The Role of Nonmuscle Myosins in Development

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，每个小鼠系的非肌肉肌球蛋白II-A基因Myh9(R702C、D1424N和E1841K)都有不同的突变。每一株都会患上与Myh9相关的疾病，与在人类患者中发现的疾病相似。在Myh9的第一个编码外显子中引入与GFP融合的R702C突变型人基因，在相应的外显子中直接引入D1424N和E1841K突变。纯合子R702C小鼠在胚胎10.5-11.5天死亡，而纯合子D1424N和E1841K小鼠存活。所有杂合子和纯合子突变小鼠都表现出大血小板减少，出血时间延长，凝块回缩缺陷，髓外巨核细胞增多。对培养的巨核细胞和骨髓中巨核细胞的活细胞成像研究表明，R702C杂合子巨核细胞形成的前血小板更少、更短，分支更少，芽更大。结果表明，破坏的前血小板形成导致了小鼠和很可能的人类的大血小板减少症。我们还观察到早发性白内障的形成，肾脏异常，包括蛋白尿，局灶性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，肌球蛋白运动或微丝形成区域突变的杂合子小鼠表现出与患有Myh9相关疾病的人类相似的血液学、眼睛和肾脏表型。除了利用这些突变小鼠研究非肌肉肌球蛋白IIA突变与疾病的关系外，我们还计划使用这些小鼠衍生的各种细胞系来研究突变对细胞基本性质的影响。这些包括胞质分裂、细胞-细胞和细胞基质黏附、细胞极性和细胞迁移。 为了更清楚地了解不同亚型的非肌肉肌球蛋白II(NMII)在正常小鼠中的分布和功能，在Myh9基因第一个编码外显子的起始密码子之前插入了增强的GFP或mCherry序列。我们已经获得了纯合子GFP或mCherry标记的NMIIA小鼠。标记的NMIIA在野生型小鼠体内的表达水平与内源性表达的未标记的NMIIA相似。这一荧光标记的NMIIA小鼠模型将阐明NMIIA在发育过程中以及在不同细胞类型、组织和器官中的功能。从小鼠来源的各种细胞系将被用来研究NM IIA在黏附、细胞极性和细胞迁移方面的调节和功能。例如，我们已经从GFP-NMIIA小鼠中分离出骨髓干细胞，并将它们送到法国居里研究所的Ana-Maria Lennon-Dumnils博士实验室，以研究NM IIA在免疫反应过程中DC细胞迁移中的作用。 替代实验的目的是了解NM II的一个亚型，特别是NM IIC1，是否可以在功能上取代第二个亚型NM IIA。NMIIC前-mRNA的选择性剪接产生几种异构体。编码8个氨基酸的另一个外显子可以结合到环1的227位氨基酸形成NM IIC1。编码41个氨基酸的另一个外显子可结合到636位氨基酸的环2中，形成NM IIC2。在少数情况下，两个插入物可以结合在一起形成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，与没有插入的NM IIC0的HMM相比，NM IIC1的酶活性片段HMM具有更高的肌动蛋白激活的镁ATPase活性和体外运动能力。在NM IIC的四种亚型中，NM IIC的肌动蛋白激活的镁ATPase活性和体外运动能力比其他亚型更接近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取代NM IIA，将NM IIC1-GFP的cDNA插入Myh9基因的第一个编码外显子，用同源重组灭活NM IIA。我们用杂合子NMIIC代替NMIIA小鼠。然而，培育杂合子突变小鼠并不能产生纯合子NMIIC来取代NMIIA小鼠。纯合子胚胎在胚胎第10.5天左右死亡。在E9.5和E10.5，AC/AC胚胎比野生型小得多，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。用时间推移显微镜观察小鼠胚胎成纤维细胞(MEF)的迁移情况。与野生型外植体相比，纯合AC/AC MEF的生长速度要快得多。MEF培养的图像显示，AC/AC MEF似乎比野生型更极化，具有更多的片状褶皱和长丝足状结构，表明细胞正在迁移。免疫组织化学染色显示，肌球蛋白IIC在AC/AC MEF中可形成细丝。AC/AC MEF中应力纤维较少，组织化程度较差。AC/AC MEF中的灶性粘连数量较少，体积较小。这些结果表明，NMHCII-C不能取代II-AS在局灶性黏附形成和成熟过程中的作用。为了进一步研究AC/AC MEF的动力学性质，还将进行其他实验，如Transwell实验。

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.