Cytoskeletal Function in C. elegans Embryos

项目摘要 25年来，我们实验室一直使用带有荧光标记的活细胞成像。 蛋白质，以及分离温度敏感(TS)和 胚胎致死线虫突变体，以研究细胞骨架功能 胚胎细胞分裂。最近，我们(I)采用了更高的吞吐量 利用Illumina DNA测序技术实现位置克隆的方法 扩大我们的努力，作为一个群体来鉴定线虫基本基因的TS突变 资源，以及(Ii)采用CRISPR/Cas9基因组编辑技术来增强我们的 遗传学和活细胞成像方法。过去五年，除了 开发更高吞吐量的位置克隆方法，我们主要关注两个 极其重要的细胞生物学过程：(一)细胞分裂轴的取向 在开发过程中建立多细胞体系结构，以及(Ii) 卵母细胞减数分裂过程中微管成核并组织成双极结构 纺锤体组装，在没有中心体微管的情况下发生 调节有丝分裂纺锤体组装的组织中心。我们的研究计划结束了 未来五年将专注于扩大我们对Essential中TS突变的识别 秀丽线虫的基因，并使它们普遍可供研究界使用， 扩展我们对以前未知但广泛保守的微管的分析- 定位细胞分裂的独立和皮质肌动球蛋白依赖机制 在动物发育过程中的轴，并提高我们对无着丝体的理解 卵母细胞减数分裂纺锤体的组装和功能。

Project Summary For 25 years now, our laboratory has used live cell imaging with fluorescently marked proteins, and the forward genetics approach of isolating temperature-sensitive (TS) and embryonic-lethal C. elegans mutants, to investigate cytoskeletal function during embryonic cell divisions. More recently, we have (i) incorporated higher throughput positional cloning methods made possible by Illumina DNA sequencing technology to expand our effort to identify TS mutations in essential C. elegans genes as a community resource, and (ii) employed CRISPR/Cas9 genome editing technology to augment our genetics and live cell imaging approaches. Over the past five years, in addition to developing higher throughput positional cloning approaches, we have focused on two fundamentally important cell biological processes: (i) the orientation of cell division axes during development to establish multicellular architectures, and (ii) the mechanisms that nucleate and organize microtubules into a bipolar structure during oocyte meiotic spindle assembly, which occurs in the absence of the centrosomal microtubule organizing centers that mediate mitotic spindle assembly. Our research program over the next five years will focus on expanding our identification of TS mutations in essential C. elegans genes and making them generally available to the research community, extending our analysis of a previously unknown but widely conserved microtubule- independent and cortical actomyosin-dependent mechanism for orienting cell division axes during animal development, and improving our understanding of acentrosomal oocyte meiotic spindle assembly and function.