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

肌动蛋白丝形成对肌球蛋白V步进行为的影响

• 批准号: 7940982
• 负责人: Takeshi Sakamoto
• 金额: $ 24.9万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-26 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940982
• 关键词: ATP Hydrolysis; Actin-Binding Protein; Actinin; Actins; Address; Affect; Area; Behavior; Binding; Bladder Neoplasm; Bundling; Cell Line; Cell model; Cells; Coupling; Cytoskeleton; Data; Disease; Electron Microscopy; Event; Filament; Film; Fluorescence; Head; Higher Order Chromatin Structure; Image; In Vitro; Label; Lateral; Lead; Length; Lipids; MYO5A gene; Measurement; Measures; Mediating; Melanosomes; Membrane; Messenger RNA; Methods; Microfilaments; Microscopy; Molecular Motors; Mono-S; Motion; Movement; Mutation; Myosin ATPase; Myosin Type V; Nature; Neck; Neurons; Nucleotides; Positioning Attribute; Proteins; Quantum Dots; Resolution; Rhodamine; Rhodamines; Running; Series; Side; Solutions; Speed; Stress Fibers; Structure; Surface; Techniques; Testing; Total Internal Reflection Fluorescent; Triton; Triton X100; Tropomyosin; Yeasts; analog; cell motility; fascin; in vivo; insight; interest; melanocyte; monolayer; mutant; nanometer; research study; trafficking; two-dimensional

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.

这项建议的重点是研究肌动蛋白的高级结构和肌动蛋白结合蛋白的作用。 用于肌球蛋白V分子的加工和运动行为。近年来，许多体外运动研究 研究了肌球蛋白V如何在肌动蛋白细丝上运动，测量了游程长度、速度等参数 和步长，但这些研究几乎总是只使用单一的肌动蛋白细丝作为轨迹。然而，肌动蛋白细丝在细胞内形成分支网络、松散的束状和高度有序的束状等多种高级有序结构。这项研究将深入了解一些基本问题，如肌球蛋白V能否从一根细丝横向步入另一根细丝？在捆绑行进的时候？这是否会导致单丝的运行长度比观察到的更长？原肌球蛋白与肌动蛋白的结合是否影响肌动蛋白的速度或行程？为了回答这些问题，我将使用三种不同的肌动蛋白构型。作为目标1，(1)单个肌动蛋白细丝 由非肌肉原肌球蛋白或肌动蛋白结合蛋白、筋膜蛋白和α-肌动蛋白装饰。(2)用α-肌动蛋白或筋膜素包扎在脂质单层上的二维肌动蛋白微丝准晶阵。(3)在溶液中形成由α-肌动蛋白或筋膜蛋白介导的三维肌动蛋白细丝束。我将测量肌球蛋白V在二维肌动蛋白束上的运行长度和速度，并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处理生长在盖片表面的细胞来去除膜，并用罗丹明鬼臼糖苷来稳定Acfin细丝，来制备Triton不溶于细胞的骨架。我们将使用全内反射荧光显微镜和一种名为FIONA(一纳米精度的荧光成像)的分析技术来测量先前标记的肌球蛋白V分子的运动。FIONA(一纳米精度的荧光成像)可以测量单个荧光体的位置，精度为1.5 nm，时间分辨率为0.5 S。在目标1和2中，1将回答肌球蛋白V如何跨过不同种类的肌动蛋白束，以及肌球蛋白V作为转运体如何沿着高度有序的肌动蛋白结构移动。目标3，1已经完成了这个项目。