A CRISPR Tagging-Based Screen Reveals Localized Players in Wnt-Directed Asymmetric Cell Division
A CRISPR Tagging-Based Screen Reveals Localized Players in Wnt-Directed Asymmetric Cell Division
复制标题
DOI：
10.1534/genetics.117.300487
复制
发表时间：
2018-03-01
期刊：
影响因子：
3.3
通讯作者：
Goldstein, Bob
中科院分区：
文献类型：
--
作者：
Heppert, Jennifer K.;Pani, Ariel M.;Goldstein, Bob
定向细胞分裂对于建立和维持细胞命运和组织结构至关重要。不同的细胞外和细胞内线索已被证明提供空间信息的有丝分裂纺锤体定位，然而，细胞外信号与细胞直接有丝分裂纺锤体定位的分子机制在很大程度上是未知的。在动物细胞中，定向细胞分裂通常是通过将产生力的运动蛋白复合物定位到离散的皮质结构域来实现的。破坏这些力产生复合物或蛋白质，全局影响微管稳定性的有丝分裂定位的缺陷的结果，无论这些蛋白质是否作为空间线索主轴方向的功能。这对传统的基因解剖过程提出了挑战。因此，作为一种替代策略，以确定关键蛋白质的下游细胞间信号传导的行为，我们筛选了定位的许多候选蛋白质直接插入到内源性基因位点的荧光标记，而不过度表达的蛋白质。我们标记了秀丽隐杆线虫中的23个候选蛋白质，并检查了每个蛋白质在四细胞阶段胚胎中的特征良好的定向细胞分裂中的定位。我们使用细胞操作和遗传实验来确定哪些细胞具有关键的定位蛋白，以及哪些信号在体内指导这些定位。我们发现，Dishevelled和腺瘤性结肠息肉病同源物在此定向细胞分裂过程中响应于Wnt信号而极化，但通常与有丝分裂纺锤体定位相关的两种蛋白质，NuMA和动力蛋白的同源物，未被可检测地极化。这些结果表明，有丝分裂纺锤体定位在这个系统中的一个意想不到的机制，他们指出关键的感兴趣的蛋白质，他们强调了基于分析内源性标记的蛋白质的本地化的筛选方法的实用性。
Oriented cell divisions are critical to establish and maintain cell fates and tissue organization. Diverse extracellular and intracellular cues have been shown to provide spatial information for mitotic spindle positioning; however, the molecular mechanisms by which extracellular signals communicate with cells to direct mitotic spindle positioning are largely unknown. In animal cells, oriented cell divisions are often achieved by the localization of force-generating motor protein complexes to discrete cortical domains. Disrupting either these force-generating complexes or proteins that globally affect microtubule stability results in defects in mitotic positioning, irrespective of whether these proteins function as spatial cues for spindle orientation. This poses a challenge to traditional genetic dissection of this process. Therefore, as an alternative strategy to identify key proteins that act downstream of intercellular signaling, we screened the localization of many candidate proteins by inserting fluorescent tags directly into endogenous gene loci, without overexpressing the proteins. We tagged 23 candidate proteins in Caenorhabditis elegans and examined each protein's localization in a well-characterized, oriented cell division in the four-cell-stage embryo. We used cell manipulations and genetic experiments to determine which cells harbor key localized proteins and which signals direct these localizations in vivo. We found that Dishevelled and adenomatous polyposis coli homologs are polarized during this oriented cell division in response to a Wnt signal, but two proteins typically associated with mitotic spindle positioning, homologs of NuMA and Dynein, were not detectably polarized. These results suggest an unexpected mechanism for mitotic spindle positioning in this system, they pinpoint key proteins of interest, and they highlight the utility of a screening approach based on analyzing the localization of endogenously tagged proteins.