New Motility Mechanism and high speed, in vivo, imaging of Motor Protein Dynamics

新的运动机制和运动蛋白动力学的高速体内成像

• 批准号: 7487568
• 负责人: Joshua Vaughan
• 金额: $ 5.13万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7487568
• 关键词: Actins; Active Biological Transport; Alzheimer' s Disease; Behavior; Biochemical; Biological Assay; Biological Models; Biomechanics; Blindness; Capsid; Caveolins; Cell division; Cell membrane; Cell physiology; Cell surface; Cells; Clathrin; Common Cold; Coxsackie Viruses; Cytokinesis; Cytoplasm; Data; Development; Disease; Dyes; Dynein ATPase; Echo Viruses; Endocytosis; Enterovirus; Fluorescence; Fluorescence Microscopy; Genome; Goals; Growth; Heart Diseases; Hepatitis A; Human Virus; Human poliovirus; Huntington Disease; Image; In Vitro; Individual; Infection; Kinesin; Label; Lead; Length; Life; Light; Measurement; Mediating; Methods; Microfilaments; Microscopy; Motion; Motor; Muscle Contraction; Myosin ATPase; Optics; Organelles; Pathway interactions; Pharmaceutical Preparations; Play; Poliomyelitis; Polioviruses; Process; Property; Protein Dynamics; Proteins; RNA; RNA Interference; RNA Viruses; Range; Rhinovirus; Role; Running; Scheme; Series; Site; Small Interfering RNA; Speed; Staging; Surface; Techniques; Testing; Time; Travel; Viral; Viral Pathogenesis; Virion; Virus; Work; analog; analytical method; base; caveolin 1; cell motility; deafness; flotillin; in vivo; insight; nanometer; nervous system disorder; novel; particle; pathogen; poliovirus receptor; protein function; research study; single molecule; size; therapeutic target; trafficking

DESCRIPTION (provided by applicant): Live cell fluorescence microscopy will be used to investigate anomalously rapid actin-dependent motility of poliovirus that is 5-10 times faster than the fastest known actin-dependent translocation of cargo. Three aspects of this behavior will be investigated. First, the hypothesis that a myosin motor protein is responsible for the motility will be tested by means of various drugs, fluorescent protein myosin analogs, and siRNA. Myosin specific drugs or siRNA will be tested for inhibition of the rapid motion and various fluorescent protein myosin analogs will be tested for colocalization with poliovirus during transport. Second, the novel motility will be characterized in vivo using imaging-based, high speed and high accuracy particle tracking with the goal of dissecting the motion to the level of individual motor protein steps. Careful analysis of particle tracking data will be used to gain insight into the mechanochemical properties that give rise to the rapid motion. Third, the role of the motility to the infection pathway of poliovirus will be evaluated. Cells which lack an endogenous poliovirus receptor will be transfected with a fluorescent protein poliovirus receptor analog in order to directly visualize whether the rapid motion of poliovirus represents the active recruitment of poliovirus receptors. A three-dimensional particle tracking method, in combination with a dual fluorescent labeling scheme in which poliovirus capsids are labeled with one dye and poliovirus RNA is labeled with a different dye, will be used to determine whether the motion functions to transport viruses to a site of genome release. Poliovirus is a model system for nonenveloped RNA viruses, a group which includes numerous viruses ranging from the common cold to hepatitis A. Elucidating the mechanisms by which polio enters and navigates cells will enhance our basic understanding of viral pathogenesis and will lead to possible targets for therapies based on the inhibition of viral entry or transport.

描述(申请人提供)：活细胞荧光显微镜将用于研究脊髓灰质炎病毒依赖肌动蛋白的异常快速移动，其速度比已知的最快的依赖肌动蛋白的货物转运快5-10倍。将对这一行为的三个方面进行调查。首先，将通过各种药物、荧光蛋白肌球蛋白类似物和siRNA来检验肌球蛋白马达蛋白负责运动性的假设。将测试肌球蛋白特定药物或siRNA对快速运动的抑制作用，并将测试各种荧光蛋白肌球蛋白类似物在运输过程中是否与脊髓灰质炎病毒共存。其次，将使用基于成像的、高速和高精度的粒子跟踪来在体内表征这种新的运动性，目标是将运动解剖到单个运动蛋白质步骤的水平。对粒子跟踪数据的仔细分析将被用来洞察导致快速运动的机械力化学特性。第三，评价运动性在脊髓灰质炎病毒感染途径中的作用。缺乏内源性脊髓灰质炎病毒受体的细胞将被转染一种脊髓灰质炎病毒受体类似物，以便直接观察脊髓灰质炎病毒的快速运动是否代表脊髓灰质炎病毒受体的积极招募。三维粒子跟踪方法，结合双重荧光标记方案，其中脊髓灰质炎病毒衣壳用一种染料标记，脊髓灰质炎病毒RNA用不同的染料标记，将被用来确定运动是否具有将病毒运送到基因组释放部位的功能。脊髓灰质炎病毒是无包膜RNA病毒的模型系统，包括从普通感冒到甲型肝炎的众多病毒，阐明脊髓灰质炎进入和导航细胞的机制将增强我们对病毒发病机制的基本理解，并将导致基于抑制病毒进入或运输的可能的治疗靶点。