Actin and myosins in organelle motility

描述(由申请人提供)： 这项拟议研究的目标是确定肌动蛋白细丝和肌球蛋白马达在鱼类视网膜色素上皮(RPE)细胞内黑素小体(色素颗粒)运输中的作用。在鱼RPE细胞中，黑素小体在体内经历了大规模的迁移，以响应光的条件。在体外分离培养的RPE细胞中，黑素小体对化学信号的反应持续存在。最近的研究得出了RPE细胞中黑素小体运动的模型。该模型假设RPE投射中黑素小体的向心性聚集是通过黑素小体与肌动蛋白细丝经历结构性逆行流动的被动结合而发生的。假设黑素小体的离心性分散是通过肌球蛋白马达沿肌动蛋白细丝运输黑素小体而发生的。为了验证这一模型的预测，拟议研究的具体目标是：1)利用荧光散斑显微镜(FSM)确定RPE根尖投射内的肌动蛋白细丝是否经历肌动蛋白逆行流动，并同时观察聚集过程中肌动蛋白流动和黑素小体的运动，以确定黑素小体的运动是否与肌动蛋白流动相协调。2)用体外运动性分析方法鉴定RPE黑素小体上的肌动蛋白马达。为此，将使用密度梯度离心法纯化黑素小体，并使用SDS-PAGE和免疫印迹鉴定与黑素体膜相关的蛋白质。依赖肌动蛋白的黑素小体的运动性将使用体外运动性分析进行测试。将使用两种分析方法：在Nitella分析中，将分离的黑素小体应用于被解剖的轮虫海藻Nitella细胞的肌动蛋白缆线上，并使用延时视频进行观看。第二种分析是滑动细丝分析，观察到荧光标记的肌动蛋白细丝在附着在底物上的黑素小体上移动。这些分析将有助于表征与黑素小体相关的肌球蛋白马达。这项拟议的研究将有助于更好地理解真核细胞的普遍特征--肌动蛋白依赖的细胞器运动。

DESCRIPTION (provided by applicant): The goals of the proposed research are to characterize the role of actin filaments and myosin motors in transport of melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells from fish. Melanosomes undergo massive migrations in fish RPE cells in vivo in response to light condition. Melanosome motility persists in dissociated, cultured RPE cells in vitro in response to chemical signals. Recent investigations resulted in a model for melanosome motility in RPE cells. The model hypothesizes that centripetal aggregation of melanosomes in RPE projections occurs by passive association of melanosomes with actin filaments undergoing constitutive retrograde flow. Centrifugal dispersion of melanosomes is hypothesized to occur by transport of melanosomes along actin filaments via myosin motors. To test the predictions of this model, the specific aims of the proposed research are 1) To determine whether actin filaments within RPE apical projections undergo retrograde actin flow using fluorescent speckle microscopy (FSM), and to simultaneously view actin flow and melanosome motility during aggregation to determine whether the movement of melanosomes is coordinated with actin flow. 2) To characterize actin-based motors on RPE melanosomes using in vitro motility assays. To accomplish this, melanosomes will be purified using density gradient centrifugation, and proteins associated with melanosome membranes will be identified using SDS-PAGE and immunoblotting. Actin-dependent motility of melanosomes will be tested using in vitro motility assays. Two assays will be used: in the Nitella assay, isolated melanosomes are applied to actin cables of dissected cells of the Characean alga, Nitella, and viewed using time-lapse video. The second assay is the sliding filament assay, where fluorescently labeled actin filaments are observed moving over melanosomes attached to a substrate. These assays will help to characterize melanosome-associated myosin motors. The proposed research will contribute to a better understanding of actin-dependent organelle motility, a universal characteristic of eukaryotic cells.