Studies of Drosophila Myosins

果蝇肌球蛋白的研究

基本信息

批准号：
10929119
负责人：
James Sellers
金额：
$ 77.93万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/10929119
关键词：
Actins Adult Affect Amoeba genus Animals Area Bacteria Binding Biological Assay Birth Blindness C-terminal Cell Adhesion Cells Centrioles Collaborations Complex Cytoplasmic Granules Cytoskeleton Defect Diffuse Dimerization Drosophila genus Embryo Event Eye Filopodia Genes Hemocytes Hemolymph Human Immunofluorescence Immunologic Kinesin Larva Length MYO7A gene Macrophage Mammals Maps Microfilaments Microtubules Molecular Motor Mutation Myosin ATPase Organism Pathway interactions Phagocytosis Photoreceptors Pigments Play Proteins RNA Interference Regulation Retinal Cone Retinitis Role Running Scanning Stains Structure Time Tissues Transgenic Organisms Yeasts base cell motility deaf deafness experience fly in vitro Assay interest knock-down lens migration mutant novel particle pericentrin protein purification residence single molecule yeast two hybrid system

项目摘要

Myosin 7a 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 previously shown that Drosophila myosin 7a that 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 7a(GFP-FLM7a) has a diffuse localization when expressed in Drosophila S2 cells in culture. The same is true when a 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. This suggests that M7BP may dimerize myosin 7a and allow it to be processive. Consistent with this, while myosin 7a alone does not move processively on actin filaments in a single molecule motility assay, it does move processively when M7BP is also added to the assay. The velocity is very slow, but the run length is around 0.5 um. Thus the residence time that the myosin-7a/M7BP complex spends on actin is very long. The most predominate translocating particle has two myosin 7a motor domains, suggesting that M7BP dimerizes the molecules. Myosin 7a and the M7BP are both expressed in larval hemocytes, which are macrophage-like cells found in the hemolymph of larva and have the ability to phagocytose bacteria. Hemocytes from flies that do not express myosin VIIa do not efficiently phagocytose 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 phagocytosed. In 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 also showed abnormal bristles. When we knockdown either M7a or M7BP in the pigment cells in the eye using RNAi lines, we observed roughness of the eye, suggesting that both M7a and M7BP interacts within the same pathway. While not a myosin, we have studied the microtubule motor, kinesin=1, from Drosophila in collaboration with the lab of Nasser Rusan. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP.

肌球蛋白7a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。我们以前已经表明，果蝇肌球蛋白7a受分子内折叠事件调节。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，当在培养物中果蝇S2细胞表达时，GFP标记的全长肌球蛋白7a（GFP-FLM7A）具有弥漫性定位。当这些细胞在这些细胞中自身表达麦克利M7BP时，情况也是如此。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。这表明M7BP可能会降低肌球蛋白7a并允许其处理。与此相一致，虽然单独的肌球蛋白7a在单个分子运动测定中的肌动蛋白丝上没有进行过程移动，但当当M7BP也添加到该测定中时，它确实会进行过程移动。速度非常慢，但是运行长度约为0.5 um。因此，肌球蛋白7A/M7BP综合体在肌动蛋白上花费的时间很长。最主要的易位粒子具有两个肌球蛋白7a运动结构域，这表明M7BP二聚体分子。肌球蛋白7a和M7bp均在幼虫血细胞中表达，这些血细胞是在幼虫的血淋巴中发现的巨噬细胞样细胞，并且具有吞噬细胞细菌的能力。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胞细菌。我们创建了一种表达GFP标记的FLM7A的转基因苍蝇，可以观察细菌被吞噬时肌球蛋白的定位。在幼虫眼盘中，M7a，M7bp和Rab 11的免疫荧光染色位于透镜分泌锥细胞中。 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也显示出异常的刷毛。当我们使用RNAi系中的色素细胞中的M7A或M7BP敲低时，我们观察到眼睛的粗糙度，这表明M7A和M7BP在同一途径中都相互作用。虽然不是肌球蛋白，但我们已经研究了果蝇与纳赛尔·鲁桑（Nasser Rusan）的实验室，研究了微管电机= 1。我们将丁香蛋白样蛋白（PLP）鉴定为一种新型的驱动蛋白-1相互作用分子，对于中心运动必不可少。体外测定表明，PLP直接与驱动蛋白1的货物结合结构域相互作用，从而使PLP可以在MT上迁移。使用纯化蛋白质的结合测定表明，驱动蛋白-1自抑制的缓解对于与PLP的相互作用至关重要。