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 是一种非常规肌球蛋白，广泛表达于从变形虫到哺乳动物的各种生物体中，已被证明在细胞粘附和吞噬作用中发挥重要作用。我们研究了在 Sf9 细胞中表达的果蝇肌球蛋白 VIIa。我们已经证明，这种肌球蛋白具有与加工马达相似的高占空比动力学，但我们也表明，即使全长分子在 Sf9 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调节。在酵母二种杂交筛选中，使用肌球蛋白的 C 端 FERM 结构域作为诱饵，鉴定了 FLM7a 的结合配偶体（称为肌球蛋白 7 结合配偶体的 M7BP）。在低浓度肌动蛋白存在的情况下，结合伴侣会激活 FLM7a 的 MgATP 酶活性。我们目前正在绘制 FLM7a 上与结合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) 在培养的果蝇 S2 细胞中表达时具有弥漫性定位。当 mCherry M7BP 在这些细胞中单独表达时，情况也是如此。然而，用 GFP-FLM7a 和 mCherry M7BP 共转染 S2 细胞会导致细胞骨架活性显着激活。细胞的板状伪足明显皱起，并长出大量的丝状伪足。 FLM7a 和 M7BP 广泛共定位于肌动蛋白丝形成区域，并且沿着丝状伪足及其尖端存在，并且可以观察到一起向丝状伪足尖端移动。肌球蛋白 VIIa 和 M7BP 均在血细胞中表达，血细胞是幼虫血淋巴中发现的一种吞噬细胞类型，可以吞噬细菌。不表达肌球蛋白 VIIa 的果蝇血细胞不能有效吞噬细菌。我们创建了一种表达 GFP 标记的 FLM7a 的转基因果蝇，并且可以在细菌被吞噬时观察肌球蛋白的定位。在幼虫眼盘中，M7a、M7BP 和 Rab 11 的免疫荧光染色定位于晶状体分泌视锥细胞。 M7a 突变体成年眼睛的电镜切片显示，初级色素细胞周围和感光细胞横纹球底部的色素颗粒缺失。 M7a突变体和M7BP突变体的眼睛扫描电镜显示异常的刚毛。 M7BP突变体是P元件插入系，我们目前正在进行切除诱变以分离更多M7BP突变体。 。 我们目前正在使用光学捕获纳米技术检查肌球蛋白 VIIa 截断的机械能力。初步结果表明，这种肌球蛋白具有较长的附着寿命和较长的动力冲程。