Akisaka Toshitaka, Yoshida Hisaho, Suzuki Reiko, Shimizu Kouichi, Takama Keiko
Department of Anatomy, Asahi University School of Dentistry, Hozumi 1851, Mizuho, Gifu 501-0296, Japan.
J Electron Microsc (Tokyo). 2003;52(6):535-43. doi: 10.1093/jmicro/52.6.535.
Physical cell-shearing resulted in various degrees of disruption of the basolateral (upper) membranes, cytoskeletons or cell organelles and exposed the protoplasmic surface of ventral (adhesion) membranes of osteoclasts that were attached to the underlying substratum, such as coverslips, mica or synthetic apatite plates. Freeze-dried replicas of the ventral membranes left behind on the substratum after cell-shearing provided three-dimensional information on the ultrastructure of the protoplasmic membrane surface of cultured osteoclasts. An extensive area of the protoplasmic surface and various amounts of cytoskeletal structures attached to the adherent ventral surface of the plasma membrane were visible. In particular, the most characteristic finding of the present study is that numerous clathrin sheets displaying various sizes, shapes and curvature were revealed on the ventral membrane. The polygon substructures of the clathrin lattices appeared to be composed of hexagons with a few pentagons interspersed. They were seen at the peripheral membranes where they were situated at the sites of close contact with the underlying substratum. In addition, clathrin lattices were never observed on the basolateral (upper) membranes. In favourable stereo views, most cytoskeletons were not in direct contact with the clathrin sheets. However, a few observations indicated possible remnants of cytoskeletons attached to clathrin lattices. Podosomes did not have a direct structural relationship to clathrin lattices. Although it is generally accepted that cytoskeletal podosomes in motile cells, such as osteoclasts, play a major role in cell adhesion, the present study indicates that membrane-associated clathrin might also function during attachment to the substrate. In this regard, clathrin is thought to be required for receptor-mediated endocytosis, but whether it might also function in cell attachment is still a matter for debate. This type of clathrin-related adhesion appears to be a previously unrecognized site of cell/substrate adhesion in osteoclasts. To assess this possible function, we focused on clathrin and related cytoskeletal elements on the ventral membranes of cultured osteoclasts.
物理性细胞剪切导致基底外侧（上部）膜、细胞骨架或细胞器出现不同程度的破坏，并使附着于下层基质（如盖玻片、云母或合成磷灰石板）的破骨细胞腹侧（黏附）膜的原生质表面暴露出来。细胞剪切后留在基质上的腹侧膜冻干复制品提供了培养破骨细胞原生质膜表面超微结构的三维信息。可见原生质表面的大片区域以及附着于质膜黏附腹侧表面的不同数量的细胞骨架结构。特别值得一提的是，本研究最具特征性的发现是在腹侧膜上发现了许多呈现出不同大小、形状和曲率的网格蛋白片层。网格蛋白晶格的多边形亚结构似乎由六边形组成，并穿插有一些五边形。它们出现在外周膜上，位于与下层基质紧密接触的部位。此外，在基底外侧（上部）膜上从未观察到网格蛋白晶格。在有利的立体视图中，大多数细胞骨架并未与网格蛋白片层直接接触。然而，有一些观察结果表明可能存在附着于网格蛋白晶格的细胞骨架残余物。足体与网格蛋白晶格没有直接的结构关系。虽然一般认为运动细胞（如破骨细胞）中的细胞骨架足体在细胞黏附中起主要作用，但本研究表明与膜相关的网格蛋白在附着于底物的过程中可能也发挥作用。在这方面，网格蛋白被认为是受体介导的内吞作用所必需的，但它是否也在细胞黏附中发挥作用仍存在争议。这种与网格蛋白相关的黏附似乎是破骨细胞中一个以前未被认识的细胞/底物黏附位点。为了评估这种可能的功能，我们重点研究了培养破骨细胞腹侧膜上的网格蛋白和相关细胞骨架成分。
