Cytokinesis separates a cell into two daughters at the end of mitosis. We show that anillin, a key component of the cytokinesis machinery, is controlled by importin binding. Active RhoA and importins coordinate to regulate neighboring domains on anillin to stabilize a conformation that is favorable for cortical recruitment.
Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Finally, we address the need for gene editing to study cytokinesis in more physiologically relevant contexts. Therefore, this Review provides a roadmap for gene editing to be used in the study of cytokinesis and other cellular processes.
Cytokinesis occurs by the ingression of an actomyosin ring that cleaves a cell into two daughters. This process is tightly controlled to avoid aneuploidy, and we previously showed that active Ran coordinates ring positioning with chromatin. Active Ran is high around chromatin, and forms an inverse gradient to cargo-bound importins. We found that the ring component anillin contains an NLS that binds to importin and is required for its function. Anillin contains a RhoAbinding domain (RBD), which we revealed autoinhibits the adjacent NLS-containing C2 domain.Here, we show that active RhoA relieves inhibition of the C2 domain. Furthermore, FRAP experiments show that the NLS regulates anillin's cortical properties, supporting feedback to the RBD. Indeed, mutations that disrupt the interface between the RBD and C2 domain disrupt anillin's localization and function. Thus, active RhoA induces a conformational change that increases accessibility to the C2 domain, which is maintained by importin-binding for recruitment to the equatorial cortex. activity vs. spindle localization and the role of microtubules remains to be clarified (Frenette et al., 2012;Lakomtsev et al.
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