STRUCTURAL AND FUNCTIONAL ANALYSIS OF DROSOPHILA MYOSIN V

果蝇肌球蛋白 V 的结构和功能分析

• 批准号: 7969057
• 负责人: James Sellers
• 金额: $ 17.53万
• 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/7969057
• 关键词: Affinity; Antibodies; Atomic Force Microscopy; C-terminal; Circular Dichroism; Development; Drosophila genus; Electron Microscopy; Elements; Escherichia coli; Event; Family; Gene Proteins; Genes; Goals; Heating; Lamin Type B; Life; MYO5A gene; Measurement; Mechanics; Molecular; Molecular Motors; Monitor; Motion; Motor; Mus; Myosin ATPase; Myosin Type V; Nuclear Envelope; Pattern; Play; Process; Proteins; Recombinant Proteins; Role; Shadowing (Histology); Spectrum Analysis; Staging; Stretching; Structure; System; Tail; Techniques; Vertebrates; Vesicle; cell type; chemical binding; expression vector; flexibility; fly; genetic analysis; interest; mammalian genome; member; mutant; retinal rods; single molecule

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. We showed 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. We are also interested in what role myosin V plays in Drosophila. To do this, we have been localizing myosin V in various stages of development. Preliminary results suggest that myosin V in some cell types localizes to the nuclear envelope where it may interact with lamin B. We are in the process of raising new antibodies and are studying the localization of lamin B in myosin V deficient mutant Drosophila. The role of the rod fragment of myosin V molecules in the processive motion of these single molecule motors is largely unknown. We compared the mechanical stability of a processive (mouse myosin Va) and nonprocessive (Drosophila myosin V) molecular motors. Rod fragments (coiled-coil regions) of mouse myosin Va and Drosophila myosin V were cloned into expression vector (pET-16b) and expressed in prokaryotic expression system (E. coli, BL21(DE3)pLysS). Recombinant proteins were purified using Ni-affinity column in denaturing conditions. The formation of proper coiled-coil structure during renaturation was monitored using circular dichroism (CD). Single molecule force spectroscopy measurements were performed using atomic force microscopy (AFM). In order to facilitate strong and specific binding, chemical handles were applied at the N- and C-terminal ends of the fragments. During the mechanical stretching, the extension of rod fragment occurs as two consecutive events: extension of flexible loops followed by extension and unfolding of coiled-coil motifs. We find that the processive and nonprocessive rod fragments display different unfolding patterns. The unfolding of coiled-coil structures occurs much later during the AFM stretch cycle for processive myosin Va than for nonprocessive Drosophila myosin V. Additionally, we find that unfolding forces of the coiled-coil structures of mouse myosin Va is higher then those in Drosophila myosin V, suggesting that the former coiled-coil region is mechanically more stable. Heat-induced denaturation CD measurements show the same result. Our observations suggest that the tether between the cargo and motor in case of myosin V molecules consists of a mechanically strong coiled-coil region combined with elastic elements, and this connection might play an important role in sustaining processive motions of single molecule motors. We have also examined the structure of the myosin V tail region by atomic force microscopy and rotary shadowed electron microscopy.

肌球蛋白V是脊椎动物中最佳特征的囊泡转运蛋白，但肌球蛋白v家族的所有成员是否具有共同的，进化上保守的作用机理，但尚不清楚。 我们表明，果蝇的肌球蛋白V与脊椎动物肌球蛋白VA的运动机制截然不同，并且是一种非过程的合奏运动。 我们也对肌动蛋白在果蝇中扮演什么角色感兴趣。 为此，我们一直在各个开发阶段将肌球蛋白V定位。 初步结果表明，某些细胞类型中的肌球蛋白V定位于可能与层粘连蛋白相互作用的核包膜。我们正在提高新抗体的过程中，并正在研究层粘连蛋白B在肌球蛋白V缺陷型突变型果蝇中的定位。 肌球蛋白V分子的杆片段在这些单分子电动机的过程中的作用是未知的。我们比较了过程（小鼠肌球蛋白VA）和非过程（果蝇肌球蛋白V）分子电机的机械稳定性。将小鼠肌球蛋白VA和果蝇肌球蛋白V的杆片段（卷曲的螺旋区域）克隆到表达载体（PET-16B）中，并在原核表达系统（E. Coli，Bl21（DE3）Plyss）中表达。在变性条件下，使用Ni-obilitial柱纯化重组蛋白。使用圆形二色性（CD）监测恢复过程中适当的盘绕结构的形成。使用原子力显微镜（AFM）进行了单分子力光谱测量。为了促进较强和特定的结合，在片段的N末端和C末端施加化学柄。在机械拉伸期间，杆片段的延伸作为两个连续事件发生：柔性环的延伸，然后扩展和盘绕螺旋序列的延伸和展开。我们发现，过程和非过程的杆片段显示出不同的展开模式。在AFM拉伸周期中，肌动蛋白VA的盘绕结构的展开发生在很晚的时间之后，而不是非过程性的果蝇肌球蛋白V。此外，我们发现，小鼠肌球蛋白VA的盘绕型结构的展开力比果蝇肌球蛋白V中的那些更高的肌动蛋白VA的结构更高，暗示了以前的固定型蛋白油。热诱导的变性CD测量结果显示出相同的结果。我们的观察结果表明，在肌球蛋白V分子的情况下，货物和电动机之间的束缚由机械强的盘绕螺旋螺旋区和弹性元素组合组成，并且这种连接可能在维持单分子电动机的过程中起重要作用。 我们还通过原子力显微镜和旋转阴影电子显微镜检查了肌球蛋白V尾部区域的结构。