The Role of Nonmuscle Myosins in Development

编码非肌肉肌球蛋白重链(NMHC)IIA的Myh9基因发生点突变的人类会出现各种综合征，包括血小板(大血小板减少症)、肾脏(肾小球肾炎)和粒细胞(包涵体)缺陷。到目前为止，已有30多种不同的Myh9突变被报道，包括错义突变和无义突变。这些研究的目的是通过为其中三个突变(运动域的R702C；杆域的D1424N和E1841K)建立小鼠模型，并研究由此产生的小鼠表型，来深入了解这些突变引起疾病的病理机制。先前的体外工作表明，位于NMHC IIA运动区的R702C突变会影响肌球蛋白的镁ATPase活性，并与肌球蛋白的运动速度有关；而杆状区的D1424N和E1841K突变可能会影响NMHC IIA的细丝形成。我们通过同源重组，用突变体R702C或D1424N替换野生型NMHC IIA，获得了R702C和D1424N突变小鼠。E1841K突变小鼠来自杜克大学的M·凯利博士。培育杂合的R702C突变小鼠并没有产生纯合子突变后代。纯合子在胚胎8.5天到10.5天之间死亡。另一方面，培育杂合的D1424N突变小鼠产生接近正常比例的纯合子突变后代，而E1841K突变小鼠也产生纯合子突变后代，这表明在胚胎发育过程中，NMHC IIA运动域的突变可能比杆状突变产生更严重的影响。有趣的是，在R702C和D1424N成年杂合小鼠的血液涂片中都发现了巨大的血小板。与野生型相比，三个突变体的平均血小板体积均显著增加(6.12+/-0.62fl，wt；10.17+/-1.50fl，杂合子R702C；10.13+/-1.66fl，杂合子D1424N；10.92+/-1.83fl，杂合子E1841K)。通过检测尿白蛋白/肌酐比值来研究肾功能。两个突变系的部分(但不是全部)成年杂合子在8-9周龄时具有较高的白蛋白/肌酐比值，这表明一些杂合子突变可能在早期就出现肾脏损害。这些初步结果表明，这些小鼠模型应该有助于理解人类Myh9相关疾病的病理生理学。 除了利用这些突变小鼠研究非肌肉肌球蛋白II-A突变与疾病的关系外，我们还计划使用这些小鼠来源的各种细胞来研究突变对细胞基本性质的影响。这些包括细胞-细胞和细胞基质的黏附，细胞的极性和细胞的迁移。 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。我们已经获得了杂合子GFP或mCherry标记的NMIIA小鼠。在两个杂合子突变体中，标记的NMIIA的表达水平都相对低于内源表达的非标记的NMIIA。我们已经建立了杂合子标记的NMIIA小鼠与CRE小鼠的繁育笼子，以去除Neo盒，因此标记的NMIIA蛋白的表达水平可以提高到正常水平。这一标记的NMIIA小鼠模型将有助于阐明NMIIA在发育中的作用。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。我们还计划将mCherry标记的NM IIA小鼠与GFP标记的NMIIB小鼠杂交。MCherry标记的NMIIA和GFP标记的NMIIB的后代可用于研究NMIIA和NMIIB在体内能否形成共聚物。从该小鼠获得的细胞系(如成纤维细胞)可以用共聚焦显微镜或TIRF显微镜研究NMIIA和NMIIB在细胞极性和迁移方面的不同动力学特性。 另一项研究的目的是了解NM II的一个亚型，特别是NM IIC，是否可以在小鼠身上在功能上取代第二个亚型NM IIA。为了用NM IIC取代NM IIA，将NM IIC-GFP的cDNA插入Myh9基因的第一个编码外显子，利用同源重组来灭活NM IIA。在NM IIA启动子的控制下，我们已经获得了几个杂合子NMIIC-GFP的阳性胚胎干细胞株。这些胚胎干细胞将用于制造嵌合体小鼠。

Humans with point mutations in MYH9, the gene encoding nonmuscle myosin heavy chain (NMHC) IIA, develop a variety of syndromes including defects in their platelets (macrothrombocytopenia), kidneys (glomerulonephritis) and granulocytes (inclusion bodies). More than 30 different mutations in MYH9 have been reported to date, including both mis-sense and nonsense mutations. The purpose of these studies is to gain insight into the pathological mechanism of the diseases caused by these mutations by creating mouse models for three of the mutations (R702C in motor domain; D1424N and E1841K in rod domain) and studying the resultant mouse phenotypes. Previous in vitro work has shown that the R702C mutation, which is in the motor domain of NMHC IIA compromises the MgATPase activity and is responsible for the movement velocity of the myosin; while mutations D1424N and E1841K in the rod domain may affect NMHC IIA filament formation. We have produced both R702C and D1424N mutant mice by using homologous recombination to replace wild type NMHC IIA with mutant R702C or D1424N in NMHC IIA. The E1841K mutant mice were obtained from Dr. M Kelley at Duke University. Breeding of heterozygous R702C mutant mice has not produced homozygous mutant offspring. The homozygotes die between embryonic day 8.5 and 10.5. On the other hand, breeding of heterozygous D1424N mutant mice