Mechanisms Establishing Oocyte Polarity

建立卵母细胞极性的机制

• 批准号: 9145723
• 负责人: Mary C. Mullins
• 金额: $ 34.08万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-17 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9145723
• 关键词: Actins; Adult; Alleles; Animals; Anterior; Balbiani Body; Binding; CRISPR/Cas technology; Caenorhabditis elegans; Cell Nucleus; Cell Polarity; Cells; Code; Cytokeratin filaments; Cytoplasmic Granules; Dependence; Disease; Dorsal; Embryo; Epithelial; Exhibits; Family; Female; Fishes; Future; Genes; Genetic; Genome; Germ; Germ Cells; Health; Human; Insecta; Link; Malignant neoplasm of ovary; Medical; Membrane; Messenger RNA; Microtubules; Mitochondria; Mitochondrial RNA; Molecular; Nature; Neoplasm Metastasis; Neurodegenerative Disorders; Oocytes; Oogenesis; Organ; Organelles; Organogenesis; Ovary; Permeability; Phase Transition; Phosphorylation; Play; Positioning Attribute; Property; Proteins; RNA; Radial; Rana; Regulation; Ribonucleoproteins; Role; Series; Spectrin; Staging; Stress; Structure; Testing; Therapeutic; Tissues; Transcript; Vertebrates; Zebrafish; crosslink; egg; genetic analysis; genome editing; in vivo; loss of function; mutant; novel; protein aggregation; retinal rods; synucleinopathy; vertebrate embryos

 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.

 描述（由申请人提供）：细胞极性对于许多动物的上皮组织、器官形成和胚胎轴形成至关重要。在大多数脊椎动物中，卵的极性决定胚胎的前后极性，并且对于胚胎轴的形成至关重要。建立胚胎背腹轴。卵子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物-植物域。脊椎动物中的第一个极化结构。卵母细胞是巴尔比亚尼体 (Bb)，这是一种从昆虫到脊椎动物都保守的结构。青蛙和鱼类中的 Bb 由核糖核蛋白、种质、线粒体聚集体以及运往卵母细胞和卵子植物极的 RNA 组成。 Bb 是一种短暂的结构，会移动到卵母细胞皮质，在那里它分离并输送其内容物并确定植物极。为了形成大多数脊椎动物胚胎的主轴，由于这些早期卵母细胞阶段难以在脊椎动物胚胎中形成，因此对脊椎动物卵母细胞极性的研究很少。 通过对斑马鱼的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：巴基球 (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发挥作用的基因。 buc 突变体的雌性在卵子发生过程中无法形成 Bb，并且在 macf1 突变体卵母细胞中也不能形成 Bb。但无法分解，破坏了卵母细胞的极性，并且细胞核的位置不对称。 Macf1 是 Spectraplakin 家族的一种非常大的细胞骨架连接蛋白。 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重复结构域以及一系列血影蛋白重复结构被抢夺并测试Macf1在卵母细胞中连接Bb内容物的双重功能。 Bb 分解过程中通过 CK 到达肌动蛋白皮质，并通过将核周 CK 连接到细胞质 MT 来定位细胞核。 Bb 是一个大的无膜细胞。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能够经历相变为颗粒形式或溶解为非颗粒形式，以及相变对本质的依赖性。无序蛋白 (IDP) 是已知 Bb 形成所需的唯一蛋白，这里研究了 Bb 颗粒动力学和特性，包括 IDP Buc 的性质。为了解 Bb 的功能如何调节以建立卵母细胞极性提供了第一个功能切入点。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，包括突触核蛋白病的神经退行性疾病。颗粒的形成、功能、调节和分解，这是该领域严重缺乏的。