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（由申请人提供）：活细胞荧光显微镜将用于研究脊髓病毒的异常快速肌动蛋白依赖性运动性，该运动性比最快的已知肌动蛋白依赖性货物的易位快5-10倍。将研究这种行为的三个方面。首先，将通过各种药物，荧光蛋白肌球蛋白类似物和siRNA测试肌球蛋白运动蛋白负责运动性的假设。将测试肌球蛋白特异性药物或siRNA以抑制快速运动，并且在运输过程中将测试各种荧光蛋白肌球蛋白类似物与脊髓灰质炎病毒共定位。其次，新型运动性将在体内使用基于成像的高速和高精度粒子跟踪来表征，以将运动剖析至单个运动蛋白步骤的水平。仔细分析粒子跟踪数据将用于深入了解引起快速运动的机械化学特性。第三，将评估运动对脊髓灰质炎病毒感染途径的作用。缺乏内源性脊髓灰质炎病毒受体的细胞将被荧光蛋白poliovirus受体类似物转染，以直接可视化脊髓灰质炎病毒的快速运动是否代表了脊髓灰质炎病毒受体的主动募集。一种三维粒子跟踪方法与双荧光标记方案结合使用，其中脊髓灰质炎病毒带有一种染料和脊髓灰质炎病毒RNA标记为不同的染料，将用于确定运动功能是否将运动功能运输到基因组释放的部位。脊髓灰质炎病毒是一种用于非发育RNA病毒的模型系统，其中包括从普通感冒到乙型肝炎的多种病毒。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理的基本理解，并将基于抑制病毒入口或运输的疗法可能导致可能的靶标。