The Function of Nonmuscle Myosin Heavy Chains

非肌肉肌球蛋白重链的功能

基本信息

项目摘要

Nonmuscle Myosin II-A is Essential for Mouse Placental and Embryonic Development In vertebrates three genes, Myh9, Myh10 and Myh14 encode three different isoforms of nonmuscle myosin II heavy chain II (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. Gene disruption studies in mice reveal dramatically different embryonic-lethal phenotypes upon knockout of NM II-A versus II-B. 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. Disrupted NM II-B causes later embryonic lethality between E14.5 and birth due to cardiac and neuronal development defects. Though our previous studies indicated that the functions of NM II-B can be partially replaced by II-A during development, it is unclear whether this is the case in the reverse situation. To investigate whether NM II-B can replace II-A and to uncover novel functions of NM II-A during development, we used homologous recombination to generate a mouse line (Ab*/Ab* mice) in which NM II-A is disrupted and cDNA encoding human NMHC II-B is placed under control of the II-A promoter. In addition, as a control mouse line, cDNA encoding human NM II-A is also inserted in the II-A locus (AmCh/AmCh). Our results show: first, normal homozygous Ab*/Ab* (II-B replacing II-A) embryos were found at embryonic day (E)6.5 indicating that II-B can rescue the visceral endoderm abnormalities found in the II-A null mice at E6.5, allowing gastrulation and organogenesis to occur. It also demonstrates that the lethality in II-A null mice was not due to a specific role of NM II-A but due to the decrease in total NM II in the embryo and the absence of any NM II at this early stage in the visceral endoderm. Second, Ab*/Ab* mice exhibit severe defects in the placenta, which result in embryonic lethality at E9.5-10.5, indicating a unique function for NM II-A in normal placental development. Third, in the mutant placentas, the fetal vessels failed to invade into the labyrinthine layer of the placenta, suggesting defects in cell migration. Abnormal actin organization, reduced focal adhesion formation and abnormal migratory behavior were found in the Ab*/Ab* mouse embryonic fibroblasts in culture, indicating that NM II-A has an important role during these processes that can not be replaced by II-B. Taken together, our results indicate the importance of an isoform-specific function for NM II-A in placenta development and highlight the role of NM II-A in cell migration and adhesion. Intact Nonmuscle Myosin II-A is Critical for Its Functions To further explore whether the isoform-specific roles of NM II-A and II-B are associated with their two functional domains (N terminal head and C terminal rod), we also generated two mouse lines in which the NM II-A is replaced by two chimeric NM IIs, one encoding the N-terminal motor domain of NMHC II-A fused to the C-terminal II-B rod domain (Aab/Aab mice) and one encoding the N-terminal domain of NMHC II-B fused to the C-terminal domain of II-A (Aba/Aba mice). Our results showed that Aba/Aba mice die at a similar 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 survived to E12.5, with lethal defects in placentation although fetal vessels were found to invade into the labyrinthine layer of the placenta. In addition, these experiments confirmed that the C-terminal rod domain of NM II-A or II-B play vital roles in determining cellular localization. Collectively, our results highlight the importance of an intact NM II-A molecule for its functions during mid-gestation. Characterizing the Biochemical Properties of Full-length Nonmuscle Myosin IIs In vertebrates three different isoforms of nonmuscle myosin heavy chain (NMHC) II have been identified. Their encoded genes in humans are referred as Myh9, Myh10, Myh14 and their protein product are called NMHC-IIA, IIB and IIC respectively. Each heavy chain has an N-terminal globular head