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:7969055
- 负责人:
- 金额:$ 37.58万
- 依托单位:
- 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:
- 关键词:AdhesionsAdultAffectAgeAlbuminsAmino AcidsBloodBlood PlateletsBreedingCell LineCell PolarityCell divisionCell-Cell AdhesionCell-Matrix JunctionCellsChimera organismCodeComplementary DNACreatinineDefectDevelopmentDiseaseES Cell LineEmbryoEmbryonic DevelopmentExonsFibroblastsFilamentFunctional disorderGenesGiant PlateletGlomerulonephritisGoalsHandHeterozygoteHomozygoteHumanImpairmentIn VitroInclusion BodiesInitiator CodonKidneyKineticsLearningLightMotorMovementMusMutant Strains MiceMutationMyosin ATPaseMyosin Heavy ChainsMyosin Type IINonmuscle Myosin Type IIANonsense MutationPhenotypePoint MutationPropertyProtein IsoformsRegulationRenal functionReportingRoleSamplingSyndromeTissuesTotal Internal Reflection FluorescentUniversitiesUrineWorkcell motilitycopolymerdisease-causing mutationembryonic stem cellgranulocytehomologous recombinationhuman diseasehuman subjectin vivoinsightmigrationmouse developmentmouse modelmutantoffspringpromoterprotein expressionresearch studyretinal rods
项目摘要
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.
MYH9(编码非肌肉肌球蛋白重链 (NMHC) IIA 的基因)出现点突变的人类会出现多种综合征,包括血小板缺陷(巨血小板减少症)、肾脏缺陷(肾小球肾炎)和粒细胞缺陷(包涵体)。 迄今为止,已报道了 30 多种不同的 MYH9 突变,包括错义突变和无义突变。这些研究的目的是通过针对其中三种突变(运动域中的 R702C;杆域中的 D1424N 和 E1841K)创建小鼠模型并研究由此产生的小鼠表型,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,NMHC IIA 运动结构域中的 R702C 突变会损害 MgATPase 活性,并负责肌球蛋白的运动速度;而杆结构域中的突变 D1424N 和 E1841K 可能会影响 NMHC IIA 丝的形成。 我们通过同源重组,用 NMHC IIA 中的突变型 R702C 或 D1424N 取代野生型 NMHC IIA,制备了 R702C 和 D1424N 突变型小鼠。 E1841K突变小鼠获自杜克大学M Kelley博士。 杂合R702C突变小鼠的繁殖并未产生纯合突变后代。纯合子在胚胎第 8.5 天到 10.5 天之间死亡。 另一方面,杂合 D1424N 突变小鼠的繁殖产生了接近正常比例的纯合突变后代,而 E1841K 突变小鼠也产生了纯合突变后代,这表明 NMHC IIA 运动结构域的突变可能比 NMHC IIA 运动结构域的突变具有更严重的影响。胚胎发育过程中的杆。 有趣的是,在 R702C 和 D1424N 成年杂合小鼠的血涂片中都发现了巨大的血小板。 与野生型同窝小鼠相比,所有三种突变体的平均血小板体积也显着更高(6.12 +/- 0.62 fL,wt;10.17 +/- 1.50 fL,杂合 R702C;10.13 +/- 1.66 fL,杂合 D1424N;10.92 +/- 1.83 fL,杂合子E1841K)。 通过检查尿液样本中的白蛋白/肌酐比率来研究肾功能。两种突变体系的一些但不是全部成年杂合子在 8-9 周时具有较高的白蛋白/肌酐比率,表明一些杂合突变体可能在早期就出现肾损伤。 这些初步结果表明这些小鼠模型应该有助于了解人类 MYH9 相关疾病的病理生理学。
除了使用这些突变小鼠研究非肌肉肌球蛋白 II-A 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来研究突变对细胞基本特性的影响。这些包括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 基因的起始密码子前面。 我们获得了带有 GFP 或 mCherry 标签的杂合 NMIIA 小鼠。 在两种杂合突变体中,带标签的 NMIIA 的表达水平相对低于内源表达的未带标签的 NMIIA。 我们将杂合标记的NMIIA小鼠与Cre小鼠建立饲养笼,去除Neo盒,使标记的NMIIA蛋白表达水平可能增加至正常水平。这个带有标签的 NMIIA 小鼠模型将揭示正在开发的 NM IIA 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。 我们还计划将 mCherry 标记的 NM IIA 小鼠与 GFP 标记的 NMIIB 小鼠杂交。 带有 mCherry 标记的 NMIIA 和 GFP 标记的 NMIIB 的后代应该有助于研究 NMIIA 和 NMIIB 是否可以在体内形成共聚物。来自该小鼠的细胞系(例如成纤维细胞)可用于通过共聚焦或 TIRF 显微镜研究 NMIIA 和 NMIIB 在细胞极性和迁移方面的独特动力学特性。
另一项研究的目的是了解 NM II 的一种亚型(特别是 NM IIC)是否可以在小鼠中功能性地替代另一种 NM IIA。为了用 NM IIC 替换 NM IIA,将使用同源重组通过将 NM IIC-GFP 的 cDNA 插入 Myh9 基因的第一个编码外显子来灭活 NM IIA。我们已经获得了几个阳性胚胎干细胞系,它们是在NM IIA启动子控制下的杂合NMIIC-GFP。 这些胚胎干细胞将用于培育嵌合体小鼠。
项目成果
期刊论文数量(0)
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Robert Adelstein其他文献
Robert Adelstein的其他文献
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