Ultrastructural investigation of the membrane modifications of osteoclasts

本研究的主要目的是探讨骨细胞，尤其是骨吸收破骨细胞的超微结构状态。目前，快速冷冻被认为是尽可能保持细胞结构自然状态的最好但困难的方法。冷冻复制品(FR)清晰地显示了膜的内部结构。FR显示不同破骨细胞膜结构域的大小、形状和分布模式不同。特定的杆状MP定位于褶皱边缘(RB)膜内。讨论了这类MP与质子泵ATPase的关系。RB的MP强度最大。在附着透明带膜上，MP呈簇状分布，可能与细胞附着有关。另一项使用快速冷冻和冷冻替代的实验显示了有关破骨细胞结构的几个有趣的发现。Rb膜由细胞骨架成分支撑。在Rb内，观察到一些闭合接触，如功能不明的自反性缝隙连接。与常规固定标本相比，快速冷冻破骨细胞显示出明显的差异；各种细胞器、膜结构的保存率显著提高。在细胞质内，常规固定不能保存的管泡状结构占据细胞质中的细胞器。从吸收的骨基质中衍生出的碎裂羟基磷灰石晶体位于管泡结构内。这一发现表明了摄取钙的可能途径。我们计划在以后发表在我们的工作中获得的各种数据，这些数据是在目前的科学研究补助金的支持下获得的。

Main purpose of our study is to investigate the morenative state of bone cell ultrastructure, especially bone resorbing osteoclasts. At the present, quick freezing is believed to be the best but difficult way to preserve cell structure in its native state as possible. Freeze replicas (FR) showed clearly the membrane interior. Membrane particles (MP) by FR showed a different size, shape and distribution pattern of various membrane domains of osteoclasts. Specific rodshaped MP were localized within the ruffled border (RB) membrane. This type of MP is discussed in relation to the proton pump ATPase. RB had the most intensive MP. In the attaching clear zone membrane, MP showed a cluster fashion which may be responsible for cell attachment. Another our experiment using quick freezing followed by freeze-substitution showed several interesting findings about osteoclast structures. RB membrane were supported by cytoskeletal components. Within RB, some closed contacts were observed like the reflexive gap junction which its functional role is obscure. Comparing with the routinely fixed samples, quick frozen osteoclasts showed remarkable differences; the preservation of various organelles, membrane structures were dramatically improved. Within the cytoplasm, tuburovesicular structure which was never preserved by routinely fixation occupied among the cell organelles in the cytoplasm. Fragmented hydroxyapatite crystals derived from resorbed bone matrix were located within the tubulovesicular structures. This finding indicates the possible pathway for up-take calcium.We are planning, hereafter, to publish various data which were obtained during our work supported by the present Grant-in-Aid for Scientific Research.