The Role of Nonmuscle Myosins in Development

非肌肉肌球蛋白在发育中的作用

基本信息

批准号：
8557928
负责人：
Robert Adelstein
金额：
$ 42.52万
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

来源：
https://reporter.nih.gov/project-details/8557928
关键词：
Actins Adhesions Affect Albuminuria Alternative Splicing Amino Acid Sequence Amino Acids Apoptosis Apoptotic Biological Assay Bleeding time procedure Bone Marrow Bone Marrow Stem Cell Breeding Cataract Cell Cycle Kinetics Cell Line Cell Polarity Cell division Cell-Cell Adhesion Cell-Matrix Junction Cells Cessation of life Clot retraction Code Complementary DNA Cytokinesis Defect Development Disease Embryo Exons Extramedullary Eye Fibroblasts Filament Filopodia Fluorescence Focal Adhesions Focal Segmental Glomerulosclerosis France Genes Goals Green Fluorescent Proteins Human Image Immigration Immune response In Vitro Initiator Codon Kidney Kidney Diseases Kinetics Learning Life Light Liver Lung Megakaryocytes Microscopy Motor Mus Mutant Strains Mice Mutation Myosin ATPase Myosin Heavy Chains Myosin Type II Nonmuscle Myosin Type IIA Organ Patients Peptide Sequence Determination Phenotype Point Mutation Property Protein Isoforms Regulation Role Speed Stress Fibers Structure Testis Brain Time Tissues Transgenic Mice Wild Type Mouse cell motility cell type cellular imaging enhanced green fluorescent protein hearing impairment homologous recombination in vitro activity in vivo insight mRNA Precursor migration mouse development mouse model mutant non-muscle myosin premature promoter research study

项目摘要

In order to generate mouse models of human MYH9-related disease, which is caused by mutations in nonmuscle myosin IIA, and to study the pathological mechanisms of the mutations in these diseases, we generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with GFP was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the bone marrow show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domains manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related diseases. In addition to using these mutant mice to study the relation between the nonmuscle myosin IIA mutation and disease, we plan to use various cells lines derived from these mice to study the effects of the mutation on basic properties of the cell. These include cytokinesis, cell-cell and cell matrix adhesion, cell polarity and cell migration. To gain clear insights into the distribution and function of different isoforms of nonmuscle myosin II (NMII) in normal mice, the enhanced GFP or mCherry sequence has been inserted in front of the start codon of the Myh9 gene in the first coding exon. We have obtained homozygous GFP or mCherry tagged NMIIA mice. The expression level of the tagged NMIIA is similar to that of the endogenously expressed untagged NMIIA in wild type mice. This fluorescence tagged NMIIA mouse model will shed light on the functions of NM IIA in development and in different cell types, tissues, and organs. Various cell lines derived from the mice will be used to study the regulation and function of NM IIA in adhesion, cell polarity and cell migration. For example, we have isolated bone marrow stem cells from GFP-NMIIA mice and sent them to Dr. Ana-Maria Lennon-Dumnils lab in Institut Curie, France to study the function of NM IIA in DC cell migration during the immune response. The purpose of the substitution experiment is to learn whether one isoform of NM II, specifically NM IIC1, can functionally replace a second one, NM IIA, in mice. Alternative splicing of pre-mRNA of NMIIC generates several isoforms. An alternative exon encoding 8 amino acids can be incorporated into loop 1 at amino acid 227 to form NM IIC1. Another alternative exon encoding 41 amino acids can be incorporated into loop 2 at amino acid 636 to form NM IIC2. In a few cases, both inserts can be incorporated to form NM IIC1C2. NM IIC1 is found in a variety of tissues such as liver, kidney, testes, brain, and lung. In vivo study also found that an enzymatically active fragment, HMM of NM IIC1 has increased actin-activated MgATPase activity and in vitro motility compared with HMM of NM IIC0, which has no insert . Among the four isoforms of NM IIC, NM IIC1s actin-activated MgATPase activity and in vitro motility are closer to NM IIA than other isoforms of NM IIC. Therefore, we chose NM IIC1 to replace Nm IIA in vivo. To replace NM IIA with NM IIC1 in the mouse model, homologous recombination was used to inactivate NM IIA by inserting the cDNA for NM IIC1-GFP into the first coding exon of the Myh9 gene. We have obtained heterozygous NM IIC replacing NM IIA mice. However, breeding of heterozygous mutant mice does not produce homozygous NM IIC replacing NM IIA mice. Homozygous embryos die around embryonic day 10.5. At both E9.5 and E10.5, AC/AC embryos are dramatically smaller than wild-type littermates and are developmentally delayed. Apoptotic cells were found in AC/AC embryos at E9.5 and E10.5, indicating the cause of embryonic death is apoptosis related. Embryonic explants from E9.5 embryos were cultured and the mouse embryo fibroblast (MEF) migration from the explants was recorded with time lapse microscopy. Compared with wild type explants, homozygous AC/AC MEFs have a much higher outgrowth speed. Images of MEF cultures show more of the AC/AC MEFs seem to be polarized than the wild type, having both more lamellipodia ruffling and long filopodia structures, indicating cells in migration. Immunostaining shows that myosin IIC can form filaments in AC/AC MEFs. Stress fibers in AC/AC MEFs were fewer and less organized. Focal adhesions in AC/AC MEFs are also fewer in number and smaller in size. These results indicate that NMHCII-C cannot replace II-As function during focal adhesion formation and maturation. Additional experiments such as transwell assays will be carried out to further study the kinetic properties in AC/AC MEFs.

