A CRISPR Tagging-Based Screen Reveals Localized Players in Wnt-Directed Asymmetric Cell Division

Genetics: A Periodical Record of Investigations Bearing on Heredity and Variation

Daniel J. Dickinson^2¹  Ariel M. Pani^2³  Bob Goldstein^4³  Jennifer K. Heppert^1⁴  Allyson M. Roberts^3⁶ 

[1] Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, North Carolina 27599^3;Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, North Carolina 27599^4;Department of Biology, University of North Carolina at Chapel Hill, North Carolina 27599;Department of Biology, University of North Carolina at Chapel Hill, North Carolina 27599^1;Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599;Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599^2

关键词: asymmetric cell division;    CRISPR;    development;    dynein;    Wnt signaling;   

DOI  :  10.1534/genetics.117.300487

学科分类：医学（综合）

来源: Genetics Society of America

【 摘 要 】

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.

【 授权许可】

CC BY