domain containing the actin and ATP binding sites required for motor activity and a long -helical, coiled coil C-terminal rod involved in dimerization and filament assembly. The biochemical properties of these isoforms have been studied by expression of an enzymatically active fragment, heavy meromyosin (HMM), which is a dimer containing the motor domains and a part of the rods. The resulting kinetic values may have some limitations in that recent studies showed intramolecular interaction between the rods and the heads involved in the switching off myosin II activity. To circumvent the limitations of HMM and assess additional biochemical properties, we have successfully produced 9 full length NM II proteins in the Sf9-baculovirus system. These proteins include full length wild type NM II-A, II-A with the N93K point mutation, II-A with the D1424N point mutation, II-A with the E1841K point mutation, wild type II-B, IIB with B2 insert (IIB-B2), and II-B with the R709C point mutation. Additionally, two chimeric NMHC II proteins with either the amino-terminal head of II-A fused to the rod of II-B (IIAB) or the II-B head preceding the II-A rod: (IIBA) were made. We have also made an enzymatically active NM II fragment, S1 which is shorter than HMM and is a monomer: IIB-B2 S1. Our initial purification experiments showed that in contrast to the previous utility of FLAG affinity chromatography in NMHC II-A HMM purification, the FLAG tag at the C-terminal of full length II-A did not aid in purification. Based on this fact, we appended the FLAG tag to the N-terminal end where it does aid in purification of these full length NM IIs. The purification and biochemical assays of these proteins are underway.
非肌肉肌球蛋白 II-A 对于小鼠胎盘和胚胎发育至关重要 在脊椎动物中,三个基因 Myh9、Myh10 和 Myh14 编码非肌肉肌球蛋白 II 重链 II (NMHC II) 的三种不同亚型。非肌肉肌球蛋白 II (NM II) 的运动活性位于 N 端球状头结构域,丝状结构位于 C 端杆状结构域。小鼠基因破坏研究揭示了 NM II-A 与 II-B 敲除后胚胎致死表型的显着不同。小鼠中 NM II-A 的消融导致 E6.5 致死,细胞间粘附缺陷且无法产生有能力的内脏内胚层。由于心脏和神经元发育缺陷,NM II-B 中断会导致 E14.5 和出生之间的胚胎死亡。虽然我们之前的研究表明NM II-B的功能在发育过程中可以被II-A部分替代,但尚不清楚在相反的情况下是否也是如此。为了研究 NM II-B 是否可以取代 II-A 并揭示 NM II-A 在发育过程中的新功能,我们使用同源重组来生成 NM II-A 被破坏的小鼠品系(Ab*/Ab* 小鼠)编码人NMHC II-B的cDNA置于II-A启动子的控制之下。 此外,作为对照小鼠系,编码人NM II-A的cDNA也被插入到II-A基因座(AmCh/AmCh)中。 我们的结果显示:首先,在胚胎第 (E)6.5 天发现正常纯合 Ab*/Ab*(II-B 取代 II-A)胚胎,表明 II-B 可以挽救 II-A 无效胚胎中发现的内脏内胚层异常。小鼠在 E6.5,允许原肠胚形成和器官发生发生。它还表明,II-A 无效小鼠的致死性并非由于 NM II-A 的特定作用,而是由于胚胎中总 NM II 的减少以及内脏早期阶段缺乏任何 NM II。内胚层。其次,Ab*/Ab* 小鼠的胎盘表现出严重缺陷,导致 E9.5-10.5 胚胎致死,表明 NM II-A 在正常胎盘发育中具有独特的功能。第三,在突变胎盘中,胎儿血管未能侵入胎盘的迷路层,表明细胞迁移存在缺陷。在培养的Ab*/Ab*小鼠胚胎成纤维细胞中发现肌动蛋白组织异常、粘着斑形成减少和异常迁移行为,表明NM II-A在这些过程中具有II-B无法替代的重要作用。总而言之,我们的结果表明 NM II-A 的异构体特异性功能在胎盘发育中的重要性,并强调了 NM II-A 在细胞迁移和粘附中的作用。 完整的非肌肉肌球蛋白 II-A 对其功能至关重要 为了进一步探讨 NM II-A 和 II-B 的亚型特异性作用是否与其两个功能域(N 末端头和 C 末端杆)相关,我们还生成了两个 NM II-A 被替换的小鼠品系由两个嵌合 NM II 组成,一种编码 NMHC II-A 的 N 端运动结构域,与 C 端 II-B 杆结构域融合(Aab/Aab 小鼠),另一种编码 NMHC II-B 融合的 N 端结构域到II-A 的 C 端结构域(Aba/Aba 小鼠)。我们的结果表明,Aba/Aba 小鼠的死亡年龄与 Ab*/Ab* 小鼠相似,表明 N 端运动结构域在 II-A 和 II-B 之间可互换,直至 E9-10,尽管 II-A 和 II-B 的动力学特性存在差异。电机。 Aab/Aab 小鼠存活至 E12.5,尽管发现胎儿血管侵入胎盘的迷路层,但胎盘存在致命缺陷。 此外,这些实验证实NM II-A或II-B的C端杆状结构域在确定细胞定位中发挥着重要作用。 总的来说,我们的结果强调了完整的 NM II-A 分子在妊娠中期发挥其功能的重要性。 全长非肌肉肌球蛋白 II 的生化特性表征 在脊椎动物中,已鉴定出三种不同的非肌肉肌球蛋白重链 (NMHC) II 亚型。它们在人类中编码的基因被称为Myh9、Myh10、Myh14,它们的蛋白质产物分别被称为NMHC-IIA、IIB和IIC。每条重链都有一个 N 端球状头结构域,其中包含运动活动所需的肌动蛋白和 ATP 结合位点,以及一个参与二聚化和细丝组装的长螺旋、卷曲线圈 C 端杆。这些同工型的生化特性已通过酶活性片段重片段肌球蛋白 (HMM) 的表达进行了研究,该片段是包含运动结构域和杆的一部分的二聚体。由此产生的动力学值可能有一些局限性,因为最近的研究表明杆和头之间的分子内相互作用参与了肌球蛋白 II 活性的关闭。为了规避 HMM 的局限性并评估其他生化特性,我们在 Sf9 杆状病毒系统中成功生产了 9 个全长 NM II 蛋白。这些蛋白质包括全长野生型 NM II-A、具有 N93K 点突变的 II-A、具有 D1424N 点突变的 II-A、具有 E1841K 点突变的 II-A、野生型 II-B、具有 B2 插入片段的 IIB( IIB-B2) 和具有 R709C 点突变的 II-B。另外,制备了两种嵌合NMHC II蛋白,其中II-A的氨基末端头部与II-B杆融合(IIAB),或者II-B头部在II-A杆之前:(IIBA)。我们还制作了一个具有酶活性的 NM II 片段 S1,它比 HMM 短并且是一个单体:IIB-B2 S1。我们的初步纯化实验表明,与之前在 NMHC II-A HMM 纯化中使用 FLAG 亲和层析相比,全长 II-A C 端的 FLAG 标签对纯化没有帮助。基于这一事实,我们将 FLAG 标签附加到 N 末端,它确实有助于纯化这些全长 NM II。这些蛋白质的纯化和生化测定正在进行中。

