The Function of Nonmuscle Myosin II Heavy Chains

非肌肉肌球蛋白II-B的靶向缺失 重链（NMHC II-B）在小鼠中引起心脏缺陷， 大脑在胚胎发育过程中和纯合子胚胎没有 未存活或存活至足月者在分娩后一天内死亡 他们出生了心脏或大脑缺陷可能是 导致了纯合子胚胎的死亡进一步了解 NMHC II-B在发展过程中的作用，我们正试图 建立特异性过表达NMHC II-B转基因小鼠， 心脏目前正在对转基因小鼠进行分析， 用于拯救基因敲除小鼠。获救的老鼠 将有望经历一个正常的心脏发育，但仍然 大脑有缺陷。因此，基因敲除小鼠被 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。截短形式的 非肌肉肌球蛋白重链II-A（NMHC II-A），缺乏1-592 氨基酸，也被用来检查其细胞功能。 将绿色荧光蛋白（GFP）融合到 全长和截短的NMHC II-A和融合蛋白 蛋白质在HeLa Tet-off细胞系中稳定表达，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 线诱导细胞变圆伴肌动蛋白丝重排 和局灶性粘连消失。这些变化被逆转了 当截短的NMHC II-A的表达被终止时， 加入强力霉素。HeLA Tet-off细胞表达 全长NMHC II-A：GFP融合蛋白没有显示出这一点 有或没有强力霉素的表型。共聚焦显微 研究表明，全长和截短的NMHC II-A 都集中在应力纤维上我们认为NMHC II-A是 通过稳定应力纤维来维持细胞形状 和局部粘连

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.