The Function of Nonmuscle Myosin II Heavy Chains

靶向缺失非肌肉肌球蛋白II-B 重链(NMHC II-B)引起小鼠心脏和心脏的缺陷 大脑在胚胎发育过程中和纯合子胚胎 没有存活或存活到足月的人在一天内死亡 他们出生了。心脏或大脑缺陷可能是原因之一。 纯合子胚胎的死亡。为了进一步了解 NMHC II-B在开发过程中的作用，我们正在努力 建立特异性高表达NMHC II-B的转基因小鼠 心脏。转基因小鼠目前正在进行分析，并将 被用来拯救基因敲除的老鼠。获救的老鼠 预计会经历正常的心脏发育，但仍然 大脑有缺陷。因此，被拯救的基因敲除小鼠 转基因小鼠应该可以让我们进一步评估 NMHC II-B在大脑中的重要性。的截断形式。 非肌肉肌球蛋白重链II-A(NMHC II-A)，缺失1-592 氨基酸，也被用来检测其细胞功能。 将绿色荧光蛋白(GFP)融合到N端 全长和截短NMHC II-A及其融合 在HeLa Tet-Off细胞系中稳定表达蛋白质，其中 内源性NMHC II-A表达，而NMHC II-B不表达。 截短型NMHC II-A在HeLa Tet-off细胞中的表达 肌动蛋白细丝重排引起的直线诱导细胞圆化 局灶性粘连消失。这些变化被逆转了 当截短的NMHC II-A的表达被 添加多西环素。HeLa Tet-Off细胞表达 全长NMHC II-A：GFP融合蛋白没有显示这一点 表型用或不用多西环素。共焦显微镜 研究表明，全长和截短的NMHC II-A 定位于应力纤维。我们建议NMHC II-A是 参与通过稳定应力纤维来维持细胞形状 和灶性粘连。

Targeted deletion of the nonmuscle myosin II-B heavy chain (NMHC II-B) in mice caused defects in the heart and brain during embryonic development and homozygous embryos did not survive or those surviving to term died within one day after they were born. Either heart or brain defects might be responsible for the death of homozygous embryos. To further understand the role of NMHC II-B during the development, we are trying to establish transgenic mice specifically overexpressing NMHC II-B in the heart. The transgenic mice are currently being analyzed and will be used in an effort to rescue the knockout mice. The rescued mice will be expected to undergo a normal heart development, but still have defects in the brain. Therefore, the knockout mice rescued by the transgenic mice should allow us to further evaluate the importance of NMHC II-B in the brain. A truncated form of the nonmuscle myosin heavy chain II-A (NMHC II-A), lacking 1-592 amino acids, has also been used to examine its cellular functions. Green fluorescent protein (GFP) was fused to the N-terminal end of both the full-length and truncated NMHC II-A and the fusion proteins were stably expressed in a HeLa Tet-off cell line, in which endogenous NMHC II-A was expressed, but NMHC II-B was not. The expression of truncated NMHC II-A in the HeLa Tet-off cell line induced cell rounding with rearrangements of actin filaments and disappearance of focal adhesions. These changes were reversed when the expression of truncated NMHC II-A was terminated by addition of doxycycline. HeLA Tet-off cells expressing the full-length NMHC II-A:GFP fusion protein did not show this phenotype with or without doxycycline. Confocal microscopic studies indicated that both full-length and truncated NMHC II-A were localized to the stress fibers. We suggest NMHC II-A is involved in maintenance of cell shape by stabilizing the stress fibers and focal adhesions.