STRUCTURAL AND FUNCTIONAL ANALYSIS OF DROSOPHILA MYOSIN V

果蝇肌球蛋白V具有与脊椎动物肌球蛋白Va截然不同的运动机制，它是一种非进行性的整体运动。我们已经独立地产生了两个多克隆抗体对肌球蛋白V。一个多克隆抗体产生对肌球蛋白V的卷曲螺旋结构域和另一个多克隆对肌球蛋白V的尾部末端的果蝇幼虫唾液腺的免疫染色本地化的两个抗体的核膜。它与果蝇核纤层蛋白C共定位，核纤层蛋白C是幼虫核膜内膜的标记物。使用核纤层蛋白C抗体的幼虫提取物的免疫沉淀实验拉下肌球蛋白V。肌球蛋白V的突变体和过度表达的肌球蛋白V在幼虫唾液腺中使用UAS-GAL 4系统显示异常核纤层蛋白C染色的核膜。使用肌球蛋白V抗体的免疫沉淀实验拉下Rab 5蛋白。Rab 5是一个小的GTP酶，已被证明定位于早期内体。在幼虫唾液腺中使用Rab 5抗体的免疫染色显示在核膜中染色，类似于核纤层蛋白C和肌球蛋白V的染色。为了确定肌球蛋白V和Rab 5的定位是在幼虫核膜的内膜还是外膜中，我们正在进行免疫金电子显微镜实验。我们假设，果蝇肌球蛋白V，Rab 5和核纤层蛋白C相互作用，以维持幼虫唾液腺核膜的结构完整性。

Drosophila myosin V has a strikingly different motor mechanism from that of vertebrate myosin Va and it is a non-processive, ensemble motor. We have independently generated two polyclonal antibody against myosin V. One polyclonal antibody was generated against the coil-coiled domain of myosin V and another polyclonal against the tail end of myosin V. Immunostaining of the Drosophila larval salivary gland localized both antibodies to the nuclear envelope. It colocalized with Drosophila lamin C, a marker for the inner membrane of the larval nuclear envelope. Immunoprecipitation experiments of larval extracts using lamin C antibody pulled down myosin V. Mutants of myosin V and over expression of myosin V in the larval salivary gland using the UAS-GAL4 system showed aberrant lamin C staining on the nuclear envelope. Immunoprecipitation experiments using myosin V antibody pulled down Rab 5 protein. Rab 5 is a small GTPase which has been shown to localize to the early endosomes. Immunostaining using Rab 5 antibody in the larval salivary gland showed staining in the nuclear envelope, similar to the staining of lamin C and myosin V. In order to determine if the localization of myosin V and Rab 5 is in the inner or outer membrane of the larval nuclear envelope, we are performing immunogold electron microscopy experiments. We postulate that Drosophila myosin V, Rab 5 and lamin C are interacting with each other to maintain the structural integrity of the nuclear envelope of the larval salivary gland.