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，它是一种非进位的整体运动。我们已经独立制备了两种针对肌球蛋白V的多克隆抗体。一种针对肌球蛋白V的卷曲结构域的多克隆抗体和另一种针对肌球蛋白V尾部的多克隆抗体。它与果蝇的层粘胶蛋白C（一种幼虫核膜的内膜标记物）共定位。利用UAS-GAL4系统对幼虫唾液腺中肌球蛋白V的突变体和过表达的肌球蛋白V进行免疫沉淀实验，发现核膜上有异常的肌球蛋白C染色。免疫沉淀实验使用肌球蛋白V抗体拉低Rab 5蛋白。rab5是一个小的GTPase，已被证明定位于早期核内体。用Rab 5抗体在幼虫的唾腺中进行免疫染色，显示核包膜中有染色，类似于层粘胶蛋白C和肌球蛋白V的染色。为了确定肌球蛋白V和Rab 5的定位是在幼虫核包膜的内膜还是外膜，我们进行了免疫金电镜实验。我们假设果蝇myosin V， Rab 5和lamin 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.