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Structural and functional analysis of cell adhesion and nuclear envelope nano-topography in cell death.

ABSTRACT: The cell death mechanisms of necrosis and apoptosis generate biochemical and morphological changes in different manners. However, the changes that occur in cell adhesion and nuclear envelope (NE) topography, during necrosis and apoptosis, are not yet fully understood. Here, we show the different alterations in cell adhesion function, as well as the topographical changes occurring to the NE, during the necrotic and apoptotic cell death process, using the xCELLigence system and atomic force microscopy (AFM). Studies using xCELLigence technology and AFM have shown that necrotic cell death induced the expansion of the cell adhesion area, but did not affect the speed of cell adhesion. Necrotic nuclei showed a round shape and presence of nuclear pore complexes (NPCs). Moreover, we found that the process of necrosis in combination with apoptosis (termed nepoptosis here) resulted in the reduction of the cell adhesion area and cell adhesion speed through the activation of caspases. Our findings showed, for the first time, a successful characterization of NE topography and cell adhesion during necrosis and apoptosis, which may be of importance for the understanding of cell death and might aid the design of future drug delivery methods for anti-cancer therapies.

SUBMITTER: Kwon HK

PROVIDER: S-EPMC4614995 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Structural and functional analysis of cell adhesion and nuclear envelope nano-topography in cell death.

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