The Role of XMAP215 in Regulating Microtubule Assembly In Vivo

微管（MTs）是真核细胞中主要的一类细胞骨架元件。它们的特点是能够以一种受管制的方式伸长或迅速拆卸。它们在细胞形状的维持或改变中起着重要作用，它们作为亚细胞器从细胞的一个地方到另一个地方的轨迹，它们在细胞分裂过程中对染色体的分裂至关重要。在胚胎发育过程中，它们也在细胞质分配中起关键作用。本项目以非洲非洲爪蟾（Xenopus laevis）为模型系统，重点研究MTs在卵母细胞的动物-植物轴和发育中的脊椎动物胚胎的背-腹（D-V）轴的规范和/或维持中所起的作用。卵子发育过程中稳定和动态MT之间的快速转换，以及D-V轴规范所需的精子aster和皮质MT的快速组装，对MT组装的调节机制产生了独特的限制。从非洲爪蟾卵母细胞和卵中分离出三种微管相关蛋白，分别为XMAP215、XMAP230和XMAP310，它们被认为在调节MT组装中起关键作用。在这些XMAPs中，XMAP 215在体外表现出促进MT +末端组装和动态的独特特性。这些研究结果表明，XMAP215在卵子发生和早期发育期间负责mt的组装和动态。本研究旨在探讨XMAP215在青蛙卵发生和发育过程中调节MT组装的功能。需要解决的具体问题包括：1、XMAP215是否与爪蟾卵发生和早期发育过程中的MTs有关？该蛋白将通过共聚焦免疫荧光显微镜和/或通过表达gfp标记或表位标记的XMAP215 mRNA微注射到卵母细胞和胚胎中来定位；在卵母细胞和胚胎发育的不同阶段发生的特定MT阵列将被检查。2、体内特定微管阵列的组装是否需要XMAP215 ？通过将抗体或反义寡脱氧核糖核苷酸注入活卵母细胞和胚胎中，XMAP215将被耗尽，这种耗尽对MT组织和发育的影响将在共聚焦显微镜下使用抗微管蛋白抗体进行检测。3、XMAP215-M和XMAP215-Z在功能上有区别吗？这个问题是基于发现爪蟾卵和胚胎中有两种形式的XMAP215 mRNA，母系形式（-M）存在于卵母细胞和原胚前胚胎中，合子形式（-Z）存在于原胚后胚胎中。它们的不同之处在于，有一个36个氨基酸的插入（Ins2）只存在于母体形式中。这两种异构体表达的发育调控表明它们可能在功能和/或调控上有所不同。作为确定两种同种异构体之间功能区别的第一步，将在体外表达蛋白质，并检查它们与MT结合并促进MT在体外组装的能力。此外，表位和/或gfp标记的合子形式将在卵母细胞和早期胚胎中异长期表达，以检查该蛋白在体内的功能。这些研究结果将进一步加深我们对XMAP215在爪蟾卵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XMAP215和/或其人类同源物在细胞或生物生命中更广泛地发挥作用提供新的见解。

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.