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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.

研究了红细胞膜骨架在红系发育过程中的“单位组装假说”，利用逆转录病毒载体，在K562和HEK293细胞中建立了稳定的红细胞膜跨膜蛋白主要成分的转染体。条带3和glycophorinCweretransportedtotheplasmamembranevia为内质网/高尔基体途径。带3结合结构域AnkN90和蛋白4.1在带3转运细胞中显示膜定位，并与ER和高尔基体标记共同定位，尽管红系分化不同。内源性膜骨架蛋白如锚蛋白、蛋白4.1和血影蛋白也呈膜性分布。这些结果表明，膜骨架蛋白的组装发生在红系发育早期的ER表面，而不是质膜内表面。带3的稳定表达使K562细胞的增殖率下降到未转染细胞的70%左右。提示红系分化的血红蛋白产生减少也被观察到。在两相液体培养体系中，牛外周血中的红系前体细胞合成并组装了含有血影蛋白的骨骼蛋白的内质网，随后合成并定位于带3的质膜，说明生理性红细胞发生了膜骨架蛋白与内质网的组装。本研究证实红细胞膜骨架的组装是红系发育早期的内质网事件。细胞内囊泡上形成的一小部分骨架整合到质膜中的确切机制尚不清楚。