The Role of Nonmuscle Myosins in Cardiac Development

先前的研究表明，小鼠非肌肉肌球蛋白重链（NMHC）II-A的消融导致细胞粘附缺陷和E-钙粘蛋白和β-连环蛋白在细胞-细胞粘附位点的定位丢失。在这里，我们研究了NMHC II-B在小鼠胚胎成纤维细胞（MEFs）中的功能，MEFs是一种通常含有肌球蛋白II-A和II-B，但不含肌球蛋白II-C的细胞系。我们比较了MEF细胞系，这是来自一个已经消融NMHC II-B的小鼠与来自野生型小鼠的细胞系。使用免疫印迹和FACS分析，与野生型细胞相比，肌球蛋白II-B消融的MEFs显示β-1和β-3-整联蛋白的含量降低。在这些细胞中α-2和α-3-整合素没有变化。值得注意的是，与野生型MEF相比，II-B消融的MEF显示出对许多细胞外基质蛋白的附着减少，所述细胞外基质蛋白包括I型胶原蛋白、纤连蛋白和层粘连蛋白。用GFP-NMHC II-B转染II-B消融的MEFs可以挽救β-1-整合素含量和细胞附着的缺陷。当使用针对NMHC II-B的siRNA来降低野生型MEFs中肌球蛋白II-B的含量时，siRNA处理的细胞中β-1-整合素减少以及细胞基质附着减少。有趣的是，MEFs，含有一个突变的肌球蛋白II-B（一个单一的氨基酸突变，半胱氨酸代替精氨酸709），具有降低肌动蛋白激活的MgATP酶活性和体外运动，不显示任何异常整合素含量或基质附着。这意味着肌球蛋白运动活性可能不是β-整联蛋白表达所必需的。我们的研究结果表明，NMHC II-B是参与附着的MEFs的细胞外基质，通过调节β-整合素的稳定性。

Previous work has shown that ablation of nonmuscle myosin heavy chain (NMHC) II-A in mice results in defects in cell adhesion and loss of E-cadherin and beta-catenin localization to cell-cell adhesion sites. Here we study the function of NMHC II-B in mouse embryonic fibroblasts (MEFs), a cell line that normally contains myosin II-A and II-B, but not myosin II-C. We compared a MEF cell line that was derived from a mouse that had been ablated for NMHC II-B with a cell line derived from a wild-type mouse. The myosin II-B ablated MEFs showed a decrease in the content of both beta-1 and beta-3-integrins compared to wild-type cells using immunoblot and FACS analysis. There was no change in alpha-2 and alpha-3-integrins in these cells. Of note, the II-B ablated MEFs showed a decreased attachment to a number of extracellular matrix proteins including type I collagen, fibronectin and laminin compared to wild-type MEFs. The defect in both beta-1-integrin content and cell attachment could be rescued by transfection of II-B ablated MEFs with GFP-NMHC II-B. When siRNA to NMHC II-B was used to lower the content of myosin II-B in wild-type MEFs, there was a reduction in beta-1-integrin as well as a decrease in cell-matrix attachment in the siRNA-treated cells. Interestingly, MEFs that contain a mutant myosin II-B (a single amino acid mutation, Cys in place of Arg 709) that has a decreased actin-activated MgATPase activity and in vitro motility, do not show any abnormality in integrin content or matrix attachment. This implies that myosin motor activity may not be required for beta-integrin expression. Our results suggest that NMHC II-B is involved in the attachment of MEFs to the extracellular matrix by regulating beta-integrin stability.