MOLECULAR INTERACTIONS IN ERYTHROID CELL MEMB

跨膜蛋白的物理性质，包括横向 流动性，旋转流动性，空间相互作用，以及高和低 亲和结合相互作用，是细胞增殖的重要决定因素。 膜的结构和功能红系细胞提供了一个理想的系统 用于研究调节这种物理机制的分子机制， 特性.最近对人类红细胞的研究已经导致了一种有效的 模型的分子机制控制的平均横向和 带3的旋转迁移率，红细胞阴离子交换剂。空间 基于血影蛋白的膜骨架提供的阻碍相互作用 似乎调节带3横向扩散的速率;低亲和力 与膜骨架蛋白锚蛋白和 蛋白4.2调节带3旋转扩散的速率;高 与锚蛋白的亲和结合相互作用，以确定 旋转固定带3;和带3寡聚化以控制 横向不动带3的部分。该模型在 目前的建议，在个别波段3分子的水平上，在三个 相关红系细胞系统：来自患有 遗传性溶血性贫血和血影蛋白，锚蛋白，带3， 蛋白质4.1和蛋白质4.2;来自小鼠品系的成熟红细胞， 遗传性溶血性贫血和血影蛋白和锚蛋白缺陷， 从具有工程缺陷的转基因小鼠品系中， 3和蛋白质4.2;以及分化人和鼠红系细胞 在文化中。主要的红细胞唾液酸糖蛋白的物理性质， 血型糖蛋白A也在成熟红细胞和 分化红细胞。单粒子跟踪用于 区分个体的四种平移运动模式 蛋白质分子，包括不受限制的扩散， 扩散、定向运动和固定。激光光镊 用于定量高和低亲和力结合的强度 涉及天然蛋白质中单个蛋白质分子的相互作用 膜环境荧光光漂白恢复和偏振 荧光消耗用于测量平均横向和 旋转迁移率，分别为跨膜蛋白。 膜 生化和超微结构与跨膜蛋白有关 在单个分子水平上的物理性质。这些研究 将首次引导人们从分子水平上理解 限制跨膜蛋白迁移的物理力。阐发 这种力量的性质和规模将有助于解释 包括遗传性球形红细胞增多症在内的膜疾病的病理生理学 和遗传性椭圆形红细胞增多症。

Physical properties of transmembrane proteins, including lateral mobility, rotational mobility, steric interactions, and high and low affinity binding interactions, are important determinants of cell membrane structure and function. Erythroid cells provide an ideal system for study of the molecular mechanisms that regulate such physical properties. Recent studies in human red blood cells have led to a working model of the molecular mechanisms controlling the average lateral and rotational mobility of band 3, the red cell anion exchanger. Steric hindrance interactions provided by the spectrin based membrane skeleton appear to regulate the rate of band 3 lateral diffusion; low affinity binding interactions with the membrane skeletal proteins ankyrin and protein 4.2 to regulate the rate of band 3 rotational diffusion; high affinity binding interactions with ankyrin to determine the fraction of rotationally immobile band 3; and band 3 oligomerization to control the fraction of laterally immobile band 3. This model is tested in the present proposal, at the level of individual band 3 molecules, in three related erythroid cell systems: mature red cells from patients with hereditary hemolytic anemias and defects in spectrin, ankyrin, band 3, protein 4.1, and protein 4.2; mature red cells from mouse strains with hereditary hemolytic anemias and defects in spectrin and ankyrin, and from transgenic mouse strains with engineered defects in ankyrin, band 3, and protein 4.2; and differentiating human and murine erythroid cells in culture. The physical properties of the major RBC sialoglycoprotein, glycophorin A, are also examined in mature red cells and in differentiating erythroid cells. Single particle tracking is used to distinguish among four modes of translational motion of individual protein molecules, including unrestricted diffusion, constrained diffusion, directed movement, and immobilization. Laser optical tweezers are employed to quantify the strength of high and low affinity binding interactions involving individual protein molecules in the native membrane environment. Fluorescence photobleaching recovery and polarized fluorescence depletion are used to measure the average lateral and rotational mobility, respectively, of transmembrane proteins. Membrane biochemistry and ultrastructure are correlated with transmembrane protein physical properties at the level of individual molecules. These studies will lead, for the first time, to a molecular understanding of the physical forces constraining transmembrane protein mobility. Elucidation of the nature and magnitude of such forces will help to explain the pathophysiology of membrane disorders including hereditary spherocytosis and hereditary elliptocytosis.