Dynamics of Kinesins, Dyneins, and Myosins

驱动蛋白、动力蛋白和肌球蛋白的动力学

基本信息

批准号：
7477204
负责人：
PAUL R SELVIN
金额：
$ 46.89万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-09-30 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7477204
关键词：
3-Dimensional Actins Actomyosin Address Affect Alzheimer&apos s Disease Amino Acid Sequence Antibodies Binding Biological Biology Brightfield Microscopy Catalytic Domain Cell Extracts Cell Line Cell Nucleus Cells Central Nervous System Diseases Cleaved cell Clinical Color Complex Condition Consumption Cysteine Data Data Analyses Development Dimerization Dominant-Negative Mutation Drosophila genus Dyes Dynein ATPase Energy Transfer Experimental Designs Fluorescence Funding Goals Grant Head Heart Diseases High Blood Pressure Homebound Persons Huntington Disease Image In Vitro Indium Intermediate Filaments KRP protein Kinesin Kinetics Label Lanthanoid Series Elements Lead Length Leucine Zippers Light Lipids Localized Lysosomes Maintenance Measurement Measures Melanophores Melanosomes Melatonin Membrane Microfilaments Microscope Microscopy Microtubules Modeling Molecular Molecular Conformation Molecular Motors Molecular Probes Motion Motor Movement Myopathy Myosin ATPase Myosin Type II Myosin Type V Neurites Numbers Optics Organelles PC12 Cells Paper Parkinson Disease Pharmaceutical Preparations Phosphorylation Play Positioning Attribute Principal Investigator Process Proteins Publishing Quantum Dots RNA Interference Rate Regulation Research Personnel Resolution Role Sampling Science Secretory Vesicles Series Signal Transduction Smooth Muscle Smooth Muscle Myosins Speed Structure System Techniques Testing Time Tubulin Universities Upper arm Walking War Water Wisconsin Work base cell motility dimer fluorescence imaging fluorophore follow-up forest improved in vivo insight millisecond monomer mutant myosin VI nanometer optical traps organelle movement peroxisome pressure programs research study retinal rods single molecule single-molecule FRET size tool

项目摘要

DESCRIPTION (provided by applicant): Molecular motors - kinesin, dynein, and myosins - play a crucial role in the maintenances and development of the organization, motility, and cell signaling, of healthy cells. Central nervous-system disorders such as Alzheimer's, Huntington's and Parkinson's Disease, and muscular diseases such as heart disease, high blood pressure, and uterine problems, all arise from molecular motors gone awry. At its most basic level, a single, isolated "head", which gives motors its ATP-dependent motility, has been fairly well studied. However, in general, motors have two heads, and there is a growing body of evidence that multiple motors can interact. Perhaps the most extreme example is our Science paper (Kural, 2005), where kinesin and dynein interact in vivo, to yield up to ~12 times their in vitro speed. The basic biology we want to address is how these motors are affected by interaction between heads within one molecular motor, via in vitro studies, and how multiple motors of different species interact, through in vivo studies. We wish to study: a) Smooth muscle myosin: To continue the study of the conformational changes within the actomyosin complex using lanthanide-based resonance energy transfer (LRET), single molecule FRET. We will utilize an improved "cysteine-light" smooth muscle myosin II. The goal is to understand the effect of actin and phosphorylation on the structure of S1, and particularly, heavymeromyosin (HMM). Does actin cause a cleft composure in myosin? Is the model of Wendt et al., involving the structure of HMM with actin, correct? Does actin undergo conformational changes upon myosin binding? (With L. Sweeney, UPenn, and I. Rayment, U. Wisconsin). b) Kinesin, Dynein, and Myosin V, Dynamics: To study the in vivo dynamics of kinesin, dynein, and myosin V in peroxisomes, melanosomes, PC12 cells, and other cell lines, using single molecule FIONA (Fluorescence Imaging with One Nanometer Accuracy), and optical trapping. This follows up on our results using FIONA in vivo, where we have achieved 1 msec and 1.5 nm temporal and spatial resolution. We found that kinesin and dynein are not engaged in a "tug-of-war", despite moving peroxisomes in opposite directions. The idea is to now optically trap a peroxisome inside a cell (which we have shown is possible), and determine, among other things: 1) the stall force of the peroxisomes in both the dynein and kinesin direction, therefore definitively showing how many of them are pulling at a time; 2) go from in vivo to in vitro studies to understand where the transformation occurs that enables the motors to pull so fast; 3) to test other systems, e.g. PC12 neurites, to see if the kinetics are similar; 4) to measure the movement of melanosomes (which are non-fluorescent), using bright-Field Imaging with One Nanometer Accuracy (bFIONA), a knock-off of FIONA, and test the kinetics under various conditions: e.g., no actin, no microtubules, no intermediate filaments..., to show how melanosomes make the transition between one motor system and another. (With V. Gelfand, Northwestern). c) Myosin VI: To study myosin VI in vivo, and to understand the transition between an anchor (monomer) and processive motor (dimer) as a function of: position within the cell; as a function of accessory proteins (Dab2, GIPC...); as a function of insertion length of the cargo-binding domain of myosin VI (with L. Sweeney, UPenn.). d) Technological developments: Optics/microscopy needs to be achieved to fulfill the above goals.

