STRUCTURAL AND FUNCTIONAL ANALYSIS OF DROSOPHILA MYOSIN

肌球蛋白V是脊椎动物中最具特征的囊泡转运蛋白，但目前尚不清楚肌球蛋白V家族的所有成员是否都具有共同的、进化上保守的作用机制。在这里，我们展示了果蝇的肌球蛋白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。我们对单头结构的稳态和瞬时动力学测量表明，单个果蝇肌球蛋白V分子的大部分机械力化学周期时间是从肌动蛋白分离出来的，因此，它必须在由几个分子组成的进程单元中发挥作用。因此，在体外运动分析中，双头果蝇肌球蛋白V需要较高的表面浓度才能显示肌动蛋白细丝的连续移位。我们在脊椎动物和苍蝇肌球蛋白V之间的比较表明，肌球蛋白V马达在细胞质运输中保存完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，并对肌球蛋白相关小泡的动力学进行了成像，以了解其在膜运输中的作用。

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.