Cellular functions of membrane skeleton proteins, band 3

膜骨架蛋白带 3 的细胞功能

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6647334
• 关键词: actins; ankyrins; band 3 protein; cell cycle; cell growth regulation; embryonic stem cell; erythroid stem cell; erythropoiesis; gene targeting; intracellular membranes; intracellular transport; laboratory mouse; membrane activity; protein binding; protein engineering; protein isoforms; protein structure function; receptor binding; spectrin; zebrafish

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质晶格，其通过血影蛋白与锚蛋白和带3的相互作用附着于上覆的脂质双层。质膜骨架也存在于非红系细胞中，并且越来越多的证据表明它们将在内部细胞结构如高尔基体、溶酶体核和囊泡运输系统中起作用。我们将研究三个红细胞膜问题。目的1主要研究锚蛋白调控域的功能和锚蛋白的冗余性。我们将完成锚蛋白1敲除，并在体内用缺乏“调节”结构域的锚蛋白或全长锚蛋白3替换任何锚蛋白1。我们将仔细测量所得小鼠的表型以及锚蛋白的这些变化对膜骨架结构、带3的移动性和红细胞膜的物理性质的影响。由于去除调节结构域将去除保守的锚蛋白“死亡结构域”，我们还将寻找对成红细胞的凋亡或抗凋亡作用。目的2是基于以下观察结果：带3，红细胞阴离子转运蛋白，集中在分裂的成红细胞的两极，并且具有retsina等位基因的斑马鱼，缺乏带3，具有增加的双核成红细胞数量。这表明带3参与成红细胞胞质分裂。我们将寻找带3与纺锤体成分或其他蛋白质的相互作用，使用“下拉”试验和双杂交筛选，我们还将确定斑马鱼retsina缺陷的温和形式，并使用这些鱼在增强子和抑制子筛选中修饰贫血的发病或程度的基因。这样的基因将是与胞质分裂带3相互作用的蛋白质的候选者。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。我们将确定囊泡，定位血影蛋白和血影蛋白结合伴侣的囊泡，囊泡上的囊泡结合位点，并调查干扰这些相互作用的后果。将特别注意的可能性，bIII血影蛋白参与ER-高尔基体贩运。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了解，并帮助我们开始了解这一重要细胞结构的多种功能。