Cellular functions of membrane skeleton proteins, band 3

红细胞膜骨架是一种主要由血影蛋白和肌动蛋白组成的六方蛋白质晶格，它通过血影蛋白与锚蛋白和带3的相互作用附着在上层脂质双层上。质膜骨架也存在于非红系细胞中，越来越多的证据表明它们将在高尔基体、溶酶体核和囊泡运输系统等细胞内部结构中发挥作用。我们将调查三个红细胞膜问题。目标1将重点介绍Ankyrin调节域的功能和Anyrins的冗余。我们将完成Ankyrin 1的敲除，并在体内用Anyrin 1替换Anyrin 1，该基因缺失“调节”结构域，或具有全长ankyrin 3。我们将仔细测量由此产生的小鼠的表型，以及ankyrin的这些变化对膜骨架结构、带3的迁移率和红细胞膜的物理性质的影响。由于去除调控结构域将移除保守的锚定蛋白“死亡结构域”，我们也将寻找对红细胞的凋亡或抗凋亡作用。目的2是基于观察到红细胞膜负离子转运体带3集中在红细胞分裂的极点，以及缺乏带3的视网膜等位基因的斑马鱼增加了双核红细胞的数量。这表明带3参与了红细胞胞质分裂。我们将使用“下拉”分析和双杂交筛选来寻找带3与纺锤体组件或其他蛋白质的相互作用，我们还将识别较轻形式的斑马鱼视网膜缺陷，并将这些鱼类用于增强和抑制筛查改变贫血开始或程度的基因。这些基因将是胞质分裂中与带3相互作用的蛋白质的候选基因。最后，在目标3中，我们将描述位于高尔基体和细胞质小泡中的BIII光谱蛋白。我们将鉴定小泡，定位小泡上的血影蛋白结合位点和小泡上的血影蛋白结合伙伴，并研究干扰这些相互作用的后果。将特别注意BIII血影蛋白参与ER到Golgi的贩运的可能性。我们还将干扰血影蛋白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，用于救援实验和其他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各种功能。

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.