Collection of Vertebrate Cell Cultures, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Prospekt, St. Petersburg, Russia, 194064.
Laboratory of Intracellular Signaling and Transport, Smorodintsev Research Institute of Influenza, 15/17 Prof. Popova Street, St. Petersburg, Russia, 197376.
Mol Biol Rep. 2020 May;47(5):3867-3883. doi: 10.1007/s11033-020-05476-6. Epub 2020 May 5.
Here, we document changes in cell motility and organization of the contractile apparatus of human umbilical cord Wharton's jelly mesenchymal stem cells (MSCWJ-1) in the process of replicative senescence. Colocalization dynamics of F-actin and actin-binding proteins (myosin-9, α-actinin-4, RhoA) were examined in the MSCWJ-1 cell line. The results show that nuclear-cytoplasmic redistribution of RhoA occurs during replicative senescence, with maximal RhoA/nucleus colocalization evident at passage 15. At that time point, decreases in colocalization, namely myosin-9/F-actin and α-actinin-4/F-actin, were seen and myosin-9 was found in cytosolic extracts in the assembly-incompetent form. Using an automated intravital confocal cytometry system and quantitative analysis of MSCWJ-1 movements, we found that changes in cytoskeletal organization correlate with cell motility characteristics over a time period from passages 9 to 38. The factors examined (cytoskeleton structure, cell motility) indicate that the process by which cells transition to replicative senescence is best represented as three stages. The first stage lasts from cell culture isolation to passage 15 and is characterized by: accumulation of actin-binding proteins in assembly-incompetent forms; nuclear RhoA accumulation; and an increase in movement tortuosity. The second stage extends from passages 15 to 28 and is characterized by: an increase in the structural integrity of the actin cytoskeleton; exit of RhoA and alpha-actinin-4 from the nucleus; and a decrease in path tortuosity. The third stage extends from passage 28 to 38 and is marked by: a plateau in actin cytoskeleton structural integrity; significant decreases in nuclear RhoA levels; and decreases in cell speed.
在这里，我们记录了人脐带华通氏胶间充质干细胞（MSCWJ-1）在复制性衰老过程中细胞迁移和收缩装置组织的变化。我们检测了 MSCWJ-1 细胞系中 F-肌动蛋白和肌动蛋白结合蛋白（肌球蛋白-9、α-辅肌动蛋白-4、RhoA）的共定位动力学。结果表明，RhoA 在复制性衰老过程中发生核质重分布，在第 15 代时可见到 RhoA/核最大共定位。此时，观察到共定位减少，即肌球蛋白-9/F-肌动蛋白和 α-辅肌动蛋白-4/F-肌动蛋白减少，并且肌球蛋白-9 以组装无能形式存在于细胞溶质提取物中。使用自动化活细胞共聚焦细胞仪系统和对 MSCWJ-1 运动的定量分析，我们发现细胞骨架组织的变化与从第 9 代到第 38 代的细胞迁移特征相关。检查的因素（细胞骨架结构、细胞迁移）表明，细胞向复制性衰老转变的过程最好表示为三个阶段。第一阶段从细胞培养分离到第 15 代，其特征是：组装无能形式的肌动蛋白结合蛋白积累；核 RhoA 积累；运动曲折度增加。第二阶段从第 15 代延伸到第 28 代，其特征是：肌动蛋白细胞骨架的结构完整性增加；RhoA 和 α-辅肌动蛋白-4 从核中逸出；路径曲折度降低。第三阶段从第 28 代延伸到第 38 代，其特征是：肌动蛋白细胞骨架结构完整性的平台期；核 RhoA 水平显著降低；以及细胞速度降低。
