NUCLEUS & CYTOSKELETON IN LATE ERYTHROID MORPHOGENESIS

拟议的研究调查与终端相关的事件 哺乳动物红细胞生成的阶段。这些事件包括核 凝聚、核挤出和新形成的成熟 网织红细胞转化为双凹面红细胞。这些研究将提供基础 关于正常红细胞生成的信息，并深入了解各种 红系疾病，如巨幼细胞性贫血，继发于 化疗和球形细胞性贫血，其中正常的核和/或 细胞骨架功能受损。体外模型系统 红细胞生成，其使用来自感染 将使用Friend病毒的贫血株。第一个具体目标是 以确定丝状细胞骨架和膜骨架的作用 在哺乳动物成红细胞去核中的作用。含肌动蛋白的 在初始网织红细胞之间的区域中发现收缩环 并将挤出的细胞核进行分离和生化分析， 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研究初始网织红细胞 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成红细胞成熟过程中的表达 将使用蛋白质印迹、北方印迹和原位印迹测定 杂交方法第二个具体目标是生物化学和 从形态学上表征网织红细胞成熟为双凹面 红细胞网织红细胞和挤出的细胞核将从 有核成红细胞和彼此。细胞内的细胞器 和质膜将在网织红细胞成熟时进行分析， 与网织红细胞形态学变化相关。第三 具体目标是确定核凝聚的机制， 识别负责启动核挤出的信号 在哺乳动物成红细胞中。合成速率、磷酸化 将定量细胞核中特定蛋白质的状态和积累 分离自成熟早期、中期和晚期的成红细胞 使用二维凝胶电泳。分离的原子核将 分离以确定那些显示出显著变化的蛋白质是否是 与染色质、核膜/孔复合体或 不溶性核基质抗体会针对蛋白质产生 被确定为可能在核凝聚或挤压中起作用 为了确定这些蛋白质在细胞核内的分布 在成红细胞成熟期间。

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.