Mechanisms Establishing Oocyte Polarity

 描述（由申请人提供）：细胞极性对于许多动物的上皮组织形成、器官形成和胚胎轴形成至关重要。在大多数脊椎动物中，卵的极性决定了胚胎的前后极性，并且对建立胚胎的背腹轴至关重要。卵的极性起源于卵子发生的早期阶段，此时在卵母细胞中建立了不同的动物-植物结构域。脊椎动物卵母细胞中的第一个极化结构是Balbiani小体（Bb），这是一种从昆虫到脊椎动物都保守的结构。青蛙和鱼类的Bb由核糖核蛋白、生殖质、线粒体聚集体和RNA组成，这些RNA被分配到卵母细胞和卵子的植物极，以及胚胎的未来生殖细胞。Bb是一个短暂的结构，它移动到卵母细胞皮层，在那里它解离提供其内容物和确定植物极。尽管对于形成大多数脊椎动物胚胎的主轴是基本的，但是脊椎动物卵母细胞极性很少被研究，这是由于这些早期卵母细胞阶段在胚胎中的不可接近性。 卵巢和成年女性的遗传分析的困难。通过在斑马鱼中进行母体效应突变体筛选，发现了两个建立卵母细胞极性的基因，即巴基球（布克）和macf 1（微管肌动蛋白交联因子1），这是已知的第一个也是唯一一个在脊椎动物卵母细胞极性中起作用的基因。雌性布克突变体在卵子发生过程中不能形成Bb，也不能建立卵母细胞的极性。在macf 1突变的卵母细胞中，Bb形成但不能分解，破坏卵母细胞的极性，并且细胞核不对称地定位。macf 1是一个非常大的spectraplakin家族的细胞骨架连接蛋白。它包含一个 肌动蛋白结合结构域、微管结合结构域（MTBD）、可与细胞角蛋白丝（CK）相互作用的Plakin重复结构域和一系列血影蛋白重复序列。据推测，并将进行测试，Macf 1在卵母细胞连接Bb内容的肌动蛋白皮层通过CK在Bb拆卸，并定位核连接核周CK细胞质的MT的双重功能。Bb是一个大的无膜细胞器或RNA颗粒，类似于应激颗粒、P体和P颗粒。这些颗粒的一个关键特性是它们经历相变成颗粒形式或溶解成非颗粒形式的能力，以及相变对固有无序蛋白质（IDP）的依赖性。布克是唯一已知的Bb形成所需的蛋白质，是IDP。在这里Bb颗粒动力学和性能的调查，包括IDP的性质布克。这些蛋白质提供了第一个功能性切入点，以了解Bb是如何调节其功能，以建立卵母细胞极性。重要的是，细胞中不适当的蛋白质颗粒聚集体是疾病的主要原因，包括突触核蛋白病的神经退行性疾病。这些研究为颗粒的形成、功能、调节和分解提供了体内范例，这在该领域是显著缺乏的。

 DESCRIPTION (provided by applicant): Cell polarity is essential to formation of epithelial tissues, organ formation, and to embryonic axis formation in many animals. In most vertebrates, polarity of the egg determines anterior-posterior polarity of the embryo, and is essential to establishing the embryonic dorsal-ventral axis. Egg polarity originates during early stages of oogenesis when distinct animal-vegetal domains are established in the oocyte. The first polarized structure in vertebrate oocytes is the Balbiani body (Bb), a structure conserved from insects to vertebrates. The Bb in frogs and fish is composed of ribonucleoproteins, germ plasm, an aggregate of mitochondria, and RNAs destined to the vegetal pole of the oocyte and egg, and to the future germ cells of the embryo. The Bb is a transient structure that moves to the oocyte cortex, where it dissociates delivering its contents and determining the vegetal pole. Although fundamental to forming the major axes of most vertebrate embryos, vertebrate oocyte polarity has been little studied due to the inaccessibility of these early oocyte stages within the ovary and the difficulty of genetic analysis in adult females. Through a maternal-effect mutant screen in the zebrafish, two genes were discovered that establish oocyte polarity, bucky ball (buc) and macf1 (microtubule actin crosslinking factor 1), the first and only genes known to function in vertebrate oocyte polarity. Females mutant for buc fail to form the Bb during oogenesis, and do not establish oocyte polarity. In macf1 mutant oocytes the Bb forms but fails to disassemble, disrupting oocyte polarity, and the nucleus is asymmetrically positioned. Macf1 is an extraordinarily large cytoskeletal linker protein of the spectraplakin family. It contains an actin binding domain, a Microtubule binding domain (MTBD), Plakin repeat domains that can interact with Cytokeratin filaments (CK), and a series of spectrin repeats. It is hypothesized and will be tested that Macf1 has dual functions in the oocyte linking Bb contents to the actin cortex via CK in Bb disassembly, and positioning the nucleus by linking perinuclear CK to cytoplasmic MTs. The Bb is a large membrane-free organelle or RNA granule, similar to stress granules, P bodies, and P granules. A key property of these granules is their ability to undergo phase transitions into granule form or dissolution into non-granule form, and the dependence of the phase transition on intrinsically disordered proteins (IDP). Buc, the only protein known to be required for Bb formation, is an IDP. Here Bb granule dynamics and properties are investigated, including the nature of the IDP Buc. These proteins provide the first functional entry points to understanding how the Bb is regulated in its function to establish oocyte polarity. Importantly, inappropriate protein granule aggregates in cells is a major cause of disease, including neurodegenerative diseases of the synucleinopathies. These studies provide an in vivo paradigm for granule formation, function, regulation, and disassembly, which is significantly lacking in this field.