The Role of XMAP215 in Regulating Microtubule Assembly In Vivo

XMAP215 在调节体内微管组装中的作用

• 批准号: 9904504
• 负责人: David Gard
• 金额: $ 39万
• 依托单位: University of Utah
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-15 至 2003-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904504&HistoricalAwards=false
• 关键词: Role; XMAP215; Regulating; Microtubule; Assembly

Microtubules (MTs) are one of the major classes of cytoskeletal elements in eukaryotic cells. They are characterized by their ability to elongate or rapidly disassemble in a regulated fashion. They play important roles in maintenance or change of cellular shape, they serve as tracks along which subcellular organelles travel from one place in the cell to another, and they are critical to the partitioning of chromosomes during cell division. They also play critical roles in cytoplasmic partitioning during embryonic development. This project focuses on the roles played by MTs in the specification and/or maintenance of both the animal-vegetal axis of oocytes and the dorsal-ventral (D-V) axis of developing vertebrate embryos, using the African frog, Xenopus laevis, as the model system. The rapid conversion between stable and dynamic MT's during oogenesis and the rapid assembly of the sperm aster and cortical MTs required for D-V axis specification place unique constraints on mechanisms regulating MT assembly. Three microtubule-associated proteins, termed XMAP215, XMAP230, and XMAP310, have been isolated from Xenopus oocytes and eggs, where they are thought to play key roles in regulating MT assembly. Of these XMAPs, XMAP 215 exhibits the unique property of promoting both the assembly and dynamics of MT plus-ends in vitro. Results from these studies suggest that XMAP215 is responsible for the assembly and dynamics of MTs during oogenesis and early development. The proposed research addresses the function of XMAP215 in vivo in regulating MT assembly during frog oogenesis and development. Specific questions to be addressed include:1, is XMAP215 associated with MTs during Xenopus oogenesis and early development? The protein will be localized by confocal immunofluorescence microscopy and/or by expression of GFP-tagged or epitope-tagged XMAP215 mRNA microinjected into oocytes and embryos; specific MT arrays that occur during various stages of oocyte and embryo development will be examined. 2, is XMAP215 required for the assembly of specific microtubule arrays in vivo? XMAP215 will be depleted by microinjection of antibodies or anti-sense oligodeoxyribonucleotides into living oocytes and embryos, and the effect of such depletion on MT organization and development will be examined by confocal microscopy using anti-tubulin antibodies. 3, Are XMAP215-M and XMAP215-Z functionally distinct? This question is based on the finding that there are two forms of XMAP215 mRNA in Xenopus eggs and embryos, a maternal form (-M) present in oocytes and pre-gastrula embryos, and a zygotic form (-Z) present in post-gastrula embryos. These differ in that there is a 36-amino acid insert (Ins2) present only in the maternal form. The developmental regulation of the expression of these two isoforms suggests that they may differ in their function and/or regulation. As a first step in defining the functional distinction between the two isoforms, the proteins will be expressed in vitro, and their ability to bind to MTs and promote MT assembly in vitro will be examined. In addition, epitope- and/or GFP-tagged versions of the zygotic form will be heterochronically expressed in oocytes and early embryos, to examine the function of this protein in vivo. The results of these studies will further our understanding of the role of XMAP215 in the spatial and temporal regulation of MT assembly and organization during oogenesis and early development in Xenopus, and will provide new insights into the role(s) that XMAP215 and/or its human homologues play more generally in the life of a cell or an organism.

微管是真核细胞中主要的细胞骨架元件之一。 它们的特征在于它们能够以调节的方式伸长或快速分解。 它们在维持或改变细胞形状方面起着重要作用，它们作为亚细胞器从细胞中的一个地方到另一个地方的沿着的轨道，并且它们对细胞分裂期间染色体的分配至关重要。 它们在胚胎发育过程中的细胞质分配中也起着关键作用。 该项目的重点是在规范和/或维护的卵母细胞的动物-植物轴和脊椎动物胚胎发育的背腹（D-V）轴中所发挥的作用，使用非洲蛙，非洲爪蟾，作为模型系统。 卵子发生过程中稳定和动态MT之间的快速转换以及D-V轴规范所需的精子星形细胞和皮质MT的快速组装对调节MT组装的机制产生了独特的限制。 三种微管相关蛋白，称为XMAP 215，XMAP 230和XMAP 310，已从非洲爪蟾卵母细胞和卵中分离出来，它们被认为在调节MT组装中起关键作用。 在这些XMAP中，XMAP 215表现出在体外促进MT+末端的组装和动力学的独特性质。 这些研究结果表明，XMAP 215是负责组装和动态的MT卵子发生和早期发育过程中。 该研究旨在探讨XMAP 215在青蛙卵子发生和发育过程中调节MT组装的体内功能。 具体问题包括：1、XMAP 215是否与非洲爪蟾卵子发生和早期发育中的MT相关？将通过共聚焦免疫荧光显微镜和/或通过将GFP标记或表位标记的XMAP 215 mRNA显微注射到卵母细胞和胚胎中来定位蛋白质;将检查卵母细胞和胚胎发育的各个阶段期间发生的特异性MT阵列。2、体内组装特异性微管阵列需要XMAP 215吗？ 将通过将抗体或反义寡脱氧核糖核苷酸显微注射到活卵母细胞和胚胎中来耗尽XMAP 215，并且将使用抗微管蛋白抗体通过共聚焦显微镜检查这种耗尽对MT组织和发育的影响。3、XMAP 215-M和XMAP 215-Z在功能上有区别吗？这个问题是基于以下发现：在非洲爪蟾卵和胚胎中存在两种形式的XMAP 215 mRNA，一种存在于卵母细胞和前原肠胚胚胎中的母体形式（-M），以及一种存在于后原肠胚胚胎中的合子形式（-Z）。 它们的不同之处在于有一个36个氨基酸的插入片段（Ins 2）仅存在于母体形式中。 这两种异构体表达的发育调节表明它们在功能和/或调节方面可能不同。 作为定义两种亚型之间的功能区别的第一步，将在体外表达蛋白质，并检查它们与MT结合并促进MT体外组装的能力。 此外，合子形式的表位和/或GFP标记的版本将在卵母细胞和早期胚胎中异时表达，以检查该蛋白在体内的功能。 这些研究的结果将进一步加深我们对XMAP 215在非洲爪蟾卵子发生和早期发育过程中MT组装和组织的时空调控中的作用的理解，并将为XMAP 215和/或其人类同源物在细胞或生物体的生命中更普遍地发挥的作用提供新的见解。