The effect of actin filament formations for the stepping behavior of myosin V

这个建议的重点是研究更高层次的肌动蛋白结构和肌动蛋白结合蛋白的作用 肌球蛋白V分子的持续合成能力和运动行为。最近，许多体外运动研究 研究了肌球蛋白V如何在肌动蛋白丝上移动，测量了运行长度、速度等参数， 和步长，但这些研究几乎总是只使用单个肌动蛋白丝的轨道。然而，肌动蛋白丝在细胞内形成许多种更高的有序结构，如分支网络、松散束和高度有序束。这项研究将深入了解基本问题，如肌球蛋白V可以横向从一个细丝到另一个？在一个包上移动时，这是否会导致比在单丝上观察到的更长的运行长度？原肌球蛋白与肌动蛋白的结合是否影响速度或行程长度？为了回答这些问题，我将使用三种不同的肌动蛋白形成。作为AIM 1，（1）单个肌动蛋白丝 由非肌肉原肌球蛋白或肌动蛋白结合蛋白、肌成束蛋白和α-辅肌动蛋白修饰。(2)肌动蛋白丝束在具有α-辅肌动蛋白或肌成束蛋白的脂质单层上的二维次晶阵列。(3)在溶液中形成由肌动蛋白或肌成束蛋白介导的三维肌动蛋白纤维束。我将测量肌球蛋白V在二维肌动蛋白束上的行程长度和速度，并将使用上述不同类型的肌动蛋白轨迹来测试肌球蛋白颈部的柔韧性。由于AIM 2，1将通过用Triton X-100处理生长在盖玻片表面上的细胞以去除膜并用罗丹明鬼笔环肽处理以稳定acfin细丝来制备Triton不溶性细胞骨架。荧光标记的肌球蛋白V分子的运动将使用全内反射荧光显微镜和称为FIONA（1纳米精度荧光成像）的分析技术进行测量，该技术测量单个荧光团的位置，精度为1.5 nm，时间分辨率为0.5 s。在AIM 1和2中，我将回答肌球蛋白V如何跨越不同种类的肌动蛋白束，以及肌球蛋白V如何作为转运蛋白沿高度有序的肌动蛋白结构沿着移动。AIM 3，1已经完成了这个项目。

The focus of this proposal is to study the effect of higher ordered actin structure and of actin binding proteins for the processivity and movement behavior of myosin V molecule. Recently, many in vitro motility studies investigated how myosin V moves on actin filament, measuring parameters such as the run length, velocity and step size, but these studies almost always have used only single actin filaments for the track. However, actin filaments form many kinds of higher ordered structures such as branching networks, loose bundles and highly ordered bundles inside cells. This study will give insight into fundamental questions such as can myosin V step laterally from one filament to another? when moving on a bundle? Does this result in longer run lengths than observed on single filaments? Does binding of tropomyosin to actin affect the velocity or run length? To answer these questions, I will use three different actin formations. As AIM 1, (1) single actin filaments decorated by nonmuscle tropomyosin, or the actin binding proteins, fascin, and a-actinin. (2) Two dimensional paracrystalline arrays of actin filaments bundled on lipid monolayers with a-actinin or fascin. (3) Three dimensional bundles of actin filaments mediated by a-actinin or fascin formed in solution. I will measured run-length and speed of myosin V on two dimentional actin bundles and will use to test for a fiexibility of the myosin neck using these above different types of actin tracks. As AIM 2,1 will prepare Triton-insoluble cytoskeletons by treatment of cells growing on a coverslip surface by treatment with Triton X-100 to remove membranes and with rhodamine phalloidin to stabilize the acfin filaments. The movement of fiuorescently labeled myosin V molecules will be measured using total internal reflection fiuorescent microscopy and an analysis technique, termed FIONA (Fluorescent Imaging at One Nanometer Accuracy) which measures the position of a single fiuorophor to 1.5 nm accuracy with 0.5 s temporal resolution. In AIM 1 and 2,1 will answer how does myosin V step over different kinds of actin bundles and how does myosin V move along a highly ordered actin structure as a transporter. AIM 3,1 have completed thi project.