为了建立由非肌肉肌球蛋白IIA突变引起的人类MYH9相关疾病的小鼠模型，并研究这些疾病突变的病理机制，我们建立了3个小鼠系，每个系在非肌肉肌球蛋白IIA中具有不同的突变。肌球蛋白 II-A 基因 Myh9（R702C、D1424N 和 E1841K）。每个品系都会产生与人类患者相似的 MYH9 相关疾病。将与GFP融合的R702C突变人cDNA引入Myh9的第一个编码外显子中，并将D1424N和E1841K突变直接引入相应的外显子中。纯合 R702C 小鼠在胚胎第 10.5-11.5 天死亡，而纯合 D1424N 和 E1841K 小鼠则可存活。所有杂合子和纯合子突变小鼠均表现出巨血小板减少症，伴有出血时间延长、凝块回缩缺陷和髓外巨核细胞增加。对培养的巨核细胞和骨髓中巨核细胞的活细胞成像的研究表明，杂合的 R702C 巨核细胞形成更少、更短的前血小板，具有更少的分支和更大的芽。结果表明，前血小板形成被破坏会导致小鼠（最有可能是人类）的巨血小板减少症。我们还观察到过早白内障形成、肾脏异常，包括蛋白尿、局灶节段性肾小球硬化和进行性肾脏疾病，以及轻度听力损失。我们的结果表明，肌球蛋白运动或丝形成结构域发生突变的杂合小鼠表现出与患有 MYH9 相关疾病的人类相似的血液学、眼睛和肾脏表型。除了使用这些突变小鼠研究非肌肉肌球蛋白IIA突变与疾病之间的关系外，我们还计划使用源自这些小鼠的各种细胞系来研究突变对细胞基本特性的影响。这些包括胞质分裂、细胞与细胞和细胞基质粘附、细胞极性和细胞迁移。为了清楚地了解正常小鼠中非肌肉肌球蛋白 II (NMII) 不同亚型的分布和功能，增强的 GFP 或 mCherry 序列已插入到第一个编码外显子中 Myh9 基因的起始密码子之前。我们获得了带有 GFP 或 mCherry 标签的纯合 NMIIA 小鼠。标记的 NMIIA 的表达水平与野生型小鼠中内源表达的未标记的 NMIIA 相似。这种荧光标记的 NMIIA 小鼠模型将揭示 NM IIA 在发育过程中以及在不同细胞类型、组织和器官中的功能。来自小鼠的各种细胞系将用于研究 NM IIA 在粘附、细胞极性和细胞迁移方面的调节和功能。例如，我们从GFP-NMIIA小鼠中分离出骨髓干细胞，并将其送往法国居里研究所的Ana-Maria Lennon-Dumnils博士实验室，研究NM IIA在免疫反应过程中DC细胞迁移中的功能。替代实验的目的是了解 NM II 的一种亚型（特别是 NM IIC1）是否可以在小鼠中功能性地替代另一种亚型（NM IIA）。 NMIIC 前 mRNA 的选择性剪接产生多种亚型。编码 8 个氨基酸的替代外显子可以并入环 1 的氨基酸 227 处，形成 NM IIC1。编码41个氨基酸的另一个替代外显子可以并入环2的氨基酸636处以形成NM IIC2。在少数情况下，两个插入片段可以合并形成 NM IIC1C2。 NM IIC1 存在于多种组织中，如肝、肾、睾丸、脑和肺。体内研究还发现，与没有插入片段的 NM IIC0 的 HMM 相比，NM IIC1 的酶活性片段 HMM 增加了肌动蛋白激活的 MgATPase 活性和体外运动性。在 NM IIC 的四种亚型中，NM IIC1 肌动蛋白激活的 MgATPase 活性和体外运动性比 NM IIC 的其他亚型更接近 NM IIA。因此，我们选择NM IIC1在体内替代Nm IIA。为了在小鼠模型中用 NM IIC1 替换 NM IIA，通过将 NM IIC1-GFP 的 cDNA 插入 Myh9 基因的第一个编码外显子，使用同源重组来灭活 NM IIA。我们获得了杂合的 NM IIC 替代 NM IIA 小鼠。然而，杂合突变小鼠的繁殖并不能产生替代 NM IIA 小鼠的纯合 NM IIC。纯合胚胎在胚胎第 10.5 天左右死亡。在 E9.5 和 E10.5 中，AC/AC 胚胎明显小于野生型同窝胚胎，并且发育迟缓。在E9.5和E10.5的AC/AC胚胎中发现了凋亡细胞，表明胚胎死亡的原因与细胞凋亡有关。培养来自 E9.5 胚胎的胚胎外植体，并用延时显微镜记录来自外植体的小鼠胚胎成纤维细胞 (MEF) 迁移。与野生型外植体相比，纯合 AC/AC MEF 具有更高的生长速度。 MEF 培养物的图像显示，更多的 AC/AC MEF 似乎比野生型极化，具有更多的片状伪足褶皱和长丝状伪足结构，表明细胞正在迁移。免疫染色显示肌球蛋白 IIC 可以在 AC/AC MEF 中形成细丝。 AC/AC MEF 中的应力纤维越来越少且组织较差。 AC/AC MEF 中的粘着斑数量较少且尺寸较小。这些结果表明，在粘着斑形成和成熟过程中，NMHCII-C 不能替代 II-As 功能。将进行 Transwell 等其他实验，以进一步研究 AC/AC MEF 的动力学特性。