Control over phase-separated germ-granules morphology, molecular patterning, positioning and function.

在某些脊椎动物和无脊椎动物中，生殖细胞的发育依赖于种质--没有被膜包围的含有RNA和蛋白质的相分离凝聚体。种质和其中的成分被称为生殖粒，它们对生殖细胞的规格和后期的发育很重要。因此，除了指导细胞承担生殖细胞的命运外，生殖质成分对于细胞迁移和分化为配子、精子和卵子也是必不可少的。在这项研究提案中，我们使用斑马鱼胚胎作为一个可访问的通用模型来研究活的脊椎动物有机体中细菌颗粒的基本生物学，并最终将这些发现与人类不孕不育联系起来。本研究的目标是：1.利用显微镜、新颖的图像分析工具和不同的生物物理方法，对观察到的颗粒数量、分布和形态的演变进行详细的描述。2.确定相分离物质与细胞内其他成分的空间关系，以及不同细胞器和细胞结构对颗粒形状和位置的影响。3.开发新的工具，以便能够研究Tdrd7蛋白的功能，这是获得正确数量和大小的生殖粒所必需的，从而了解控制种质这些特征的机制。4.研究了与分子分布有关的相分离材料的图案化，考察了颗粒大小对细胞器内组织的影响。5.定义控制Tdrd7功能的蛋白质结构域，并确定这些结构域中的突变是否会影响生殖细胞的发育，并与人类的生育能力有关。

The development of germ cells in certain vertebrate and invertebrate organisms depends on germ plasm - RNA and protein-containing phase-separated condensates that are not surrounded by a membrane. Germ plasm and components that reside within it are termed germ granules and these are important for the specification of the germ cells and their development at later stages. Thus, in addition to directing cells to assume the germ-cell fate, germ plasm components are also essential for the migration and differentiation of the cells into gametes, sperm, and egg. In this research proposal, we use zebrafish embryos as an accessible general model for studying the basic biology of germ granules in the live vertebrate organism and eventually link the findings to infertility in humans. The objectives of the proposed research are- 1. To provide a detailed description of the evolution of the observed number, distribution, and morphology of the granules employing microscopy, novel image analysis tools, and different biophysical methods in the live embryo. 2. To determine the spatial relationships between the phase-separated material and other components within the cell and the effect of the different organelles and cellular structures on the granule shape and position. 3. Development of new tools which will allow for investigating the function of the Tdrd7 protein, which is required for attaining the correct number and size of the germ granules, thereby learning about the mechanisms controlling these features of the germ plasm. 4. Study the patterning of the phase-separated material concerning the molecules' distribution and examine the effect granule size has on the intra-organelle organization. 5. Define protein domains that control the function of Tdrd7 and determine if mutations within those domains affect the development of germ cells and can be linked to fertility in humans.