Cellular functions of membrane skeleton proteins, band 3

红细胞膜骨架是一个主要由谱蛋白和肌动蛋白组成的六边形蛋白质晶格，通过谱蛋白与锚蛋白和带3的相互作用附着在脂质双分子层上。质膜骨架也存在于非红细胞中，越来越多的证据表明，它们将在细胞内部结构中发挥作用，如高尔基体、溶酶体核和囊泡运输系统。我们将探讨三个红细胞膜问题。目的1将重点介绍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的敲除，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。我们将仔细测量由此产生的小鼠的表型，以及这些锚蛋白变化对膜骨架结构、带3的迁移率和红细胞物理性质的影响。由于去除调控结构域将去除一个保守的锚蛋白“死亡结构域”，我们还将寻找对红母细胞的凋亡或抗凋亡作用。目的2是基于观察到带3，红细胞阴离子转运体，集中在分裂的红母细胞的两极，并且具有retsina等位基因的斑马鱼缺乏带3，双核红母细胞数量增加。这表明带3参与红母细胞分裂。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，我们还将识别轻度形式的斑马鱼视网膜缺陷，并将这些鱼用于改善贫血发病或程度的基因的增强和抑制筛选。这些基因将成为在细胞质分裂中与带3相互作用的蛋白质的候选基因。最后，在Aim 3中，我们将表征bIII谱蛋白，它位于高尔基体和细胞质囊泡中。我们将识别囊泡，定位囊泡在spectrin上的结合位点以及在囊泡和囊泡上的spectrin结合伙伴，并研究干扰这些相互作用的后果。将特别注意bIII spectrin参与ER-to-Golgi贩运的可能性。我们还将破坏spectrin bIII基因以评估功能表型的丧失，并分离缺乏spectrin bIII的成纤维细胞和造血细胞系进行拯救实验和其他bIII spectrin功能的测试。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.