Studies Of Myosin V

肌球蛋白 V 的研究

• 批准号: 10699698
• 负责人: James Sellers
• 金额: $ 28.01万
• 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/10699698
• 关键词: ATP phosphohydrolase; Actins; Adopted; Atomic Force Microscopy; Biological Assay; Calcium; Complementary DNA; Cryoelectron Microscopy; Data; Diffusion; Engineering; F-Actin; Genes; Goals; Hand; Head; Image; Image Analysis; In Vitro; Length; Mammals; Microfilaments; Molecular; Motor; Myosin ATPase; Myosin Type V; Negative Staining; Organelles; Positioning Attribute; Property; Regulation; Research Personnel; Running; Structure; Surface; Techniques; Total Internal Reflection Fluorescent; Walking; arm; cell motility; interest; microscopic imaging; nanomachine; optical traps; particle; single molecule; vesicle transport

There are three myosin V genes in mammals, termed Va, Vb and Vc. Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used negative staining and cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. Wild type myosin V molecules move processively along actin attached to coverslip surfaces in vitro. The average run length is about 1 um in length. Many investigators have been interested in using myosin V as a motor in nanomachines to move along engineered tracks of actin filaments. In order to increase the run lengths of myosin V, we have cloned cDNA constructs that drive the expression of coiled-coils that form trimeric, tetrameric and pentameric structures fused to the lever arm and motor domain of myosin V. Preliminary data show that we have successfully made a three-headed myosin and that this molecule has a longer run length than seen with the two-headed wild type molecule.

哺乳动物中有三个肌球蛋白V基因，称为VA，VB和VC。最近的研究提供了有力的证据，表明单级V肌球蛋白分子沿F-肌动蛋白进行过程中运输囊泡和细胞器，并在每次扩散遭遇中采取了几个36 nm的步骤，手工握手。我们证明了肌球蛋白的ATPase活性需要钙才能达到最大活性，并表明在没有钙的情况下，肌球蛋白V采用了折叠的，无活性的结构。我们已经使用了阴性染色和冷冻电子显微镜检查在肌动蛋白上行走的肌球蛋白V的结构。这些图像给出了肌球蛋白V分子的清晰图像，并附有两个头部附着在肌动蛋白上，并且可以使我们对杠杆臂的位置和刚度进行观察。野生型肌球蛋白V分子在体外沿附着在盖玻片表面的肌动蛋白进行过程移动。 平均运行长度约为1 UM。 许多调查人员对使用肌球蛋白V作为纳米机器的发动机感兴趣，以沿肌动蛋白丝的工程轨道移动。为了增加肌球蛋白V的运行长度，我们具有克隆的cDNA构建体，这些cDNA构造可以驱动形成三聚体，四聚体和五聚体结构的表达，这些结构与肌球蛋白V的杆臂和运动域融合在一起。初步数据表明，我们已经成功地构成了三头肌蛋白，并且与两种摩尔蛋白相比，我们已经成功地构成了两种摩托蛋白。