Admin supplemental funds to purchase a Cherry Biotech CherryTemp temperature-controlled stage for live cell imaging of temperature-sensitive C. elegans mutants with oocyte meiotic spindle defects.

项目摘要 卵母细胞减数分裂以两轮细胞分裂结束，产生一个具有单倍体的卵子。 基因组虽然有丝分裂使用双极纺锤体，但卵母细胞 在没有中心体的情况下组装双极纺锤体。虽然组装和功能的 有丝分裂纺锤体已被广泛研究，无中心体卵母细胞纺锤体组装仍然存在， 知之甚少，C. elegans提供了一个有吸引力的模型系统， 控制这一过程的机制。此外，我们对卵母细胞纺锤体组装的理解 在所有广泛使用的模型系统中仍然不完整，直到最近才有活细胞成像 开始阐明纺锤体组装的动力学，以及一些零碎的遗传学， 研究提供了一个部分但不完整的重要参与者目录。我们 提出了一个系统的和定量的分析卵母细胞减数分裂纺锤体组装和纺锤体 双极性，使用一个有价值的收集温度敏感的突变体，我们已经分离。我们 还将使用RNA干扰来识别在卵母细胞发育过程中起作用的基因和途径， 主轴组件在C.沿着活细胞荧光视频显微镜和基因组 编辑提供一个更完整的和机械的理解，这从根本上 重要的过程。2014年，我们发表了一篇手稿，描述了我们在卵母细胞方面的初步工作。 细胞分子生物学中的纺锤极组装，以及2015年《细胞生物学》中的另一份手稿。 细胞生物学杂志，并描述了我们的证据，微管解聚酶KLP- 7/MCAK通过动粒促进卵母细胞纺锤体极的聚结。这些 结果，连同我们鉴定的额外的温度敏感突变体与卵母细胞 主轴缺陷和文献的全面调查，涵盖了最广泛使用的模型 系统，为我们现在提出的具体目标提供基础，以推进我们的 了解无中心体卵母细胞减数分裂纺锤体组装。 尽管有丝分裂纺锤体广泛依赖于中心体来组织双极纺锤体， 卵母细胞减数分裂和体细胞有丝分裂共用无核粒体途径， 组织微管。因此，研究卵母细胞纺锤体组装将提高我们的 了解导致癌症的异常有丝分裂增殖。此外，人类 卵母细胞在纺锤体装配中非常容易出错， 受精后发育异常的卵母细胞。对卵母细胞减数分裂的认识 因此，纺锤体组装与人类生育力和发育有关。

Project Summary Oocyte meiosis ends with two rounds of cell division that produce an egg with a haploid genome. While mitosis uses bipolar spindles that assemble using two centrosomes, oocytes assemble bipolar spindles in the absence of centrosomes. While the assembly and function of mitotic spindles have been studied extensively, acentrosomal oocyte spindle assembly remains poorly understood, and C. elegans provides an appealing model system for investigating the mechanisms that govern this process. Moreover, our understanding of oocyte spindle assembly remains incomplete in all widely used model systems, with live cell imaging only recently beginning to shed light on the dynamics of spindle assembly, and somewhat piecemeal genetic studies having provided a partial but incomplete catalogue of the important players. We propose a systematic and quantitative analysis of oocyte meiotic spindle assembly and spindle bipolarity, using a valuable collection of temperature-sensitive mutants we have isolated. We also will use RNA interference to identify the genes and pathways that operate during oocyte spindle assembly in C. elegans, along with live-cell fluorescent video-microscopy and genome editing to provide a more complete and mechanistic understanding of this fundamentally important process. In 2014, we published a manuscript describing our initial work on oocyte spindle pole assembly in Molecular Biology of the Cell, and in 2015 another manuscript in The Journal of Cell Biology and describing our evidence that the microtubule depolymerase KLP- 7/MCAK acts through kinetochores to promote the coalescence of oocyte spindle poles. These results, together with our identification of additional temperature-sensitive mutants with oocyte spindle defects and a thorough survey of the literature covering the most widely used model systems, provide the foundation for the specific aims we now propose to advance our understanding of acentrosomal oocyte meiotic spindle assembly. Even though mitotic spindles rely extensively on centrosomes to organize bipolar spindles, oocyte meiosis and somatic mitosis share the use of acentrosomal pathways for nucleating and organizing microtubules. Thus investigating oocyte spindle assembly will improve our understanding the abnormal mitotic proliferations that result in cancer. Moreover, human oocytes are remarkably prone to errors in spindle assembly and often produce aneuploid oocytes that upon fertilization develop abnormally. A better understanding of oocyte meiotic spindle assembly is therefore relevant to human fertility and development.