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是一种非传统的肌球蛋白，广泛表达于从阿米巴到哺乳动物的生物体中，已被证明在细胞粘附和吞噬中发挥重要作用。我们研究了果蝇肌球蛋白VIIa在Sf 9细胞中的表达。我们已经表明，这种肌球蛋白具有类似于进行性马达的高占空比动力学，但我们也表明，即使全长分子在Sf 9细胞中表达，它也不容易二聚化。全长果蝇肌球蛋白VIIa的酶活性受分子内折叠事件的调节。在酵母双杂交筛选中，使用肌球蛋白的C-末端FERM结构域作为诱饵，鉴定了FLM 7a的结合伴侣（称为肌球蛋白7结合伴侣的M7 BP）。结合伴侣在低浓度肌动蛋白存在下激活FLM 7a的MgATPase活性。我们目前正在绘制FLM 7a上与结合伴侣相互作用的区域，反之亦然。我们发现GFP标记的全长肌球蛋白VIIa （GFP-FLM 7a）在培养的果蝇S2细胞中表达时具有弥散定位。当mCherry M7 BP自身在这些细胞中表达时也是如此。然而，用GFP-FLM 7a和mCherry M7 BP共转染S2细胞导致细胞骨架活性的显著活化。细胞经历明显的板状伪足褶皱，并生长出许多丝状伪足。FLM 7 a和M7 BP广泛共定位于肌动蛋白丝形成的区域，并且沿着和在丝状伪足的尖端处存在，并且可以观察到一起向丝状伪足尖端移动。肌球蛋白VIIa和M7 BP都在血细胞中表达，血细胞是一种在幼虫血淋巴中发现的吞噬细胞类型，可以吞噬细菌。来自不表达肌球蛋白VIIa的苍蝇的血细胞不能有效地吞噬细菌。我们已经建立了一个转基因果蝇，表达GFP标记的FLM 7a，并可以观察到的定位肌球蛋白时，细菌被吞噬在幼虫的眼盘，M7 a，M7 BP和Rab 11的免疫荧光染色定位于晶状体分泌锥细胞。M7 a突变体的成年眼睛的EM切片显示在初级色素细胞周围和感光细胞的横纹肌基部缺失色素颗粒。扫描电镜观察M7 a和M7 BP突变体的眼毛异常。M7 BP突变体是P元件插入系，我们目前正在进行切除突变以分离更多M7 BP突变体。. 我们目前正在研究的机械能力，肌球蛋白VIIa截断使用光学捕获纳米。初步结果表明，这种肌球蛋白具有较长的附着寿命和较长的动力冲程。