Marginal Band Formation and Cellular Morphogenesis in Nucleated Erythrocytes

有核红细胞被用作研究细胞骨架生物发生及其与细胞形态发生关系的模型系统。重点研究微管边缘带（MB）的形成及其在红细胞球形向扁平椭球细胞转变过程中的作用。一个主要的目标是分析MT捆绑作为MB形成的基础，使用最近开发的体外系统。在这里，通过低温提取细胞骨架或分离的mb获得的MT蛋白重组过程中，实时视频显微镜跟踪MT捆绑。分析包括MT-MT交联相关蛋白（MAPs）的鉴定和超微结构定位，以及抗体作为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MB生物发生阶段及其功能与细胞形状变化的相关性。这项工作利用贫血的非洲爪蟾红细胞在循环中分化。在这种贫血青蛙的血液循环中发现了单尖和双尖的红细胞，其中含有尖的，不完整的MBs；这些可能代表形态发生的中间产物。实验包括BrdU核标记以确定体内和体外的形态发生序列，测试尖细胞中预测的MT极性，以及研究；MB结构和细胞形状之间的因果关系。这项工作有望有助于解决目前关于MT束形成和功能的争议，并提高对真核细胞细胞骨架功能的普遍理解。非哺乳动物脊椎动物的红细胞与哺乳动物的红细胞有很大不同，哺乳动物红细胞在成熟过程中失去细胞核，而非哺乳动物红细胞保留细胞核。在青蛙红细胞分化过程中，红细胞的形态发生了明显的变化，由红母细胞前体的圆球状向成熟红细胞的扁平椭球状转变。这种形状的变化与扁平平面上一组微管的周向束的发展有关，称为边缘带（MB），其功能可能是维持这些细胞的独特形状。该提案解决了两个关于MBs发展和功能机制的问题：1，哪些非微管蛋白参与了捆绑过程；二、MB在细胞形态发生中的作用是什么？除了增加我们对非哺乳动物脊椎动物红细胞发育的理解外，该项目还可能揭示微管捆绑和微管与相关蛋白相互作用的一般原理。

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.