Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, UK.
Goodman Cancer Research Centre, Department of Human Genetics, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2021;2329:291-309. doi: 10.1007/978-1-0716-1538-6_21.
The cell and cilia cycles are inextricably linked through the dual functions of the centrioles at both the basal body of cilia and at mitotic centrosomes. How cilia assembly and disassembly, either through slow resorption or rapid deciliation, are coordinated with cell cycle progression remains unclear in many cell types and developmental paradigms. Moreover, little is known about how additional cilia parameters including changes in ciliary length or frequency of distal tip shedding change with cell cycle stage. In order to explore these questions, we have developed the Arl13bCerulean-Fucci2a tricistronic cilia and cell cycle biosensor (Ford et al., Dev Cell 47:509-523.e7, 2018). This reporter allowed us to document the heterogeneity in ciliary behaviors during the cell cycle at a population level. Without the need for external stimuli, it revealed that in several cell types and in the developing embryo cilia persist beyond the G1/S checkpoint. Here, we describe the generation of stable cell lines expressing Arl13bCerulean-Fucci2a and open-source software to aid morphometric profiling of the primary cilium with cell cycle phases, including changes in cilium length. This resource will allow the investigation of multiple morphometric questions relating to cilia and cell cycle biology.
细胞周期和纤毛周期通过中心粒在纤毛的基底体和有丝分裂中心体的双重功能紧密相连。在许多细胞类型和发育范例中，纤毛的组装和拆卸（通过缓慢吸收或快速去纤毛）如何与细胞周期进程相协调仍然不清楚。此外，对于包括纤毛长度变化或远端尖端脱落频率变化在内的其他纤毛参数如何随细胞周期阶段而变化，人们知之甚少。为了探索这些问题，我们开发了 Arl13bCerulean-Fucci2a 三顺反子纤毛和细胞周期生物传感器（Ford 等人，Dev Cell 47:509-523.e7，2018）。该报告器使我们能够在群体水平上记录细胞周期中纤毛行为的异质性。无需外部刺激，它揭示了在几种细胞类型和发育中的胚胎中，纤毛在 G1/S 检查点之后仍然存在。在这里，我们描述了稳定表达 Arl13bCerulean-Fucci2a 的细胞系的生成，并提供了开源软件来辅助细胞周期阶段的初级纤毛的形态计量分析，包括纤毛长度的变化。该资源将允许研究与纤毛和细胞周期生物学相关的多个形态计量问题。
