The Role of Nonmuscle Myosins in Cardiac Development

以往的工作表明，消融小鼠非肌肉肌球蛋白重链(NMHC)II-A会导致细胞黏附缺陷，并导致E-钙粘附素和β-连环素在细胞-细胞黏附部位的定位丢失。在这里，我们研究了NMHC II-B在小鼠胚胎成纤维细胞(MEF)中的功能，该细胞系通常含有肌球蛋白II-A和II-B，但不含肌球蛋白II-C。我们比较了来自NMHC II-B消融小鼠的MEF细胞系和来自野生型小鼠的细胞系。免疫印迹和流式细胞仪分析显示，肌球蛋白II-B消融的MEF与野生型细胞相比，β-1和β-3-整合素的含量都降低了。这些细胞中的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与包括I型胶原、纤维连接蛋白和层粘连蛋白在内的许多细胞外基质蛋白的粘附性降低。通过将GFP-NMHC II-B导入II-B消融的MEF，可以挽救β-1-整合素含量和细胞贴壁的缺陷。当使用针对NMHC II-B的siRNA来降低野生型MEF中肌球蛋白II-B的含量时，在siRNA处理的细胞中，β1整合素减少，细胞与基质的附着减少。有趣的是，含有突变的肌球蛋白II-B(单个氨基酸突变，半胱氨酸取代精氨酸709)的MEF在肌动蛋白激活的镁ATPase活性和体外运动能力降低的情况下，在整合素含量或基质附着方面没有显示任何异常。这意味着β-整合素的表达可能不需要肌球蛋白的运动活性。我们的结果提示NMHC II-B通过调节β-整合素的稳定性参与MEF与细胞外基质的附着。

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.