Studies of Drosophila Myosin VII

Myosin VIIa是一种非常规的肌球蛋白，广泛表达于从变形虫到哺乳动物的生物体中，已被证明在细胞粘附和吞噬中起重要作用。我们研究了Sf9细胞中表达的果蝇myosin VIIa。我们已经证明，这种肌球蛋白具有与进程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via的酶活性受分子内折叠事件的调节。利用肌凝蛋白的c端FERM结构域作为诱饵，在酵母双杂交筛选中鉴定出FLM7a的结合伙伴（称为肌凝蛋白7结合伙伴M7BP）。在低浓度的肌动蛋白存在下，结合伙伴激活FLM7a的MgATPase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，用GFP-FLM7a和mCherry M7BP共同转染S2细胞可显著激活细胞骨架活性。细胞经历明显的板足褶皱，并生长出许多丝状足。FLM7a和M7BP广泛共定位于肌动蛋白丝形成区域，并沿丝状伪足的尖端存在，可以观察到它们一起向丝状伪足尖端移动。Myosin VIIa和M7BP都在幼虫的血细胞中表达，这些血细胞是幼虫血淋巴中发现的巨噬细胞样细胞，具有吞噬细菌的能力。苍蝇体内不表达肌球蛋白VIIa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的转基因苍蝇，并且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的免疫荧光染色定位于晶状体分泌锥细胞。M7a突变体成体眼睛的EM切片显示，在原代色素细胞周围和光感受器横纹肌基部缺失色素颗粒。M7a突变体和M7BP突变体的眼扫描电镜也显示异常刚毛。当我们使用RNAi细胞系敲除眼睛色素细胞中的M7a或M7BP时，我们观察到眼睛的粗糙度，这表明M7a和M7BP在同一途径中相互作用。

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 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. Myosin VIIa 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.