Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome‐wide screens have identified a number of proteins that exhibit cell line‐specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the 4‐subunit centromere‐localized CENP‐O complex, whose precise function has been difficult to define. Our results support a model in which the CENP‐O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell‐state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease.

与其他核心生物过程类似，绝大多数细胞分裂成分对于人类细胞系的生存能力至关重要。然而，最近的全基因组筛选已经确定了许多表现出细胞系特异性必要性的蛋白质。定义这些蛋白质的行为对于我们理解复杂的生物过程至关重要。在这里，我们利用差异必要性来揭示 4 亚基着丝粒定位的 CENP-O 复合物的贡献，其精确功能难以定义。我们的结果支持一个模型，其中 CENP-O 复合物和 BUB1 以平行途径起作用，以将阈值水平的 PLK1 募集到有丝分裂动粒，确保准确的染色体分离。我们证明对任一途径的有针对性的变化使细胞对另一种成分的损失敏感，导致细胞状态依赖性要求。这种方法还突出了比较不同细胞系的表型以定义可用于疾病靶向治疗的关键功能贡献和行为的优势。