Podosome structures as a focal adhesion appeared in osteoclasts in culture

破骨细胞是专门用于降解矿化基质的大型多核细胞。在与成骨细胞骨形成相协调的过程中，破骨细胞性骨吸收是维持骨内环境动态平衡的关键。我们目前研究的目的是可视化地观察质膜是如何被修饰的，以及细胞骨架如何与附着物相互作用，以及重新吸收破骨细胞的边缘区域是如何皱折的。为了观察破骨细胞膜和相关细胞骨架的表面修饰，我们采用细胞剪切结合快速冷冻和旋转复制的方法，暴露和复制了与人工合成磷灰石接触的破骨细胞表面膜的细胞质表面的大片区域。与磷灰石相对的膜由3个不同的区域组成：附着区、褶皱边缘和剩余部分。在附着区，高度组织化的肌动蛋白细丝网络形成点状、富含F-肌动蛋白的粘附点，即所谓的足体和肌动蛋白环。足体的细胞骨架细丝和肌动蛋白环似乎与细胞膜的细胞质表面直接接触。在肌动蛋白环内，单个可识别的足体被保存得很好，这表明肌动蛋白环可能是由足体融合而来的。细胞剪切法结合快速冷冻显微镜可以提供更详细的附着区和褶皱边缘区域细胞膜表面的信息。

Osteoclasts are large, multinucleated cells specialized for the degradation of mineralized matrix. In the harmony with osteoblastic bone formation, osteoclastic bone resorption is essential for the dynamic equilibrium of bone homeostasis. The aim of our present research was to visualize how the plasma membrane is modified and how the cytoskeleton interacts with the attachment, and ruffled border regions of resorbing osteoclasts. In order to view the surface modification of membranes and associated cytoskeleton, we employed the method of cell shearing combined with quick freezing and rotary replication to expose and replicate an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. The membrane apposed to the apatite was composed of 3 different domains : the attachment zone, ruffled border, and the remainder. In the attachment zone, a highly organized actin filament network formed dot-shaped, F-actin rich adhesion sites, so-called podosomes, and the actin ring. The cytoskeletal filament of podosomes and actin ring appeared to be in direct contact with the cytoplasmic surface of the underlying membrane. Within the actin ring, individually recognizable podosomes were well preserved, which indicates that the actin ring was probably derived from the fusion of podosomes.Cell shearing combined with quick-freezing microscopy was used as we considered that this method would give more detailed information on the cytoplasmic surface of membrane in the attachment zone and ruffled border region.