Meinig School of Biomedical Engineering, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, United States of America.
PLoS One. 2018 Apr 12;13(4):e0195664. doi: 10.1371/journal.pone.0195664. eCollection 2018.
Recent in vitro and in vivo studies have highlighted the importance of the cell nucleus in governing migration through confined environments. Microfluidic devices that mimic the narrow interstitial spaces of tissues have emerged as important tools to study cellular dynamics during confined migration, including the consequences of nuclear deformation and nuclear envelope rupture. However, while image acquisition can be automated on motorized microscopes, the analysis of the corresponding time-lapse sequences for nuclear transit through the pores and events such as nuclear envelope rupture currently requires manual analysis. In addition to being highly time-consuming, such manual analysis is susceptible to person-to-person variability. Studies that compare large numbers of cell types and conditions therefore require automated image analysis to achieve sufficiently high throughput. Here, we present an automated image analysis program to register microfluidic constrictions and perform image segmentation to detect individual cell nuclei. The MATLAB program tracks nuclear migration over time and records constriction-transit events, transit times, transit success rates, and nuclear envelope rupture. Such automation reduces the time required to analyze migration experiments from weeks to hours, and removes the variability that arises from different human analysts. Comparison with manual analysis confirmed that both constriction transit and nuclear envelope rupture were detected correctly and reliably, and the automated analysis results closely matched a manual analysis gold standard. Applying the program to specific biological examples, we demonstrate its ability to detect differences in nuclear transit time between cells with different levels of the nuclear envelope proteins lamin A/C, which govern nuclear deformability, and to detect an increase in nuclear envelope rupture duration in cells in which CHMP7, a protein involved in nuclear envelope repair, had been depleted. The program thus presents a versatile tool for the study of confined migration and its effect on the cell nucleus.
最近的体外和体内研究强调了细胞核在控制通过受限环境迁移中的重要性。模拟组织狭窄细胞间隙的微流控装置已成为研究受限迁移过程中细胞动力学的重要工具，包括核变形和核膜破裂的后果。然而，虽然在电动显微镜上可以自动进行图像采集，但对于核通过孔的相应延时序列的分析以及核膜破裂等事件目前仍需要手动分析。除了高度耗时之外，这种手动分析还容易受到人与人之间的差异影响。因此，对于比较大量细胞类型和条件的研究，需要自动化的图像分析来实现足够高的通量。在这里，我们提出了一种自动化的图像分析程序，用于注册微流道的限制并进行图像分割以检测单个细胞核。该 MATLAB 程序可跟踪核的迁移随时间的变化，并记录限制过渡事件、过渡时间、过渡成功率和核膜破裂。这种自动化减少了分析迁移实验所需的时间，从数周减少到数小时，并消除了不同人类分析者之间出现的差异。与手动分析的比较证实，限制过渡和核膜破裂都被正确和可靠地检测到，并且自动化分析结果与手动分析黄金标准非常匹配。将该程序应用于特定的生物学示例，我们证明了它能够检测具有不同核膜蛋白 lamin A/C 水平的细胞之间的核迁移时间差异，lamin A/C 控制核变形性，并且能够检测到核膜修复蛋白 CHMP7 耗尽的细胞中核膜破裂持续时间的增加。因此，该程序为研究受限迁移及其对细胞核的影响提供了一种通用的工具。
