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

基本信息

批准号：
8127805
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
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-26 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8127805
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 Running Series Side Solutions Speed Stress Fibers Structure Surface Techniques Testing Total Internal Reflection Fluorescent Triton Triton X100 Tritons Tropomyosin Yeasts analog cell motility fascin in vivo insight interest melanocyte monolayer mutant nanometer research study trafficking two-dimensional

项目摘要

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.

该建议的重点是研究较高有序肌动蛋白结构和肌动蛋白结合蛋白的影响用于肌球蛋白V分子的加工性和运动行为。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，测量参数，例如运行长度，速度和步骤大小，但是这些研究几乎总是只使用单肌动蛋白丝进行曲目。但是，肌动蛋白丝形成了许多高排序的结构，例如分支网络，松动的束和高度有序的细胞内部束。这项研究将深入了解基本问题，例如肌球蛋白V可以从一个细丝到另一个细丝的横向一步吗？当捆绑上移动时？这是否会导致长度长于单细丝上的长度？肌动蛋白对肌动蛋白的结合会影响速度或延伸长度吗？为了回答这些问题，我将使用三种不同的肌动蛋白形成。作为目标1，（1）单肌动蛋白丝由非肌肉肌球蛋白或肌动蛋白结合蛋白，fascin和a-肌动蛋白装饰。（2）肌动蛋白丝的二维旁晶阵列捆绑在脂质单层上，带有a-肌动蛋白或fascin。（3）溶液中形成的A-肌动蛋白或FASTIN介导的肌动蛋白丝的三维束。我将在二维肌动蛋白束上测量肌球蛋白V的跑步速度和速度，并将使用这些上述不同类型的肌动蛋白轨道来测试肌球蛋白颈的屈光度。作为AIM 2,1，将通过用Triton X-100处理在盖玻片表面上生长的细胞来制备Triton-不溶性细胞骨架，以去除膜和杜鹃明粘类素来稳定Acfin细丝。将使用总的内部反射曲线显微镜和一种分析技术来测量被标记为肌球蛋白V分子的运动，称为fiona（一种纳米精度，荧光成像），以0.5 s Permutal Sountolutions衡量单个fiuorophor的位置为1.5 NM精度。在AIM 1和2,1中，将回答肌球蛋白V如何在不同种类的肌动蛋白束上逐步发展，以及肌球蛋白V如何沿着高度有序的肌动蛋白结构作为转运蛋白移动。 AIM 3,1已经完成了Thi项目。