Department of Biology, University of North Carolina at Chapel Hill, North Carolina 27599.
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599.
Genetics. 2018 Mar;208(3):1147-1164. doi: 10.1534/genetics.117.300487. Epub 2018 Jan 18.
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 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 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.
定向细胞分裂对于建立和维持细胞命运和组织组织至关重要。已经表明，各种细胞外和细胞内线索为有丝分裂纺锤体定位提供了空间信息；然而，细胞外信号与细胞通信以指导有丝分裂纺锤体定位的分子机制在很大程度上尚不清楚。在动物细胞中，定向细胞分裂通常通过将产生力的马达蛋白复合物定位到离散的皮质域来实现。破坏这些产生力的复合物或全局影响微管稳定性的蛋白质都会导致有丝分裂定位缺陷，无论这些蛋白质是否作为纺锤体定向的空间线索发挥作用。这对这个过程的传统遗传分析提出了挑战。因此，作为鉴定作用于细胞间信号下游的关键蛋白的替代策略，我们通过将荧光标签直接插入内源性基因座，而不过表达蛋白，筛选了许多候选蛋白的定位。我们在 中标记了 23 个候选蛋白，并在具有特征的四细胞期胚胎中定向细胞分裂中检查了每个蛋白的定位。我们使用细胞操作和遗传实验来确定哪些细胞含有关键的定位蛋白，以及哪些信号指导这些定位。我们发现，Dishevelled 和腺瘤性结肠息肉病同源物在这个定向细胞分裂中是极化的，这是对 Wnt 信号的反应，但两个通常与有丝分裂纺锤体定位相关的蛋白，NuMA 和 Dynein 的同源物，没有明显的极化。这些结果表明该系统中纺锤体定位的一种意外机制，它们指出了感兴趣的关键蛋白，并突出了基于分析内源性标记蛋白定位的筛选方法的实用性。
