The Function of Nonmuscle Myosin Heavy Chains

非肌肉肌球蛋白重链的功能

基本信息

批准号：
7734985
负责人：
ROBERT ADELSTEIN
金额：
$ 37.61万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

In vertebrates three genes, Myh9, Myh10 and Myh14 encode three different isoforms of the nonmuscle myosin II heavy chain (NMHC II). The motor activity of nonmuscle myosin II (NM II) resides in the N-terminal globular head domain and filament formation resides in the C-terminal rod domain. Previous work has shown that ablation of NM II-A in mice results in lethality by E6.5 with defects in cell-cell adhesion and a failure to produce a competent visceral endoderm. To understand the function of NM II-A during development we used homologous recombination to generate 4 different mouse lines:1-we replaced NM II-A with NM II-B by "knocking in" cDNA encoding human NMHC II-B into the II-A locus, thereby ablating II-A and placing NMHC II-B under control of the endogenous II-A promoter (Ab*/Ab* mice). 2-we replaced endogenous NM II-A by knocking in two chimeric NMHCs, one encoding the N-terminal motor domain of NMHC II-A fused to the C-terminal II-B rod domain (Aab/Aab mice), 3-and another encoding the N-terminal domain of NMHC II-B fused to the C-terminal domain of II-A (Aba/Aba mice). 4-For control mice we inserted cDNA encoding NMHC II-A into the II-A locus. Replacing NM II-A with II-B (Ab*/Ab*) allows normal development of the visceral endoderm, gastrulation, organogenesis and survival to E9-10. These mice show a delay in embryonic turning with defects in the vasculature and endocardium. Aba/Aba mice die at the same age as Ab*/Ab* mice indicating that the N-terminal motor domains are interchangeable between II-A and II-B up to E9-10 despite differences in the kinetic properties of the motors. Aab/Aab mice survive beyond E12 with a normal vasculature but with a hypoplastic heart and an aorta overriding a ventricular septal defect. Thus, the N-terminal II-A motor domain has extended the developmental period from E9-10 to E13, displaying its importance for the early embryonic development.

在脊椎动物中，三个基因MyH9，MyH10和MyH14编码了非肌动蛋白II重链的三种不同同工型（NMHC II）。非肌肉肌球蛋白II（NM II）的运动活性存在于N端球形头域和细丝形成中，位于C末端杆域中。先前的工作表明，小鼠中NM II-A的消融导致E6.5的致死性，并且细胞细胞粘附缺陷和未能产生胜任的内脏内胚层。为了了解开发过程中NM II-A的功能，我们使用同源重组来生成4种不同的鼠标线：1-WE通过“敲打”编码人NMHC II-B的NM II-A代替NM II-A，从而在II-A基因座中置于II-A基因座中，从而在II-A中散发II-A，并在NMHC II-A中放置NMHC II-B，并将其放置在内源性II-A II-A-a-a促进剂（Ab* Mim）下。 2-WE通过在两个嵌合NMHC中敲击内源性NM II-A，一个编码NMHC II-A的N末端运动结构域与C-末端II-B杆域（AAB/AAB/AAB小鼠）融合，3-及另一个编码NMHC II-B Fused TO ton TO t ton TO c-terminical ton/aba的NMHC II-B型号的N端子域（II-BA）。 4-For对照小鼠我们将编码NMHC II-A的cDNA插入II-A基因座。用II-B（Ab*/ab*）代替NM II-A，可以正常发展内胚层，胃胃，器官发生和生存率为E9-10。这些小鼠在脉管系统和内膜内部有缺陷显示胚胎转弯的延迟。 ABA/ABA小鼠在与AB*/AB*小鼠的年龄相同的年龄中死亡，表明尽管电动机的动力学特性差异，但N末端运动域在II-A和II-B之间仍可互换至E9-10。 AAB/AAB小鼠的生存在E12之外，具有正常的脉管系统，但心脏不良和主动脉覆盖了心室间隔缺陷。因此，N末端II-A运动域已将发育时期从E9-10扩展到E13，表明了其对早期胚胎发育的重要性。