Studies of Drosophila Myosin VII

果蝇肌球蛋白 VII 的研究

• 批准号: 8746626
• 负责人: James Sellers
• 金额: $ 46.67万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746626
• 关键词: Actins; Adult; Affect; Amoeba genus; Animals; Area; Bacteria; Binding; Birth; Blindness; C-terminal; Cell Adhesion; Cells; Cytoplasmic Granules; Defect; Diffuse; Drosophila genus; Embryo; Event; Excision; Eye; Filopodia; Genes; Hearing Impaired Persons; Hemocytes; Hemolymph; Human; Immunofluorescence Immunologic; Kinetics; Larva; Length; MYO7A gene; Mammals; Maps; Mechanics; Microfilaments; Motor; Mutagenesis; Mutation; Myosin ATPase; Organism; Phagocytes; Phagocytosis; Phosphorus; Photoreceptors; Pigments; Play; Power stroke; Regulation; Retinal Cone; Retinitis; Role; Scanning; Staining method; Stains; Structure; Tissues; Transfection; Transgenic Organisms; Yeasts; base; cell type; deafness; experience; fly; lens; mutant; optical traps; yeast two hybrid system

Myosin VIIa is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. We have studied Drosophila myosin VIIa that was expressed in Sf9 cells. We have shown that this myosin has high duty ratio kinetics similar to that of processive motors, but we also showed that it did not easily dimerize even if the full length molecule was expressed in Sf9 cells. The enzymatic activity of full length Drosophila myosin VIIa is regulated by an intramolecular folding event. A binding partner (termed M7BP for myosin 7 binding partner) for FLM7a was identified using the C-terminal FERM domain of the myosin as a bait in a yeast two hybrid screen. The binding partner activates the MgATPase activity of FLM7a in the presence of low concentrations of actin. We are currently mapping the areas on FLM7a that interact with the binding partner and vice versa. We find that a GFP-tagged full length myosin VIIa (GFP-FLM7a) has a diffuse localization when expressed in Drosophila S2 cells in culture. The same is true when an mCherry M7BP is expressed by itself in these cells. However, co-transfection of S2 cells with GFP-FLM7a and mCherry M7BP results in a marked activation on cellular cytoskeletal activity. The cells experience marked ruffling of the lamellipodia and grow numerous filopodia. FLM7a and M7BP are extensively co-localized in the regions of actin filament formation and are present along and at the tips of filopodia and can be observed moving together toward filopodial tips. Myosin VIIa and the M7BP are both expressed in hemocytes, a phagocytic cell type found in the hemolymph of larva which can phagocytize bacteria. Hemoctyes from flies that do not express myosin VIIa do not efficiently phagocytize bacteria. We have created a transgenic fly that expresses a GFP-tagged FLM7a and can observe the localization of the myosin when bacteria are being phagocytizedIn the larval eye disc, immunofluorescence staining of M7a, M7BP and Rab 11 are localized to the lens-secreting cone cells. EM sections of the adult eyes of the M7a mutants showed missing pigment granules surrounding the primary pigment cells and at the base of the rhabdomeres of the photoreceptor cells. Scanning EM of the eye of M7a mutant and M7BP mutant showed abnormal bristles. The M7BP mutant is a P-element insertion line and we are presently performing excision mutagenesis to isolate more M7BP mutants. . We are currently examining the mechanical ability of myosin VIIa truncations using optical trapping nanometry. Preliminary results reveal that this myosin has a long attachment lifetimes and a long power stroke.

肌球蛋白VIIA是一种非常规的肌球蛋白，在从变形虫到哺乳动物的生物中广泛表达，已显示出在细胞粘附和吞噬作用中起着至关重要的作用。我们研究了在SF9细胞中表达的果蝇肌球蛋白VIIA。我们已经表明，该肌球蛋白具有与过程中电动机相似的高功效比率动力学，但是我们也表明，即使在SF9细胞中表达了全长分子，也不容易降低。全长果蝇肌球蛋白VIIA的酶活性受分子内折叠事件的调节。使用肌球蛋白的C末端FERM结构域（称为肌球蛋白7结合伙伴称为M7BP）的结合伙伴（称为肌球蛋白7结合伴侣），作为肌球蛋白的C末端FERM结构域作为酵母两杂种筛网中的诱饵。在肌动蛋白浓度低的情况下，结合伴侣激活FLM7A的MGATPase活性。我们目前正在绘制FLM7A上与绑定伙伴相互作用的区域，反之亦然。我们发现GFP标记的全长肌球蛋白VIIA （GFP-FLM7A）在培养果蝇S2细胞中表达时具有弥漫性定位。当麦克利M7BP在这些细胞中自身表达时，也是如此。然而，S2细胞与GFP-FLM7A和MCHERRY M7BP共转染导致细胞骨骼活性的显着激活。这些细胞经历了标志着薄片的皱纹，并生长许多丝状虫。 FLM7A和M7BP在肌动蛋白丝形成区域进行了广泛的共定位，并且沿丝状尖端存在，并且可以观察到朝向丝状尖端。肌球蛋白VIIA和M7BP都在血细胞中表达，这是一种在幼虫的血淋巴中发现的吞噬细胞类型，可以吞噬细菌。来自未表达肌球蛋白VIIA的苍蝇的血肿不会有效地吞噬细菌。我们创建了一种表达GFP标记的FLM7A的转基因蝇，当细菌被吞噬素时，可以观察肌球蛋白的定位，幼虫眼盘，M7a，M7BP和RAB 11的免疫荧光染色位于晶状体切片锥细胞上。 M7a突变体的成年眼睛的EM部分显示出围绕原代色素细胞和光感受器细胞的横纹器的底部的色素颗粒。 M7a突变体和M7BP突变体的扫描EM显示出异常的刷毛。 M7BP突变体是P元素插入线，我们目前正在执行切除诱变，以分离更多的M7BP突变体。 。 我们目前正在使用光学诱捕纳米测定法检查肌球蛋白VIIA截断的机械能力。初步结果表明，这种肌球蛋白具有很长的依恋寿命和长时间的中风。