Actin and myosins in organelle motility

细胞器运动中的肌动蛋白和肌球蛋白

基本信息

批准号：
6555622
负责人：
CHRISTINA A KING-SMITH
金额：
$ 12.16万
依托单位：
SAINT JOSEPH'S UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-01 至 2006-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6555622
关键词：
SDS polyacrylamide gel electrophoresis actins cell motility density gradient ultracentrifugation fish fluorescence microscopy intracellular transport melanosomes microfilaments microinjections myosins retinal pigment epithelium transmission electron microscopy western blottings

项目摘要

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.

描述（由申请人提供）：拟议的研究的目标是表征肌动蛋白丝和肌球蛋白电动机在视网膜色素上皮（RPE）细胞中从鱼类中的黑素体（色素颗粒）运输中的作用。黑素体在体内经体在体内经历大规模迁移，以响应光条件。响应化学信号的分解的，培养的RPE细胞中，黑色素体运动持续存在。最近的研究导致了RPE细胞中黑色素体运动的模型。该模型假设RPE投影中黑素体的中心偏聚聚集是由黑色素体与肌动蛋白丝的被动关联发生的。假设黑素体的离心分散剂是通过肌球蛋白电动机沿肌动蛋白丝的运输而发生的。为了测试该模型的预测，提出的研究的具体目的是1）确定RPE顶端预测中的肌动蛋白丝使用荧光斑点显微镜（FSM）进行逆行肌动蛋白流量（FSM），并同时观察肌动蛋白流动和在聚集过程中的黑色素体动力以确定梅拉素体的运动是否与Atcins conflows conflows conflow一起进行。 2）使用体外运动测定法表征基于肌动蛋白的电动机。为此，将使用密度梯度离心纯化黑色素体，并使用SDS-PAGE和免疫印迹鉴定与黑色素体膜相关的蛋白质。黑色素体的肌动蛋白依赖性运动将使用体外运动测定测试。将使用两种测定法：在NITELLA测定中，将分离的黑色素体应用于Characean Alga，Nitella的解剖细胞的肌动蛋白电缆，并使用延时视频观察。第二个测定是滑动丝测定法，其中观察到荧光标记的肌动蛋白丝在附着在底物上的黑素体上移动。这些测定将有助于表征与黑色素体相关的肌球蛋白电机。拟议的研究将有助于更好地理解肌动蛋白依赖性细胞器运动性，这是真核细胞的普遍特征。