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.

红细胞膜骨骼骨骼是一种主要由光谱和肌动蛋白组成的六角蛋白晶格，它通过谱素与脚Ankyrin and band 3的相互作用附着在上覆的脂质双层。质膜膜骨架也越来越有证据表明它们在内部细胞中也会在内部细胞中发挥作用，例如它们在内部细胞中的作用，例如它们在内部细胞中的作用。囊泡运输系统。我们将研究三个红细胞膜问题。 AIM 1将重点放在氨基林调节结构域的功能上，并将其冗余性的冗余。我们将完成一个arnkyrin 1敲除和体内替代任何kinkyrin 1的脚踝缺乏“调节性”域，或使用全长的Ankyrin 3。我们将仔细测量所得小鼠的表型，以及这些变化的Ankyrin对膜骨架的结构的影响，这些变化的影响是膜骨架的结构。由于去除调节域将去除保守的Ankyrin“死亡结构域”，因此我们还将寻找对红细胞的凋亡或抗凋亡作用。 AIM 2基于观察结果，即红细胞阴离子转运蛋白频段3集中在分裂的红细胞分裂的极点，而缺乏频带3的斑马鱼与RETSINA等位基因的斑马鱼具有增加数量的二核细胞粉红细胞。这表明频带3参与了红细胞细胞因子。我们将使用“下拉”测定法和两种杂交屏幕来寻找带3与主轴组件或其他蛋白质的相互作用，我们还将确定斑马鱼RETSINA缺陷的温和形式，并在增强剂和抑制器筛选中使用这些FISH的基因，以修改贫血的发作或程度。此类基因将是与细胞因子中与带3相互作用的蛋白质的候选物。最后，在AIM 3中，我们将表征位于高尔基体和细胞质囊泡中的BIII光谱。我们将识别囊泡，在光谱上定位囊泡结合位点，并在囊泡和囊泡上定位光谱蛋白结合伴侣，并研究干扰这些相互作用的后果。特别关注BIII Spectrin参与急诊室贩运的可能性。我们还将破坏Spectrin BIII基因，以评估功能表型的丧失，并分离缺乏用于救援实验的光谱BIII的成纤维细胞和造血细胞系和BIII光谱函数的其他测试。这些实验将扩大我们对膜骨骼的了解，并帮助我们开始理解这种重要的细胞结构的各种功能。