Automated analysis of cell migration and nuclear envelope rupture in confined environments.

Joshua J Elacqua,Alexandra L Mcgregor,Jan Lammerding,Ramani Ramchandran

doi:10.1371/journal.pone.0195664

Abstract

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.

Highlights

Cell migration is necessary for a number of important physiological processes including immune response, wound healing, and cancer metastasis
Cell migration is important in the context of cancer metastasis, which is responsible for the vast majority of cancerrelated deaths, including over 90% of breast cancer deaths [1]
We have developed and validated a MATLAB program for the automated and robust analysis of nuclear activity as cells migrate through microfluidic devices

Summary

Introduction

Cell migration is necessary for a number of important physiological processes including immune response, wound healing, and cancer metastasis. Out of the 50 verified constriction transit events present and analyzed in the two image sequences, 46 were identified by the program prior to manual verification/correction, resulting in a “miss” rate of 8%. To assess whether the program could detect differences in constriction transit times in cell lines other than those used for the ‘training’ and ‘test’ data, we performed experiments with A549 human lung carcinoma cells treated with siRNA against lamin A/C or a non-target control (S5 Fig). Consistent with the previous reports, the automated image analysis of our experiments found that lamin A/C-depleted cells passed faster through 1- and 2-μm wide constrictions than the non-target controls (p < 0.05) Both groups had comparable transit times (p = 0.34) when passing through 15-μm wide control channels that do not require nuclear deformation (Fig 6A).

Discussion

Findings

Materials and methods