Cytoskeletal Function in C. elegans Embryos

项目摘要 25年来，我们的实验室一直使用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C.线虫突变体，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 通过Illumina DNA测序技术实现的定位克隆方法， 扩大我们的努力，以确定TS突变的必要C。线虫基因作为一个群落 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。在过去的五年里，除了 为了开发更高通量的定位克隆方法，我们重点研究了两种 基本上重要的细胞生物学过程：（i）细胞分裂轴的方向 在发育过程中建立多细胞结构，和（ii）机制， 在卵母细胞减数分裂过程中使微管成核并组织成双极结构 纺锤体组装，这发生在中心体微管的情况下 介导有丝分裂纺锤体组装的组织中心。我们的研究计划结束了 接下来的五年将集中在扩大我们对TS突变的识别， C.线虫基因，并使它们普遍提供给研究界， 扩展了我们对以前未知但广泛保守的微管的分析， 独立和皮质肌动球蛋白依赖性细胞分裂定向机制 轴在动物发育过程中，并提高我们的理解acentrosomal 卵母细胞减数分裂纺锤体组装和功能。

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.