Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells

Abstract

Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This process occurs via the ingression of an actomyosin ring that assembles in anaphase and pulls in the overlying plasma membrane as it constricts. Mechanistic studies have uncovered different pathways that regulate the assembly and position of the ring in mammalian cells, but the majority of these studies were done using HeLa cells with overexpressed transgenes, and the relative requirement for these mechanisms among the majority of cell types is not known. Here, we used CRISPR/Cas9 gene editing to endogenously tag cytokinesis proteins, anillin, Ect2 and RhoA, as well as other cellular components, with fluorescent proteins. These tools enabled the visualization of cytokinesis by live imaging to quantitatively study these proteins at endogenous levels. As a proof-of-concept, tagging anillin in multiple mammalian cell lines revealed cytokinetic diversity, which will be useful for studies of how mechanisms controlling cytokinesis vary among cell types. We also successfully tagged multiple cellular components in the same cell line, demonstrating the versatility of these tagging tools.