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，在从阿米巴到哺乳动物的各种生物中广泛表达，已被证明在细胞黏附和吞噬过程中发挥重要作用。我们研究了在Sf9细胞中表达的果蝇肌球蛋白VIIa。我们已经证明了这种肌球蛋白具有类似于前进马达的高占空比动力学，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。果蝇全长肌球蛋白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，用肌球蛋白的C-末端FERM结构域作为诱饵，鉴定了一个与FLM7a结合的伙伴(称为M7BP，肌球蛋白7结合伙伴)。在低浓度的肌动蛋白存在下，结合伙伴激活FLM7a的镁ATPase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也是如此。然而，GFP-FlM7a和mCherry M7BP共转染S2细胞后，细胞骨架活性显著增强。细胞经历明显的片状皱褶，并生长出大量的丝状足细胞。FLM7a和M7BP广泛共定位于肌动蛋白细丝形成区，沿丝状足端和在丝状足端存在，可以观察到它们一起向丝状足端移动。肌球蛋白VIIa和M7BP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。不表达肌球蛋白VIIa的果蝇的血细胞不能有效地吞噬细菌。我们已经创造了一种表达GFP标记的FLM7a的转基因果蝇，当细菌被吞噬时，可以观察到肌球蛋白的定位。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锥细胞。M7a突变体成人眼的电子显微镜切片显示，初级色素细胞周围和感光细胞横纹肌基底部的色素颗粒缺失。M7a突变体和M7BP突变体的扫描电镜显示异常刷毛。M7BP突变体是一个P元件插入线，我们目前正在进行切除突变，以分离出更多的M7BP突变体。。我们目前正在使用光学捕获纳米技术检测肌球蛋白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功率行程。