CAREER: Remodeling the egg extracellular matrix at fertilization

职业：受精时重塑卵子细胞外基质

• 批准号: 1846563
• 负责人: Sara Olson
• 金额: $ 82.8万
• 依托单位: Pomona College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1846563&HistoricalAwards=false
• 关键词: CAREER; Remodeling; egg; extracellular; matrix

Fertilization represents a major biological challenge, where sperm and egg must recognize and bind each other to create new life. However, an equally serious challenge soon awaits. Immediately after fertilization, the new embryo must rapidly remodel its surface so that additional sperm can no longer bind, and it must create an impenetrable barrier that will protect the embryo during development. The goal of this project is to explore protective barrier formation in the nematode worm, C. elegans, which offers several advantages over other organisms typically used to study this process (humans, mice, sea urchins). The work will be conducted entirely by undergraduate and high school students at Pomona College, a small liberal arts college dedicated to training the next generation of exceptional scientists. A diverse group of students in the investigator's research lab, as well as students in an Advanced Cell Biology course, will use genetic, biochemical, and cell biological approaches to investigate eggshell assembly in C. elegans. These studies will harness the power of C. elegans as a model system to explore both widely-conserved and species-specific strategies that contribute to protective barrier assembly in nematodes. Common features will lend insight into evolutionarily-conserved reproductive strategies, while species-specific features could identify new therapeutic targets to fight parasitic nematode infection of humans, plants, and animals. Importantly, this award will also create a mentorship pipeline for students historically underrepresented in science to support their persistence in STEM fields.Before fertilization, the C. elegans vitelline layer (analogous to the mammalian zona pellucida, fish chorion, and sea urchin vitelline layer) forms on the oocyte surface and is comprised of at least three proteins with an interdependent relationship. CBD-1 functions at the top of the hierarchy and recruits PERM-2 and PERM-4, who in turn maintain each other's localization. Interestingly, CBD-1 also organizes a separate protein complex involved in fertilization and egg activation (the EGG complex). After fertilization, the PERM complex is remodeled into the outermost eggshell layer, while the EGG complex is internalized. Little is understood about the mechanism of vitelline layer remodeling, or how the relationships among vitelline layer proteins promote these events. In Aim 1 of this project, students will examine the interdependence of the PERM and EGG structural scaffolds at the oocyte surface before fertilization by analyzing null mutants and domain variants generated by CRISPR/Cas9 gene editing. In Aim 2, students will use biochemical and phenotypic analyses to investigate how PERM-2 and PERM-4 contribute to the structural and functional changes to the vitelline layer after fertilization. In Aim 3, they will explore whether similarities exist between nematode and mammalian vitelline layer structure and assembly. Together, these studies will provide mechanistic insight into conserved and divergent processes in protective barrier assembly across distantly-related phyla.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

受精是一个重大的生物挑战，精子和卵子必须相互识别和结合才能创造新的生命。然而，同样严峻的挑战即将到来。受精后，新胚胎必须迅速重塑其表面，使额外的精子不能再结合，它必须创造一个不可穿透的屏障，在发育过程中保护胚胎。本项目的目的是探索保护性屏障的形成，在线虫，C。线虫，它提供了几个优势，比其他生物体通常用于研究这一过程（人类，小鼠，海胆）。这项工作将完全由波莫纳学院的本科生和高中生进行，波莫纳学院是一所致力于培养下一代杰出科学家的小型文理学院。在研究者的研究实验室的学生，以及学生在高级细胞生物学课程，将使用遗传，生物化学和细胞生物学方法来研究蛋壳组装在C。优美的这些研究将利用C.线虫作为一个模型系统，探索广泛保守的和物种特异性的战略，有助于保护性屏障组装线虫。共同特征将有助于深入了解进化保守的生殖策略，而物种特异性特征可以确定新的治疗靶点，以对抗人类，植物和动物的寄生线虫感染。重要的是，该奖项还将为历史上在科学领域代表性不足的学生创造一个导师管道，以支持他们在STEM领域的坚持。秀丽线虫卵黄层（类似于哺乳动物的卵透明膜、鱼绒毛膜和海胆卵黄层）形成于卵母细胞表面，并且由具有相互依赖关系的至少三种蛋白质组成。CBD-1在层次结构的顶部起作用，并招募PERM-2和PERM-4，后者反过来维护彼此的本地化。有趣的是，CBD-1还组织了一个独立的蛋白质复合物，参与受精和卵子激活（EGG复合物）。受精后，PERM复合体被重塑为最外层的蛋壳层，而EGG复合体被内化。关于卵黄层重塑的机制，或者卵黄层蛋白之间的关系如何促进这些事件的了解很少。在本项目的目标1中，学生将通过分析CRISPR/Cas9基因编辑产生的无效突变体和结构域变体，研究受精前卵母细胞表面PERM和EGG结构支架的相互依赖性。在目标2中，学生将使用生物化学和表型分析来研究PERM-2和PERM-4如何促进受精后卵黄层的结构和功能变化。在目标3中，他们将探索线虫和哺乳动物卵黄层结构和组装之间是否存在相似性。总之，这些研究将提供机械洞察保守和不同的过程中，保护屏障组装在遥远的相关phyla.This奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。