Functional Analysis of the Dual Specificity Kinase NEK1 in Mammalian Meiosis

项目摘要 在减数分裂过程中，同源染色体相互寻找，然后通过染色体连接在一起。 联会复合体（SC）和姐妹染色单体凝聚，没有同源重组和 减数分裂不能发生。编码任何SC组分或粘附素的基因突变的小鼠 显示减数分裂配对缺陷，并且在大多数情况下是不育的。SC蛋白，FKBP 6，这是必不可少的 雄性小鼠减数分裂前期I的完成，与一种新型减数分裂激酶NEK 1（NIMA相关）相互作用 激酶1）。NEK 1是一种具有丝氨酸/苏氨酸和酪氨酸双重活性的激酶，在生殖细胞中高度表达， 特别是进入和通过前期I的狭窄窗口。Nek1 突变小鼠显示出严重的发育缺陷，不仅在它们的生育能力方面，而且还显示出生长缺陷， 颅面畸形和多囊肾病。这个提议的核心假设是NEK 1 是前期I到中期进展所必需的，因为它将关键SC事件与涉及 姐妹染色单体凝聚力为了验证这一假设，我将分析Nek 1 kat 2 J系的减数分裂进程， 含有单核苷酸插入和随后的过早终止的小鼠，导致 蛋白质产物和无效表型。提出了两个具体目标：（1）深入研究 FKBP 6、NEK 1和cohesin蛋白在小鼠精母细胞中的直接关系，并比较 直接与卵母细胞中的作用，以确定减数分裂表型中的任何性二态性，以及（2） 评估野生型和突变型NEK 1的丝氨酸/苏氨酸和酪氨酸激酶活性 直接在小鼠生殖细胞中。这些实验将为NEK 1在以下方面的作用提供新的和令人兴奋的数据： 减数分裂进程，以及前期I结束时粘着蛋白去除的机制，这是一个关于 这方面的报道很少。 我在分子生物学和细胞遗传学方面都有很强的背景（ 强调哺乳动物配子发生）;我的目标是加强我的蛋白质组学技能，以执行 这个项目，走向独立研究。四年来，我一直是一名博士后研究员， 康奈尔大学，其中两年由HD基金会资助博士后奖。在康奈尔大学， 在Paula Cohen博士的实验室进行了研究，他的实验室在记录 主要交叉途径小鼠减数分裂。我的近期职业目标包括在高影响力的出版物 期刊，在康奈尔大学之外建立科学网络，并在学术机构展示研究成果;我的 我的长期职业目标是在一个高水平的机构获得终身职位，在那里我可以专注于 独立研究以及未来研究人员的培训。我在这方面的进展将是 通过与我的共同导师定期会议，跨部门研讨会对我的研究进行评估， 最终，我的出版记录。

Project Summary During meiosis, homologous chromosomes seek each other out and are then tethered together by the synaptonemal complex (SC) and sister chromatid cohesion, without which homologous recombination and meiotic division cannot occur. Mice with mutations in genes encoding any of the SC components or cohesins show meiotic pairing defects and, in most cases, are sterile. The SC protein, FKBP6, which is essential for completion of meiotic prophase I in male mice, interacts with a novel meiotic kinase, NEK1 (NIMA-related kinase 1). NEK1 is a dual activity serine/threonine and tyrosine kinase, and is highly expressed in germ cells, particularly the narrow window encompassing the entry into, and progression through, Prophase I. Nek1 mutant mice show severe developmental defects, not only in their fertility, but also show growth defects, cranial-facial abnormalities and polycystic kidney disease. The central hypothesis of this proposal is that NEK1 is required for prophase I to metaphase progression, as it links key SC events with those involving sister chromatid cohesion. To test this hypothesis, I will analyze meiotic progression in a line of Nek1kat2J miceharboring a single nucleotide insertion and a subsequent premature stop, resulting in truncation of the protein product and a null phenotype. Two specific aims are proposed: (1) to perform an in-depth study of the relationship between FKBP6, NEK1 and cohesin proteins directly in mouse spermatocytes, and compare this directly with the action in oocytes, to determine any sexual dimorphism in the meiotic phenotype and (2) to assess the serine/threonine and tyrosine kinase activities of both the wild type and mutant forms of NEK1 directly in mouse germ cells. These experiments will provide novel and exciting data on the role of NEK1 in meiotic progression, as well as on the mechanisms of cohesin removal at the end of prophase I, a subject on which there is very little reported data. I approach this project with a strong background in both molecular biology and cytogenetics (with an emphasis on mammalian gametogenesis); my goal is to strengthen my proteomics skills in order to execute this project and move toward independent research. For four years I have been a postdoctoral researcher at Cornell University, two of these years funded by an HD foundation postdoctoral award. At Cornell I have conducted research in the laboratory of Dr. Paula Cohen, whose lab has been instrumental in documenting the major crossover pathways mouse meiosis. My immediate career goals include publishing in high-impact journals, establishing a science network outside of Cornell and to present research at academic institutions; my long-term career goal is to obtain a tenure-track position at a high caliber institution where I can focus on independent research as well as the training of future researchers. My progress in this direction will be assessed by regular meetings with my co-mentors, inter-departmental seminars on my research and, ultimately, by my publication record.