The Role of Nonmuscle Myosins in Development

编码非肌肉肌球蛋白重链（NMHC） IIA的基因MYH9发生点突变的人会出现多种综合征，包括血小板（巨血小板减少症）、肾脏（肾小球肾炎）和粒细胞（包涵体）缺陷。迄今为止，MYH9已报道了40多种不同的突变，包括错义突变和无义突变。这些研究的目的是通过建立三种突变的小鼠模型（运动结构域的R702C；杆结构域的D1424N和E1841K）并研究由此产生的小鼠表型，从而深入了解这些突变引起的疾病的病理机制。先前的体外研究表明，位于NMHC IIA运动结构域的R702C突变损害了MgATPase活性，并负责肌凝蛋白的运动速度；杆状结构域的D1424N和E1841K突变可能影响NMHC IIA丝的形成。我们产生了三个小鼠系，每个系在非肌肉肌球蛋白II-A基因Myh9 （R702C， D1424N和E1841K）中具有不同的突变。每个品系都会产生与人类患者相似的myh9相关疾病。将融合GFP的R702C突变体人cDNA导入Myh9的第一个编码外显子，将D1424N和E1841K突变体直接导入相应的外显子。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均表现出大量血小板减少，出血时间延长，凝块收缩缺陷和髓外巨核细胞增加。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，杂合的R702C巨核细胞形成的前血小板更少、更短、分枝更少、芽更大。结果表明，破坏血小板前形成有助于大血小板减少在小鼠和最有可能在人。我们还观察到过早的白内障形成，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。我们的研究结果表明，在肌球蛋白运动或丝形成结构域发生突变的杂合小鼠与myh9相关疾病的人表现出相似的血液学、眼睛和肾脏表型。

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 40 different mutations in MYH9 have been reported to date, including both missense 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 the motor domain; D1424N and E1841K in the 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 generated three 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 while 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 likely 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 domain manifest similar hematological, eye and kidney phenotypes to humans with MYH9-related diseases. These mouse models will be useful in understanding the pathophysiology of human MYH9-related diseases and should also be useful in designing and developing therapies. 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 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 both heterozygous and 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 both heterozygous mutants. This tagged NMIIA mouse model will shed light on the function of NM IIA in development. 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. 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. fibroblasts) derived from this mouse can be used to study the various biological 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 was 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 heterozygous NM IIC replacing NM IIA mice. However, breeding of heterozygous mutant mice does not produce homozygous NM IIC replacing NM IIA mice. We plan to investigate at which embryonic stage the homozygous mice die and the impact of NM IIC replacing NM IIA on cell polarization and migration.