Functional analysis of myosin superfamily in blood cells

血细胞肌球蛋白超家族的功能分析

• 批准号: 15591024
• 负责人: HIGASHIHARA Masaaki
• 金额: $ 2.24万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591024/
• 关键词: myosin; myosin sunerfamily; may-Hegglin anomaly; proplatelet; MYH-9; mutant; 軽鎖; アイソフォーム; GFP; rho; rho-kinase; バキュロウイルス

It has been demonstrated that MYH-9 (non-muscle myosin IIA) is responsible for the hereditary macrothrombocytopenia, such as May-Hegglin Anomaly (MHA), Fechtner syndrome (FS) and Sebastian syndrome (SS). We identified the E1841K mutation of MYH-9 gene of a patient with May-Hegglin Anomaly. Immunofluorescent staining of her peripheral blood smear samples revealed that non-muscle myosin IIA and actin filaments were co-localized at the Dohle-like inclusion bodies of neutrophils. To investigate the mechanism by which the E1841K mutation causes the phenotype of MHA, we first expressed GFP-tagged wild type and E1841K mutant MYH-9 cDNA in NIH3T3 cells. The mutant was able to form filaments and some inclusion bodies such as Dohle-like bodies, although it seemed to make no influences on the shape of the fibroblasts. Next we expressed the GFP-tagged wild and E1841K mutant MYH-9 in HEL cells and induced differentiation with TPA. HEL cells expressing the E1841K mutant failed in full differentiatio … More n. They could not produce the proplatelet-like projections as the cells overexpressing wild type of MYH-9 did. These results demonstrate that overexpression of this mutant can induce some phenotype similar to MHA. To clarify what molecular characteristics of the mutant myosin can cause the MHA phenotype, we expressed the recombinant wild and mutant proteins of non-muscle myosin IIA using Baculo-virus system. The mutant myosin failed to dissolve even in high ionic strength, suggesting this mutant can assemble in cytoplasm even under the condition, in which wild type myosin changes the conformation and exchanges dynamically assembly to disassembly. Next we induced myosin filaments in HEL cells by over-expressing the mutant of myosin light chain (DD mutant), which mimics the double-phosphorylated form. The DD mutant of myosin light chain also inhibited the proplatelet-like projections. Moreover silencing of MYH-9 gene with siRNA could have no inhibitory effects on the differentiation of HEL cells, producing rather more proplatelet-like projections than the control. These results indicated that filament formation of myosin is not critical for proplatelet-like projections, but disassembly of myosin is rather essential. Collectively, the E1841K mutant inhibits the disassembly of myosin to prevent the proplatelet-like formation of HEL cells, and the assembled myosin forms the Dohle-like inclusion bodies probably with actin filaments. Less

MYH-9（non-muscle myosin IIA，非肌肉肌球蛋白IIA）与遗传性巨血小板减少症（macroplateletia，巨血小板减少症）如May-Hegglin异常（May-Hegglin Anomaly，MHA）、Fechtner综合征（Fechtner syndrome，FS）和塞巴斯蒂安综合征（Sebastian syndrome，SS）等密切相关。我们在一例May-Hegglin异常患者的MYH-9基因上发现了E1841 K突变。她的外周血涂片样本的免疫荧光染色显示，非肌肉肌球蛋白IIA和肌动蛋白丝共定位于中性粒细胞的道尔样包涵体。为了研究E1841 K突变导致MHA表型的机制，我们首先在NIH 3 T3细胞中表达GFP标记的野生型和E1841 K突变体MYH-9 cDNA。该突变体能够形成纤维丝和一些包涵体，如Dohle样体，但似乎对成纤维细胞的形状没有影响。接下来，我们在HEL细胞中表达GFP标记的野生型和E1841 K突变型MYH-9，并用TPA诱导分化。表达E1841 K突变体的HEL细胞不能完全分化， ...更多信息 n.它们不能像过表达野生型MYH-9的细胞那样产生前血小板样突起。这些结果表明，该突变体的过表达可以诱导一些类似于MHA的表型。为了阐明突变型肌球蛋白的分子特征能够导致MHA表型，我们利用杆状病毒系统表达了非肌肉肌球蛋白IIA的重组野生型和突变型蛋白。突变体肌球蛋白在高离子强度下也不能溶解，表明突变体肌球蛋白在野生型肌球蛋白构象发生变化，动态地从组装到分解的条件下仍能在胞质中组装。接下来，我们通过过表达肌球蛋白轻链的突变体（DD突变体），其模拟双磷酸化形式，在HEL细胞中诱导肌球蛋白细丝。肌球蛋白轻链的DD突变体也抑制前血小板样突起。siRNA沉默MYH-9基因对HEL细胞的分化没有抑制作用，但产生了更多的前血小板样突起。这些结果表明，肌球蛋白的细丝形成是不关键的proplatelet-like投影，但肌球蛋白的拆卸是相当必要的。总之，E1841 K突变体抑制了HEL细胞肌球蛋白的分解，从而阻止了前血小板样的形成，并且组装的肌球蛋白可能与肌动蛋白丝形成Dohle样包涵体。少