Clathrin sheets and cytoskeletal podosomes on the cytoplasmic side of ventral membranes of cultured osteoclasts

培养破骨细胞腹膜细胞质侧的网格蛋白片和细胞骨架足体

基本信息

批准号：
11671828
负责人：
AKISAKA Toshitaka
金额：
$ 1.73万
依托单位：
Asahi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Using a cell shearing method combined with freeze-drying and rotary shadowing replicas after shearing cells open with a stream of buffer, we examined the ultrastructure of clathrin sheets and cytoskeletal podosomes of cultured osteoclasts. Further, to visualize the confocal images for podosomes and clathrin sheets of isolated osteoclasts, we stained F, G-actin, and clathrin. Optical sections from confocal z-scanning with F-, G-actin staining showed the podosomes to be rosette in shape. From xy-scanning the F-actin was distributed in the core of the podosomes as a dot, whereas G-actin was present in a ring-like fashion. An immunofluorescence staining for clathrin was seen at the paramarginal area and central part of osteoclasts. Both the clathrin sheets and cytoskeletal podosomes survived the shearing process. The cytoplasmic side of the ventral membranes of the osteoclasts was characterized by the presence of clathrin sheets displayed as pentagons or hexagons. Podosomes were composed of cytoskeletal framework mediating widespread association of the microfilaments with the cytoplasmic side of the membrane. Some microfilaments in podosomes seem to be closely associated with the ventral membrane via small particles, but others appeared to terminate directly on the membrane. Podosomes are well known to be a specific cell-substratum attachment apparatus for osteoclasts. Besides, the present result suggests that clathrin functions are essential for not only receptor-mediated vesicles trafficking but also cell attachment.

使用细胞剪切方法与冻干和旋转阴影复制品结合使用，在用缓冲液流剪切细胞后，我们检查了网格蛋白片的超微结构和培养骨细胞的细胞骨架上的超微结构。此外，为了可视化孤立破骨细胞的足体和网状蛋白片的共聚焦图像，我们染色了F，G-肌动蛋白和网状蛋白。与F-，G-肌动蛋白染色的共聚焦Z扫描的光学切片表明，足以表现为玫瑰花结。从XY扫描的F-肌动蛋白作为点分布在足体的核心中，而G-肌动蛋白以环状方式存在。在骨细胞的临界区域和中心部分可见网状蛋白的免疫荧光染色。网格蛋白片和细胞骨架上的足迹都在剪切过程中幸存下来。破骨细胞的腹膜的细胞质侧的特征是存在于五角龙或己糖的存在。足体由细胞骨架框架组成，将微丝与膜的细胞质侧介导了广泛的关联。足体中的一些微丝似乎通过小颗粒与腹膜密切相关，但另一些似乎直接终止于膜上。足体众所周知是破骨细胞的特定细胞肌膜附着设备。此外，目前的结果表明，网格蛋白功能不仅对于受体介导的囊泡运输和细胞附着至关重要。