , Ryan S Stowers 1 , Lei S Qi 3, 4, 5 , Ovijit Chaudhuri 1
Scientific Reports ( IF 3.8 ) Pub Date : 2019-11-20 , DOI: 10.1038/s41598-019-53659-4
Joanna Y Lee 1, 2 , Antonia A Dominguez 3, 4, 5 , Sungmin Nam 1 , Ryan S Stowers 1 , Lei S Qi 3, 4, 5 , Ovijit Chaudhuri 1
Affiliation
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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.
中文翻译：
细胞环境依赖性 YAP 相关蛋白的鉴定揭示了 β1 和 β4 整合素介导 YAP 易位，与细胞扩散无关。
Yes 相关蛋白 (YAP) 是一种转录调节因子和力转导因子，可将细胞外基质 (ECM) 硬度传递到二维培养物中的增殖基因表达。先前的研究表明，YAP 激活依赖于 F-肌动蛋白应力纤维介导的核孔开放，但 YAP 易位的蛋白质介导者仍不清楚。在这里，我们表明 YAP 在激活条件下与 F-肌动蛋白共定位，例如稀疏铺板和在坚硬的 2D 基质上培养。为了鉴定介导 YAP 易位的蛋白质，我们对在激活条件下与 YAP 存在差异相关的蛋白质进行了免疫共沉淀，然后进行质谱 (co-IP/MS)。有趣的是，YAP 在激活条件下优先与 β1 整合素结合，在失活条件下优先与 β4 整合素结合。在激活条件下，β1 整合素 (ΔITGB1) 的 CRISPR/Cas9 敲除 (KO) 导致细胞面积减少，这与 YAP 核定位减少相关。ΔITGB1 并没有显着影响 YAP 核定位与面积之间的相关性斜率，但确实降低了总体核 YAP，与细胞扩散无关。相反，β4整合素KO（ΔITGB4）细胞显示细胞面积没有变化，并且核YAP同样降低。这些结果揭示了在激活过程中与 YAP 差异相关的蛋白质，这可能有助于调节 YAP 核易位。
更新日期：2019-11-20
中文翻译：
细胞环境依赖性 YAP 相关蛋白的鉴定揭示了 β1 和 β4 整合素介导 YAP 易位，与细胞扩散无关。
Yes 相关蛋白 (YAP) 是一种转录调节因子和力转导因子，可将细胞外基质 (ECM) 硬度传递到二维培养物中的增殖基因表达。先前的研究表明，YAP 激活依赖于 F-肌动蛋白应力纤维介导的核孔开放，但 YAP 易位的蛋白质介导者仍不清楚。在这里，我们表明 YAP 在激活条件下与 F-肌动蛋白共定位，例如稀疏铺板和在坚硬的 2D 基质上培养。为了鉴定介导 YAP 易位的蛋白质，我们对在激活条件下与 YAP 存在差异相关的蛋白质进行了免疫共沉淀，然后进行质谱 (co-IP/MS)。有趣的是，YAP 在激活条件下优先与 β1 整合素结合，在失活条件下优先与 β4 整合素结合。在激活条件下，β1 整合素 (ΔITGB1) 的 CRISPR/Cas9 敲除 (KO) 导致细胞面积减少，这与 YAP 核定位减少相关。ΔITGB1 并没有显着影响 YAP 核定位与面积之间的相关性斜率，但确实降低了总体核 YAP，与细胞扩散无关。相反，β4整合素KO（ΔITGB4）细胞显示细胞面积没有变化，并且核YAP同样降低。这些结果揭示了在激活过程中与 YAP 差异相关的蛋白质，这可能有助于调节 YAP 核易位。
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