Cellular functions of membrane skeleton proteins, band 3

膜骨架蛋白带 3 的细胞功能

基本信息

批准号：
6647334
负责人：
SAMUEL E LUX
金额：
$ 24.14万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-07-01 至 2003-06-30
项目状态：
已结题

项目摘要

The red cell membrane skeleton is a hexagonal protein lattice composed principally of spectrin and actin, which is attached to the overlying lipid bilayer through interactions of spectrin with ankyrin and band 3. Plasma membrane skeletons also exist in non-erythroid cells and there is increasing evidence that they will function in internal cellular structures such as the Golgi, lysosomes nucleus and vesicular transport systems. We will investigate three red cell membrane questions. Aim 1 will focus on the function of ankyrin regulatory domain and the redundancy of the ankyrins. We will complete an ankyrin 1 knockout and in vivo replacement of anykyrin 1 with ankyrin lacking a "regulatory" domain, or with full-length ankyrin 3. We will carefully measure the phenotype of the resulting mice and the effects of these changes in ankyrin on the structure of the membrane skeleton, the mobility of band 3, and the physical properties of the red cell membrane. Since removal of the regulatory domain will remove a conserved ankyrin "death domain", we will also look for apoptotic or anti-apoptotic effect on erythroblasts. Aim 2 is based on the observations that band 3, the red cell anion transporter, concentrates at the poles of dividing erythroblasts, and that zebrafish with the retsina allele, who lack band 3, have increased numbers of binucleate erythroblasts. This suggests that band 3 is involved in erythroblast cytokinesis. We will look for interaction of band 3 with spindle components or other proteins, using "pull-down" assays and two-hybrid screens, and we will also identify milder forms of the zebrafish retsina defect and use these fish in enhancer and suppressor screens for genes that modify the onset or degree of anemia. Such genes will be candidates for proteins that interact with band 3 in cytokinesis. Finally, in Aim 3, we will characterize bIII spectrin, which is located in the Golgi and in cytoplasmic vesicles. We will identify the vesicles, locate the vesicle binding site on spectrin and the spectrin-binding partner on the vesicles, and vesicles, and investigate the consequences of interfering with these interactions. Particular attention will be paid to the possibility that bIII spectrin is involved in ER-to-Golgi trafficking. We will also disrupt the spectrin bIII gene to assess the loss of function phenotype and to isolate fibroblast and hematopoietic cell lines lacking spectrin bIII for rescue experiments and other tests of bIII spectrin function. These experiments will broaden our knowledge of the membrane skeleton and help us begin to understand the diverse functions of this important cellular structure.

红细胞膜骨架是主要由血影蛋白和肌动蛋白组成的六边形蛋白晶格，通过血影蛋白与锚蛋白和带 3 的相互作用附着在覆盖的脂质双层上。质膜骨架也存在于非红细胞中，并且有越来越多的证据它们将在高尔基体、溶酶体核和囊泡运输系统等内部细胞结构中发挥作用。我们将研究三个红细胞膜问题。目标 1 将重点关注锚蛋白调节域的功能和锚蛋白的冗余。我们将完成锚蛋白 1 敲除，并用缺乏“调节”结构域的锚蛋白或全长锚蛋白 3 体内替换锚蛋白 1。我们将仔细测量所得小鼠的表型以及锚蛋白中这些变化对小鼠的影响。膜骨架的结构、带 3 的迁移性以及红细胞膜的物理特性。由于去除调节结构域将去除保守的锚蛋白“死亡结构域”，因此我们还将寻找对成红细胞的凋亡或抗凋亡作用。目标 2 基于以下观察结果：带 3（红细胞阴离子转运蛋白）集中在分裂红细胞的两极，而带有 retsina 等位基因的斑马鱼（缺乏带 3）的双核红细胞数量增加。这表明带 3 参与成红细胞胞质分裂。我们将使用“下拉”测定和双杂交筛选来寻找带 3 与纺锤体成分或其他蛋白质的相互作用，我们还将识别斑马鱼视网膜缺陷的较温和形式，并在增强子和抑制子筛选中使用这些鱼改变贫血发作或程度的基因。这些基因将是与胞质分裂中的带 3 相互作用的蛋白质的候选基因。最后，在目标 3 中，我们将表征 bIII 血影蛋白，它位于高尔基体和细胞质囊泡中。我们将识别囊泡，定位血影蛋白上的囊泡结合位点和囊泡上的血影蛋白结合配偶体，以及囊泡，并研究干扰这些相互作用的后果。将特别关注 bIII 血影蛋白参与 ER 至高尔基体运输的可能性。我们还将破坏血影蛋白 bIII 基因，以评估功能表型的丧失，并分离缺乏血影蛋白 bIII 的成纤维细胞和造血细胞系，用于拯救实验和其他 bIII 血影蛋白功能测试。这些实验将拓宽我们对膜骨架的认识，并帮助我们开始了解这一重要细胞结构的多种功能。