produced homozygous mutant offspring at close to normal ratios, and E1841K mutant mice also have homozygous mutant offspring, suggesting that mutations in the motor domain of NMHC IIA may have a more severe effect than mutations in the rod during embryonic development. Interestingly, giant platelets were found in the blood smears from both R702C and D1424N adult heterozygous mice. All three mutants also have significantly higher mean platelet volumes compared to their wild type littermates (6.12 +/- 0.62 fL, wt; 10.17 +/- 1.50 fL, heterozygous R702C; 10.13 +/- 1.66 fL, heterozygous D1424N; 10.92 +/- 1.83 fL, heterozygous E1841K). Kidney function was studied by examining the albumin/creatinine ratio in urine samples. Some but not all adult heterozygotes of both mutant lines have higher albumin/creatinine ratios at 8-9 weeks, indicating that kidney impairment may develop in some heterozygous mutants at an early age. These preliminary results suggest that these mouse models should be useful in understanding the pathophysiology of human MYH9-related diseases. In addition to using these mutant mice to study the relation between the nonmuscle myosin II-A mutation and disease, we plan to use various cells derived from these mice to study the effects of the mutation on basic properties of the cell. These include 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 mouse, 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 the heterozygous GFP or mCherry tagged NMIIA mice. The expression level of the tagged NMIIA is relatively lower than the endogenously expressed untagged NMIIA in both heterozygous mutants. We have set up breeding cages of the heterozygous tagged NMIIA mice with Cre mice to remove the Neo cassette so the tagged NMIIA protein expression level may increase to the normal level. This tagged NMIIA mouse model will shed light on the function of NM IIA in development. Various cell lines derived from the mouse will be used to study the regulation and function of NM IIA in adhesion, cell polarity and cell migration. We also plan to cross mCherry tagged NM IIA mice with GFP tagged NMIIB mouse. The offspring with mCherry tagged NMIIA and GFP tagged NMIIB should be useful to study if NMIIA and NMIIB can form copolymers in vivo. The cell lines (e.g. fibroblast) derived from this mouse can be used to study the distinct kinetic properties of NMIIA and NMIIB in cell polarity and migration with confocal or TIRF microscopy. The purpose of an additional study is to learn whether one isoform of NM II, specifically NM IIC, can functionally replace a second one, NM IIA, in mice. To replace NM IIA with NM IIC, homologous recombination will be used to inactivate NM IIA by inserting the cDNA for NM IIC-GFP into the first coding exon of the Myh9 gene. We have obtained several positive embryonic stem cell lines which are heterozygous NMIIC-GFP under the control of NM IIA promoter. These embryonic stem cells will be used to produce chimera mice.