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

Robert Adelstein其他文献

Robert Adelstein的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ truncateString('Robert Adelstein', 18)}}的其他基金

The Role Nonmuscle Myosin II Isoforms in Focal Adhesions
非肌肉肌球蛋白 II 亚型在局灶性粘连中的作用
  • 批准号:
    8557934
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
Transgenic Core
转基因核心
  • 批准号:
    8344984
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
The Role of Nonmuscle Myosins in Development
非肌肉肌球蛋白在发育中的作用
  • 批准号:
    7969055
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
Pathology Core
病理学核心
  • 批准号:
    8940155
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
Alternative Splicing of Nonmuscle Myosin Heavy Chains
非肌肉肌球蛋白重链的选择性剪接
  • 批准号:
    9557299
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
Conditional Ablation and Mutation of Nonmuscle Myosins
非肌肉肌球蛋白的条件性消融和突变
  • 批准号:
    8149503
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
Conditional Ablation and Mutation of Nonmuscle Myosins
非肌肉肌球蛋白的条件性消融和突变
  • 批准号:
    7969068
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
The Role of Nonmuscle Myosins in Development
非肌肉肌球蛋白在发育中的作用
  • 批准号:
    8557928
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
Nonmuscle Myosin II and Upstream and Downstream Signaling
非肌肉肌球蛋白 II 和上下游信号传导
  • 批准号:
    8557932
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:
The Role of Nonmuscle Myosin 2B In Vivo
非肌肉肌球蛋白 2B 在体内的作用
  • 批准号:
    10008768
  • 财政年份:
  • 资助金额:
    $ 35.04万
  • 项目类别:

相似国自然基金

微波敏感型铁死亡纳米放大器的构建及其增敏肝癌消融-免疫联合治疗的应用与机制研究
  • 批准号:
    82302368
  • 批准年份:
    2023
  • 资助金额:
    30 万元
  • 项目类别:
    青年科学基金项目
低密度中性粒细胞促进早期乳腺癌微波消融治疗后复发转移的作用及机制研究
  • 批准号:
    82303710
  • 批准年份:
    2023
  • 资助金额:
    30 万元
  • 项目类别:
    青年科学基金项目
纳米刀消融通过METTL5介导的核糖体18S rRNA m6A修饰募集MDSC促进肝癌复发的作用及机制研究
  • 批准号:
    82373004
  • 批准年份:
    2023
  • 资助金额:
    49 万元
  • 项目类别:
    面上项目
典型草原不同退化类型雪水消融过程水分转换效率研究
  • 批准号:
    32360295
  • 批准年份:
    2023
  • 资助金额:
    32 万元
  • 项目类别:
    地区科学基金项目
基于荷顺铂温敏纳米凝胶载KU135介入栓塞联合射频消融治疗肝癌的实验研究
  • 批准号:
    82302331
  • 批准年份:
    2023
  • 资助金额:
    30 万元
  • 项目类别:
    青年科学基金项目

相似海外基金

ArpC3-mediated actin remodeling in insulin granule exocytosis and diabetes
ArpC3 介导的肌动蛋白重塑在胰岛素颗粒胞吐作用和糖尿病中的作用
  • 批准号:
    10583734
  • 财政年份:
    2023
  • 资助金额:
    $ 35.04万
  • 项目类别:
Tubulin modifications and cytoskeletal alterations in aging
衰老过程中的微管蛋白修饰和细胞骨架变化
  • 批准号:
    10590128
  • 财政年份:
    2023
  • 资助金额:
    $ 35.04万
  • 项目类别:
Receptor tyrosine kinase signaling in astrocyte migration and angiogenesis
星形胶质细胞迁移和血管生成中的受体酪氨酸激酶信号传导
  • 批准号:
    10734124
  • 财政年份:
    2023
  • 资助金额:
    $ 35.04万
  • 项目类别:
Mechanotransduction via LIM Domain Protein Mechanosensing
通过 LIM 结构域蛋白机械传感进行机械转导
  • 批准号:
    10735689
  • 财政年份:
    2023
  • 资助金额:
    $ 35.04万
  • 项目类别:
Altered nucleus-cytoskeleton coupling in dystrophic muscle
营养不良性肌肉中核-细胞骨架耦合的改变
  • 批准号:
    10615087
  • 财政年份:
    2023
  • 资助金额:
    $ 35.04万
  • 项目类别:
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了