THE MEMBRANE SKELETON OF BONE CELLS

说明(改编自调查员摘要)： 拟议的工作是检验质膜和质膜 细胞内细胞器膜具有多组分膜骨架 这对正常骨组织的生产和维护至关重要。第一 目的是确定血影蛋白和锚蛋白在血管紧张素转换酶中的作用 破骨细胞在骨吸收过程中的黏附/黏附机制 非再吸纳期。这一目标是基于免疫定位的 锚蛋白与原位破骨细胞的清晰区和与之相关的结构 与破骨细胞的细胞-底物界面在培养中。第二个目标， 锚定蛋白在破骨细胞细胞器中的分布和相互作用 和囊泡，是基于在体内发现多种锚定蛋白亚型 破骨细胞裂解物与细胞器和囊泡的免疫细胞化学定位 膜。将从20日龄的雏鸡胚胎中分离出破骨细胞并进行发育 来自一种小鼠骨髓培养系统。这些细胞将被药物处理。 已知会改变吸收行为或干扰钙粘素功能 免疫定位和免疫沉淀研究。第三个目标是 利用小鼠锚蛋白-1建立锚蛋白在骨细胞中的作用 缺乏突变体，nb/nb，与之相比，骨密度显著降低 到杂合子的产仔对照。破骨细胞和成骨细胞培养将是 用突变小鼠和正常产仔对照动物建立。 骨细胞活性参数的初步研究包括抗酒石酸 酸性磷酸酶染色与吸收窝的定量评价 破骨细胞和碱性磷酸酶染色形成和产生 成骨细胞产生的骨钙素和I型胶原。骨骼的像差 生理学反映在出生时和以后明显的先天缺陷上。 发病条件，如骨质疏松。拟议的研究将产生基本的 关于破骨细胞和成骨细胞生理学的信息将长期存在 在设计有效的治疗策略方面的价值 骨细胞活动。

DESCRIPTION (adapted from the Investigator's abstract): The goal of the proposed work is to test the hypothesis that the plasma membrane and intracellular organelle membranes possess a multicomponent membrane skeleton that is crucial to production and maintenance of normal bone tissue. The first aim is to establish the role that spectrin and ankyrin play in the attachment/adhesion mechanism of osteoclasts during resorptive and non-resorptive periods. This objective is based on the immunolocalization of ankyrin to the clear zone of osteoclasts in situ and to a structure associated with the cell-substrate interface in osteoclasts in culture. The second aim, to establish the distribution and interaction of ankyrin in osteoclast organelles and vesicles, is based on the finding of multiple ankyrin isoforms in osteoclast lysates and immunocytochemical localization to organelle and vesicle membranes. Osteoclasts will be isolated from 20-day chick embryos and developed from a murine marrow culture system. The cells will be treated with agents known to alter resorptive behavior or interfere with cadherin function prior to immunolocalization and immunoprecipitation studies. The third aim is to establish the role of ankyrin in bone cells using the murine ankyrin-1 deficient mutant, nb/nb, which has significant low bone density when compared to heterozygous littermate controls. Osteoclast and osteblast cultures will be established using the mutant mice and the normal littermate control animals. Initial studies of bone cell activity parameters include tartrate-resistant acid phosphatase staining and quantitative assessment of resorption pit formation by osteoclasts and alkaline phosphatase staining and production of osteocalcin and type I collagen by osteoblasts. Aberrations in skeletal physiology are reflected in congenital defects apparent at birth and in later onset conditions such as osteoporosis. The proposed studies will generate basic information about osteoclast and osteoblast physiology that will have long-term value in the design of effective therapeutic strategies aimed at modulating bone cell activity.