核仁是大型核子区室，核糖体组装等重要过程在此发生。技术障碍仍然限制了我们对核仁蛋白在细胞稳态和癌症发病机制中的生物学功能的理解。由于大多数核仁蛋白都是必需的，因此无法通过常规方法消除它们。此外，许多核仁蛋白的生物活性与其生理浓度有关。因此，人工过度表达可能无法完全概括其内源功能。基于蛋白水解的方法，例如生长素诱导降解子 (AID) 系统与 CRISPR/Cas9 敲入基因编辑相结合，有可能克服这些限制，为内源核仁蛋白的生物活性提供前所未有的表征。我们将该系统应用于内源性核仁素（NCL）（最丰富的核仁蛋白之一），并表征了其急性消耗对三阴性乳腺癌（TNBC）细胞行为的影响。消除内源性 NCL 会减少增殖并导致胞质分裂缺陷，从而产生双核四倍体细胞。对患者数据的生物信息分析以及使用我们的实验性 NCL 耗尽模型进行的定量蛋白质组学分析表明，NCL 水平与参与染色体分离的蛋白质丰度相关。结合其对姐妹染色单体动力学的影响，NCL 的废除增强了有丝分裂调节剂（例如后期促进复合物）的化学抑制剂的抗增殖作用。总之，使用 AID 系统结合 CRISPR/Cas9 进行内源基因编辑，我们的研究结果表明 NCL 在支持 TNBC 模型中细胞分裂的完成中具有新的作用，并且其废除可以增强有丝分裂的治疗活性。进展抑制剂。

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.