In Vivo Function of Nonmuscle Myosin II-A

脊椎动物非肌肉肌球蛋白II在细胞运动、胞质分裂、细胞-基质和细胞-细胞黏附等多种细胞过程中发挥重要作用。以前的工作已经显示了NMHC II-A消融小鼠的细胞组织和细胞黏附缺陷。这些缺陷可能是由于在培养的A-/A-ES细胞和完整的胚胎中，E-钙粘附素和β-连环素在细胞黏附部位的定位丢失。我们正在将这些研究扩展到培养中特征良好的上皮细胞，如MDCK细胞。我们已经在这些细胞中定位了非肌肉肌球蛋白，并正在研究肌球蛋白轻链磷酸化、细胞迁移和细胞黏附之间的关系。此外，我们正在用共聚焦显微镜研究NMHC II-B和II-C的心脏定位，特别是关于心肌细胞Z线和间盘的成分。 为了使转基因小鼠模仿人类的May？Hegglin综合征，其中MNHC II-A存在点突变，我们正在分离包含NMHC II-A基因座第一个编码外显子的基因组克隆。该克隆还将用于产生内源性NMHC II-A上带有荧光标记的小鼠，以及NMHC II-B取代II-A并受II-A启动子控制的小鼠。

Vertebrate nonmuscle myosin IIs have been shown to play important roles in a variety of cellular processes such as cell motility, cytokinesis, and both cell-matrix and cell-cell adhesion. Previous work has shown cell organization and cell adhesion defects in mice ablated for NMHC II-A. These defects may be explained by loss of E-cadherin and beta-catenin localization from cell adhesion sites in A-/A- ES cells grown in culture and in the intact embryos. We are extending these studies to well-characterized epithelial cells in culture such as MDCK cells. We have localized nonmuscle myosins in these cells and are studying the relationship between myosin light chain phosphorylation, cell migration, and cell adhesion. In addition, we are characterizing the cardiac localization of NMHC II-B and II-C by confocal microscopy, especially with regard to components of the myocyte Z-lines and intercalated disks. In order to make transgenic mice that mimic the human syndrome known as May?Hegglin in which there are point mutations in MNHC II-A, we are isolating a genomic clone which contains the first coding exon of the NMHC II-A locus. This clone will also be used to generate mice with a fluorescent tag on the endogenous NMHC II-A as well as mice in which NMHC II-B replaces II-A and is under control of the II-A promoter.