NUCLEUS & CYTOSKELETON IN LATE ERYTHROID MORPHOGENESIS

拟议的研究调查了与航站楼相关的事件 哺乳动物红细胞生成的各个阶段。这些事件包括核事件 凝聚、核挤压和新形成的成熟化 网织红细胞转变为双凹红细胞。这些研究将提供基本的 有关正常红细胞生成的信息，并提供对各种 红系疾病，如巨幼细胞性贫血、继发性贫血 化疗和正常核和/或球细胞性贫血 细胞骨架功能受损。一种体外模型系统的建立 小鼠脾成红细胞在感染猪瘟病毒感染后的应用 将使用Friend病毒的贫血株。第一个具体目标是 确定丝状细胞骨架和膜骨架的作用 在哺乳动物红细胞去核过程中。含有肌动蛋白的 在早期网织红细胞之间的区域发现收缩环 并对挤压出的细胞核进行生化和化学分析。 免疫学手段。细胞骨架在分离血影蛋白中的作用 将使用肌动蛋白抑制剂来研究对早期网织红细胞的影响 细丝和微管。细胞骨架蛋白水平的变化 以及它们各自基因在红细胞成熟过程中的表达 将使用Western blotts、Northern blotts和原位杂交来确定 杂交。第二个具体目标是通过生物化学和 网织红细胞成熟为双凹的形态特征 红血球。网织红细胞和突出的核将从 有核的红细胞和彼此之间的相互作用。胞内细胞器 网织红细胞成熟后的质膜将被分析。 并与网织红细胞形态变化相关。第三 具体目的是确定核凝聚和核凝聚的机制 识别引发核挤压的信号 在哺乳动物的红细胞中。合成速率、磷酸化的变化 特定蛋白质的状态和积累将在细胞核中进行量化 从成熟早、中、晚期的红细胞中分离得到 采用双向凝胶电泳法。孤立的原子核将是 以确定那些出现显著变化的蛋白质是否 与染色质、核膜/孔复合体或 不溶于水的核基质。将会产生针对蛋白质的抗体 被确定为可能在核凝聚或挤出中发挥作用 为了确定这些蛋白质在细胞核内的分布 在红细胞成熟的过程中。

The proposed research investigates events associated with the terminal stages of mammalian erythropoiesis. These events include nuclear condensation, nuclear extrusion and maturation of newly formed reticulocytes into biconcave erythrocytes. These studies will provide basic information about normal erythropoiesis, and give insights into a variety of erythroid diseases such as megaloblastic anemia, anemia secondary to chemotherapy and spherocytic anemia in which normal nuclear and/or cytoskeletal function is impaired. An in vitro model system of erythropoiesis employing erythroblasts from spleens of mice infected with the anemia-strain of Friend virus will be used. The first specific aim is to determine the role of the filamentous cytoskeleton and membrane skeleton in the enucleation of mammalian erythroblasts. The actin-containing constriction ring found in the region between the incipient reticulocyte and extruding nucleus will be isolated and analyzed by biochemical and immunological means. The role of the cytoskeleton in segregating spectrin to the incipient reticulocyte will be studied using inhibitors of actin filaments and microtubules. Changes in the levels of cytoskeletal proteins and expression of their respective genes during erythroblast maturation will be determined using Western blots, Northern blots and in situ hybridization. The second specific aim is to biochemically and morphologically characterize the maturation of reticulocytes into biconcave erythrocytes. Reticulocytes and extruded nuclei will be separated from nucleated erythroblasts and from each other. The intracellular organelles and plasma membrane will be analyzed in reticulocytes as they mature in vitro and correlated with changes in reticulocyte morphology. The third specific aim is to determine the mechanisms of nuclear condensation and identification of the signals responsible for initiating nuclear extrusion in mammalian erythroblasts. Changes in synthesis rate, phosphorylation state and accumulation of specific proteins will be quantitated in nuclei isolated from erythroblasts at early, middle and late stages of maturation using two dimensional gel electrophoresis. Isolated nuclei will be fractionated to determine if those proteins which show marked changes are associated with chromatin, the nuclear envelope/pore complex or the insoluble nuclear matrix. Antibodies will be raised against proteins identified as having a possible role in nuclear condensation or extrusion in order to determine the distribution of those proteins within the nucleus during the maturation of erythroblasts.