Mechanisms Establishing Oocyte Polarity

 描述(申请人提供)：在许多动物中，细胞极性对于上皮组织的形成、器官的形成以及胚轴的形成都是必不可少的。在大多数脊椎动物中，卵子的极性决定了胚胎的前后极性，并且对于建立胚胎的背腹轴是必不可少的。卵极性起源于卵子发生的早期阶段，此时卵母细胞中建立了不同的动植物结构域。脊椎动物卵母细胞的第一个极化结构是Balbiani小体(BB)，这种结构从昆虫到脊椎动物都很保守。青蛙和鱼中的BB由核糖核蛋白、种质、线粒体聚合体和RNAs组成，这些RNAs定位于卵母细胞和卵子的营养极，以及未来胚胎的生殖细胞。BB是一种暂时的结构，它移动到卵母细胞皮质，在那里它分离传递其内容物并确定植物极点。尽管是形成大多数脊椎动物胚胎主轴的基础，但脊椎动物卵母细胞的极性很少被研究，因为这些早期卵母细胞阶段在 成年女性的卵巢和遗传分析的困难。通过在斑马鱼中进行母体效应突变筛选，发现了两个建立卵母细胞极性的基因--Bucky ball(Buc)和macf1(微管肌动蛋白交联因子1)，这是第一个也是唯一一个已知在脊椎动物卵母细胞极性中起作用的基因。Buc的雌性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macf1突变卵母细胞中，BB形成但不能解体，破坏了卵母细胞的极性，核的位置不对称。Macf1是spectraplakin家族中一种非常大的细胞骨架连接蛋白。它包含一个 肌动蛋白结合域、微管结合域(MTBD)、能与细胞角蛋白细丝(CK)相互作用的Plakin重复结构域，以及一系列的血影蛋白重复序列。在BB分解过程中，Macf1在卵母细胞中通过CK将BB含量连接到肌动蛋白皮质，并通过连接核周CK和胞质MTS来定位细胞核，这是假设和验证的双重功能。BB是一种大的无膜细胞器或RNA颗粒，类似于应激颗粒、P小体和P颗粒。这些颗粒的一个关键特性是它们能够经历相变为颗粒形式或溶解为非颗粒形式的能力，以及相变对固有无序蛋白质(IDP)的依赖。BUC是目前已知的唯一一种形成BB所必需的蛋白质，它是一种IDP。这里研究了BB颗粒的动力学和性质，包括IdP Buc的性质。这些蛋白质提供了第一个功能切入点，以了解BB如何在其功能中被调节以建立卵母细胞的极性。重要的是，细胞中不适当的蛋白质颗粒聚集是疾病的主要原因，包括神经退行性疾病的联核病。这些研究为颗粒的形成、功能、调节和分解提供了体内范例，这在该领域是明显缺乏的。

 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.