Marginal Band Formation and Cellular Morphogenesis in Nucleated Erythrocytes

有核红细胞的边缘带形成和细胞形态发生

• 批准号: 9118773
• 负责人: William Cohen
• 金额: $ 32.51万
• 依托单位: CUNY Hunter College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-15 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9118773&HistoricalAwards=false
• 关键词: Marginal; Band; Formation; Cellular; Morphogenesis

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.

成核红细胞用于研究细胞骨架的生物发生及其与细胞形态发生的关系。重点是形成微管（MT）的边缘条带（MB）及其在影响细胞形状从球体红细胞变为平坦的椭圆形的变化方面的功能。 一个主要的目的是使用最近开发的体外系统分析MT捆绑作为MB形成的基础。 在这里，MT捆绑在重新组装MT蛋白过程中，仅通过细胞骨架或分离的MBS的低温提取获得的MT蛋白进行实时视频显微镜。 该分析包括与MT-MT跨桥相关的MT相关蛋白（MAP）的鉴定和超微结构定位，以及将抗体用作潜在捆绑抑制剂的使用。 第二个主要目标是测试我们的工作假设，以了解MB生物发生阶段及其与细胞形状变化的功能相关性。 这项工作利用了贫血的植物，其中红细胞在循环中有区别。 在这种贫血的青蛙的循环中发现了单一且包含尖的，不完全MB的细胞细胞。这些可能代表形态发生的中间体。 实验包括BRDU核标记，以确定体内形态发生序列和体外，测试尖细胞中预测的MT极性的测试，并研究； MB结构与细胞形状之间的因果关系。 预计这项工作将有助于解决有关MT束形成和功能的当前争议，并增强对真核细胞中细胞骨架功能的一般理解。 非哺乳动物脊椎动物的红细胞与哺乳动物的红细胞完全不同，因为哺乳动物红细胞在成熟过程中失去了核，而非乳腺红细胞则保留了其核。在青蛙红细胞分化过程中，从红细胞前体的圆形球形形状到成熟的红细胞的扁平椭圆形形状有明显的形状变化。 这种形状的变化与扁平平面中的一组微管的圆周束的发展有关，称为边缘带（MB），这大概可以起作用以维持这些细胞的独特形状。 该提案解决了MBS开发和功能的机制的两个问题：1，捆绑过程中涉及哪些非微管蛋白； 2，MB在细胞形态发生中的作用是什么？ 除了增加我们对非乳腺脊椎动物红细胞的理解之外，该项目还可能对微管捆绑和与相关蛋白的微管相互作用的一般原理发明新的启示。