Molecular mechanisms for the assembly of the red cell membrane skeleton during erythroid cell development

红细胞发育过程中红细胞膜骨架组装的分子机制

基本信息

批准号：
14360187
负责人：
INABA Mutsumi
金额：
$ 9.47万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360187/
关键词：
erythroid development red blood cell membrane skeleton band 3 ankyrin membrane trafficking intracellular vesicles cell culture 赤芽球系分化赤血球膜膜骨格タンパク質スペクトリン牛

项目摘要

The "unit-assembly hypothesis" for red cell membrane skeleton during erythroid development was investigated.Stable transfectants of major constituents of red cell transmembrane proteins were developed in K562 and HEK293 cells using retroviral vectors. Band 3 and glycophorinCweretransportedtotheplasmamembranevia the ER/Golgi pathway. Band 3-binding domain of ankyrin (AnkN90) and protein 4.1 showed membranous localization in the cytoplasm with co-localization with markers for the ER and the Golgi apparatus in band 3-transfeted cells despite of the difference in erythroid differentiation. Endogenous membrane skeletal proteins including ankyrin, protein 4.1, and spectrin also exhibited membranous distribution. These results indicate that the assembly of membrane skeletal proteins occurs at an early stage of erythroidal development on the surface of the ER but not on the inner surface of the plasma membrane.Stable expression of band 3 caused a reduction in a proliferation rate of K562 cells to approximately 70% that of non-transfected cells. Reduced production of hemoglobin suggestive of erythroid differentiation was also observed. However, no significant changes in intracellular localization of skeletal proteins and cell morphology was detected Synthesis and assembly to the ER of skeletal proteins involving spectrin and subsequent synthesis and localization to the plasma membrane of band 3 were observed in erythroid precursor cells from peripheral blood of cattle in two phase liquid culture system, indicating that the assembly of membrane skeletal proteins to the ER occurs in physiolosical erythropoiesis.The present study demonstrated that the assembly of the red cell membrane skeleton is an event which takes place at early stages of erythroid development on the ER. The exact mechanism by which a small unit of the ekeleton formed on intracellular vesicles are integrated in the plasma membrane remained to be clarified.

研究了红细胞发育过程中红细胞膜骨骼的“单位组装假设”。使用逆转录病毒载体在K562和HEK293细胞中开发了红细胞跨膜蛋白的主要成分的稳定转染物。频段3和糖磷酸果皮旋转eplastlasmambranevia er/golgi途径。 arkyrin（Ankn90）和蛋白质4.1的带3结合结构域显示在细胞质中与ER的标记和标记物和高尔基体在带3转基的细胞中的膜片定位，尽管ER肌缩微分化的差异有所差异。内源性膜骨骼蛋白在内，包括Ankyrin，蛋白4.1和Spectrin也表现出膜分布。这些结果表明，膜骨骼蛋白的组装发生在ER表面上的红斑发育的早期阶段，而不是在质膜内表面上的稳定表达。稳定的表达导致K562细胞增殖速率降低，从而降低了未转移的细胞的70％。还观察到了暗示性红细胞分化的血红蛋白产生的降低。然而，未检测到骨骼蛋白质的细胞内定位和细胞形态的显着变化，并在涉及光谱的骨骼蛋白质以及随后的合成以及对带3的质膜定位的骨骼蛋白的合成和组装中，观察到在两个阶段液体培养物中，在两个阶段的液体培养物中，表明量的skleter sepranter semprol sembrote y y经孔，以表明何种量，以表明在两个阶段的液体中，这表明是量的量子，这表明何种量，表明量的量，该量是量的，该量是量的，该量是量的，该量是量的，该量是量的，该量是在何种量中的组合细胞。本研究表明，红细胞膜骨骼的组装是在ER上红骨发育的早期发生的事件。在质膜中整合在细胞内囊泡上形成的小单位的ekeleton的确切机制仍有待阐明。