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

采用冷冻干燥和旋转阴影相结合的细胞剪切法，对体外培养破骨细胞的网状蛋白片层和细胞骨架足体的超微结构进行了观察。此外，为了显示分离破骨细胞的足体和网状蛋白的共聚焦图像，我们对F、G-肌动蛋白和网状蛋白进行了染色。共聚焦Z扫描F-，G-肌动蛋白染色光镜切片显示，足体呈花环状。XY扫描显示，F-肌动蛋白呈点状分布在足小体的核心，而G-肌动蛋白呈环状分布。免疫荧光染色显示破骨细胞的边缘旁和中央部有网状蛋白表达。笼状蛋白片层和细胞骨架足体都在剪切过程中幸存下来。破骨细胞腹膜的细胞质一侧可见呈五角形或六角形的网状蛋白片层。足体由细胞骨架组成，介导微丝与膜的细胞质一侧的广泛联系。足体中的一些微丝似乎通过小颗粒与腹膜紧密相连，但另一些微丝似乎直接终止于膜上。足体是破骨细胞的一种特殊的细胞-基质附着装置。此外，本研究结果表明，笼蛋白功能不仅对受体介导的囊泡运输是必要的，而且对细胞附着也是必要的。

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.