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

描述（由申请方提供）：活细胞荧光显微镜将用于研究脊髓灰质炎病毒的异常快速肌动蛋白依赖性运动，其速度比已知最快的肌动蛋白依赖性转运快5-10倍。我们将从三个方面来研究这种行为。首先，假设肌球蛋白运动蛋白是负责的运动将通过各种药物，荧光蛋白肌球蛋白类似物，和siRNA的手段进行测试。将测试肌球蛋白特异性药物或siRNA对快速运动的抑制，并测试各种荧光蛋白肌球蛋白类似物在运输过程中与脊髓灰质炎病毒的共定位。第二，新的运动性将在体内使用基于成像的，高速和高精度的粒子跟踪进行表征，目标是将运动分解到单个运动蛋白步骤的水平。对粒子跟踪数据的仔细分析将用于深入了解引起快速运动的机械化学性质。第三，评价脊髓灰质炎病毒运动性在脊髓灰质炎病毒感染途径中的作用。将用荧光蛋白脊髓灰质炎病毒受体类似物转染缺乏内源性脊髓灰质炎病毒受体的细胞，以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主动募集。一种三维粒子跟踪方法，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，将用于确定运动功能是否将病毒运送到基因组释放位点。脊髓灰质炎病毒是无包膜RNA病毒的模型系统，无包膜RNA病毒包括从普通感冒到甲型肝炎的许多病毒。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转运的治疗的可能目标。

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.