ABSTRACT

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.

Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H⁺-translocating ATPase; WAPL: WAPL cohesion release factor.

 抽象的

溶酶体作为主要的降解细胞器，是包括巨自噬在内的不同聚合途径的终点。迄今为止，溶酶体的降解功能主要在间期细胞中进行研究，但其在有丝分裂过程中的作用仍存在争议。有丝分裂决定遗传物质在世代之间的忠实传递，有丝分裂的干扰会导致染色体不稳定，这是癌症的一个标志。迄今为止，正确的有丝分裂进程依赖于有丝分裂因子的精心策划的降解，这主要归因于泛素触发的蛋白酶体依赖性降解。在这里，我们发现有丝分裂转变也依赖于溶酶体依赖性降解，因为溶酶体的损伤会增加有丝分裂时间并导致有丝分裂错误，从而促进染色体不稳定。此外，我们还确定了有丝分裂细胞中的几个假定的溶酶体靶点。其中，WAPL（一种粘连蛋白调节蛋白）作为一种新型 SQSTM1 相互作用蛋白出现，用于靶向溶酶体降解。最后，我们描述了一种非典型核表型，即环形核，作为基因毒性筛查的新型生物标志物。我们的结果表明，溶酶体依赖性降解是防止染色体不稳定的重要事件。

缩写： 3D：三维； APC/C：后期促进复合物； ARL8B：ADP 核糖基化因子，如 GTPase 8B； ATG：自噬相关； BORC：BLOC-one相关复合物； CDK：细胞周期蛋白依赖性激酶； CENPE：着丝粒蛋白E； CIN：染色体不稳定； ConcA：刀那霉素A； CQ：氯喹； DAPI：4,6-二脒基-2-戊亚油基； FTI：法呢基转移酶抑制剂； GFP：绿色荧光蛋白； H2B：组蛋白2B； KIF：驱动蛋白家族成员； LAMP2：溶酶体相关膜蛋白2； MAP1LC3/LC3：微管相关蛋白1轻链3； MEF：小鼠胚胎成纤维细胞； MTOR：雷帕霉素激酶的机制靶点； PDS5B：PDS5粘连蛋白相关因子B； SAC：主轴装配检查点； PLEKHM2：pleckstrin同源性和含有M2的RUN结构域； SQSTM1: 隔离体 1; TEM：透射电子显微镜； ULK1：unc-51 样自噬激活激酶 1； UPS：泛素-蛋白酶体系统； v-ATP酶：液泡型H ⁺转位ATP酶； WAPL：WAPL 内聚释放因子。