STRUCTURAL AND FUNCTIONAL ANALYSIS OF DROSOPHILA MYOSIN

果蝇肌球蛋白的结构和功能分析

• 批准号: 7158522
• 负责人: JAMES R. SELLERS
• 金额: --
• 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/7158522
• 关键词: Drosophilidae; actins; biochemical evolution; biomechanics; green fluorescent proteins; intracellular transport; molecular /cellular imaging; myosins; protein isoforms; protein localization; protein protein interaction; protein structure function; protein transport; protoplasm motility; tissue /cell culture; transport proteins; vesicle /vacuole

Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. Here we show that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va and it is a non-processive, ensemble motor. Our steady-state and transient kinetic measurements on single-headed constructs reveal that a single Drosophila myosin V molecule spends most of its mechanochemical cycle time detached from actin, therefore, it has to function in processive units that comprise several molecules. Accordingly, in in vitro motility assays, double-headed Drosophila myosin V requires high surface concentrations in order to exhibit a continuous translocation of actin filaments. Our comparison between vertebrate and fly myosin Vs demonstrates that the well-preserved function of myosin V motors in cytoplasmic transport can be accomplished by markedly different underlying mechanisms. We are also expressing GFP-tagged full-length myosin V and fragments of myosin V in Drosophila S2 cells and imaging the dynamics of myosin-associated vesicles in order to understand its role in membrane trafficking.

肌凝蛋白V是脊椎动物中最具特征的囊泡转运蛋白，但是否所有肌凝蛋白V家族成员都具有共同的、进化保守的作用机制尚不清楚。在这里，我们表明来自果蝇的肌凝蛋白V与脊椎动物的肌凝蛋白Va具有明显不同的运动机制，它是一种非进位的整体运动。我们对单头结构体的稳态和瞬态动力学测量表明，单个果蝇肌球蛋白V分子的大部分机械化学循环时间与肌动蛋白分离，因此，它必须在包含多个分子的过程单元中起作用。因此，在体外运动试验中，双头果蝇肌球蛋白V需要高表面浓度才能表现出肌动蛋白丝的连续易位。我们对脊椎动物和苍蝇肌球蛋白V的比较表明，肌球蛋白V马达在细胞质运输中保存良好的功能可以通过明显不同的潜在机制来完成。我们还在果蝇S2细胞中表达了gfp标记的全长肌球蛋白V和肌球蛋白V片段，并对肌球蛋白相关囊泡的动力学进行了成像，以了解其在膜运输中的作用。