Marginal Band Formation and Cellular Morphogenesis in Nucleated Erythrocytes

有核红细胞被用作模型系统， 研究细胞骨架的生物发生及其与 细胞形态发生重点是边缘的形成 微管带（MB）及其在影响细胞增殖中的作用 成红细胞形态由球形变为扁平 椭圆体 一个主要目标是分析MT捆绑作为基础 MB的形成，使用最近开发的体外系统。 在这里，MT捆绑之后是实时视频显微镜， 通过低温简单获得的MT蛋白的重组 提取细胞骨架或分离的MB。 该分析包括 结核分枝杆菌相关蛋白鉴定及超微结构定位 涉及MT-MT交联的蛋白质（MAP），以及它们的用途， 抗体作为潜在的捆绑抑制剂。 第二主 目的是检验我们对MB分期的假设 生物发生及其与细胞形状的功能相关性 变化 这项工作利用贫血的非洲爪蟾，其中红细胞 在流通中有所区别。 单个红细胞 和双指向的，并且包含指向的、不完整的MB， 在这种贫血的青蛙的循环中发现;这些可能 代表形态发生中间体。 实验包括BrdU 核标记以确定体内形态发生序列 以及在体外，在尖细胞中测试预测的MT极性， MB结构与细胞间的因果关系 形状 这项工作有望帮助解决目前的争议 关于MT束的形成和功能，并加强一般 了解真核细胞的细胞骨架功能。 非哺乳类脊椎动物的红细胞是完全不同的 与哺乳动物不同，哺乳动物的红细胞失去了它们的 而非哺乳动物红细胞保留其 原子核。在蛙红细胞分化过程中， 从成红细胞的圆形、球形形状的形状变化 成熟的扁平椭圆形的前体 红细胞 这种形状变化与 一组周向的微管束在 扁平化，被称为边缘带（MB），据推测 维持这些细胞的独特形状。 这 该提案解决了与该机制有关的两个问题， 哪种微管形成和发挥作用：1，哪些非微管蛋白 参与捆绑过程;以及2， 细胞形态发生中的MB 除了增加我们的 了解非哺乳类脊椎动物红细胞如何发育， 这个项目可能会揭示新的一般原则， 微管集束和微管与相关的相互作用 proteins.

Nucleated erythrocytes are utilized as a model system for studying biogenesis of the cytoskeleton and its relationship to cellular morphogenesis. The focus is on formation of the marginal band (MB) of microtubules (MTs) and its function in effecting the change of cell shape from spheroidal erythroblast to flattened ellipsoid. A major objective is to analyze MT bundling as a basis for MB formation, using a recently developed in vitro system. Here, MT bundling is followed by real-time video microscopy during reassembly of MT protein obtained simply by low temperature extraction of cytoskeletons or isolated MBs. The analysis includes identification and ultrastructural localization of MT-associated proteins (MAPs) involved in MT-MT crossbridging, and the use of antibodies as potential bundling inhibitors. The second major objective is to test our working hypothesis for stages of MB biogenesis and their functional correlation with cell shape changes. This work exploits anemic Xenopus in which erythroblasts differentiate in the circulation. Erythroid cells which are singly and doubly pointed and which contain pointed, incomplete MBs have been discovered in the circulation of such anemic frogs; these may represent morphogenetic intermediates. Experiments include BrdU nuclear labelling to determine the morphogenetic sequence in vivo and in vitro, testing of predicted MT polarity in pointed cells, and study of ;the causal relationship between MB structure and cell shape. The work is expected to help settle current controversy regarding MT bundle formation and function, and to enhance general understanding of cytoskeletal function in eukaryotic cells. The red blood cells of nonmammalian vertebrates are quite different from those of mammals, in that mammalian red cells lose their nuclei during maturation while nonmammalian red cells retain their nuclei. During frog red cell differentiation, there is a marked shape change from the rounded, spheroid shape of the erythroblast precursor to the flattened ellipsoid shape of the mature erythrocyte. This shape change is associated with the development of a set of circumferential bundles of microtubules in the plane of flattening, known as the marginal band (MB), which presumably functions to maintain the unique shape of these cells. This proposal addresses two questions with respect to the mechanism by which MBs develop and function: 1, what non-microtubular proteins are involved in the bundling process; and 2, what is the role of the MB in cellular morphogenesis? In addition to increasing our understanding of how nonmammalian vertebrate erythrocytes develop, this project may shed new light on general principles of microtubule bundling and microtubule interactions with associated proteins.