描述（由申请人提供）：分子马达——驱动蛋白、动力蛋白和肌球蛋白——在健康细胞的组织、运动和细胞信号传导的维持和发展中发挥着至关重要的作用。阿尔茨海默病、亨廷顿病和帕金森病等中枢神经系统疾病，以及心脏病、高血压和子宫问题等肌肉疾病，都是由分子马达出了问题引起的。在最基本的层面上，一个单一的、孤立的“头部”，它赋予马达依赖于 ATP 的运动能力，已经得到了相当好的研究。然而，一般来说，电机有两个头，并且越来越多的证据表明多个电机可以相互作用。也许最极端的例子是我们的《科学》论文（Kural，2005），其中驱动蛋白和动力蛋白在体内相互作用，产生高达其体外速度的约 12 倍。我们想要解决的基本生物学问题是，通过体外研究，这些电机如何受到一个分子电机内头部之间相互作用的影响，以及通过体内研究，不同物种的多个电机如何相互作用。我们希望研究： a) 平滑肌肌球蛋白：使用基于镧系元素的共振能量转移 (LRET)、单分子 FRET 继续研究肌动球蛋白复合物内的构象变化。我们将利用改进的“半胱氨酸轻”平滑肌肌球蛋白 II。目标是了解肌动蛋白和磷酸化对 S1，特别是重粒肌球蛋白 (HMM) 结构的影响。肌动蛋白会导致肌球蛋白的分裂吗？ Wendt 等人涉及 HMM 与肌动蛋白结构的模型正确吗？肌动蛋白结合肌球蛋白后会发生构象变化吗？（与宾夕法尼亚大学的 L. Sweeney 和威斯康星大学的 I. Rayment）。 b) 驱动蛋白、动力蛋白和肌球蛋白 V，动力学：使用单分子 FIONA（一纳米精度荧光成像）研究过氧化物酶体、黑素体、PC12 细胞和其他细胞系中的驱动蛋白、动力蛋白和肌球蛋白 V 的体内动力学）和光捕获。这是我们在体内使用 FIONA 的结果的后续，我们已经实现了 1 毫秒和 1.5 纳米的时间和空间分辨率。我们发现，驱动蛋白和动力蛋白并未参与“拔河比赛”，尽管过氧化物酶体朝相反方向移动。现在的想法是在细胞内光学捕获过氧化物酶体（我们已经证明这是可能的），并确定以下各项：1）过氧化物酶体在动力蛋白和驱动蛋白方向上的失速力，从而明确显示有多少他们同时拉动； 2）从体内研究到体外研究，了解使电机拉得如此之快的转变发生在哪里； 3）测试其他系统，例如PC12神经突，看动力学是否相似； 4) 使用一纳米精度的明场成像 (bFIONA)（FIONA 的仿制品）测量黑素体（非荧光）的运动，并测试各种条件下的动力学：例如，无肌动蛋白、无肌动蛋白微管，没有中间丝......，以展示黑素体如何在一个运动系统和另一个运动系统之间进行转换。（与西北大学 V. Gelfand 合作）。 c) 肌球蛋白 VI：研究体内肌球蛋白 VI，并了解锚定蛋白（单体）和加工运动蛋白（二聚体）之间的转变与以下因素的关系：细胞内的位置；作为辅助蛋白（Dab2、GIPC...）的功能；作为肌球蛋白 VI 货物结合域插入长度的函数（与 L. Sweeney, UPenn.）。 d) 技术发展：需要实现光学/显微镜才能实现上述目标。