DNA protection from mechanical stress using microfluidics: a short review

Sara A. Wickström’s group in Helsinki

In a 2020 Cell paper, Michele M. Nava and Yekaterina A. Miroshnikova, from Sara A. Wickström’s group in Helsinki, along with co-authors, demonstrate the ability of nucleus, cells and tissues to rapidly adapt to mechanical stress and avoid DNA damage. Genome integrity is preserved via two independent mechanisms. Rapid DNA protection is first ensured by transient softening of the nucleus independently from known cellular mechanosensors. Secondly, long-term stresses are further mitigated by tissue rearrangement to best bear mechanical load.

Abstract: Tissues protect their DNA under mechanical stress

A quite remarkable feature of our tissues and cells is their ability to endure mechanical stress in our daily lives. Any stretching, pulling, or compression could potentially lead to cell and tissue rupture and DNA damage, but this type of damage is rare. This raises the question: what are the mechanisms protecting our genome and cells from such external challenges? 

Mechanisms of DNA protection in response to mechanical stress has recently been deeply studied by the group of Sara Wickström from the Helsinki Institute of Life Science and Wihuri Research Institute at the University of Helsinki. Using a range of biomechanical and microfluidics reactors allowing to mimic the stretching and compression in vivo, the authors show how cells and tissues make up two biologically and temporally distinct mechanisms allowing DNA protection and preservation of genome integrity.

Introduction

All cells and tissues do not react to mechanical stress in the same way. Cancer cells, for instance, are known to show high DNA damage upon mechanical deformation while epithelial tissues form strong physical barriers against external forces.

Inside the cells, DNA is isolated and protected from the rest of the cell by a double lipid bilayer structure, the nuclear envelope. On the inner nuclear membrane, fibrous nuclear lamin proteins maintain nuclear shape and structure. Importantly, the nuclear envelope and its components act as a hub for cytoskeleton components of the cell, themselves forwarding the mechanically induced external stresses.

Inside the nucleus, DNA is dynamically condensed into euchromatin (transcriptionally active and low density) and heterochromatin, respectively low and high condensation levels of the double-stranded DNA. Importantly in the context of this study, dense DNA regions of heterochromatin are associated to the lamina.

While microenvironment mechanical stress has been reported in literature, mechanisms explaining how chromatin responds and how cells dissipate such forces is not well understood. Using microfluidics to mimic this stressed physiological cellular status, Nava, Miroshnikova and co-authors demonstrate how cells react both in short-term and long-term cales.

Methods: Microfluidic compression and cells/tissue stretching to mimic mechanical stress-induced DNA damage

It was exciting to realize that we could alter the mechanical properties of chromatin simply by exerting mechanical forces on the stem cells. Even more striking was that if we experimentally prevented these changes in chromatin mechanics, the stem cells now acquired DNA damage, indicating that we had discovered an important protective mechanism.

Yekaterina Miroshnikova, author.

Two types of mechanical forces have been applied to cells and tissue in order to report the responses to mechanical forces both at the cellular level (short term response) and at the tissue level (long-term adaptation)

Nuclear compression

To perform this experimentation, Nava, Miroshnikova and co-authors grew skin epidermis or mesenchymal stem cells inside a microfluidic cell confiner system. This system has first been published by Le Berre et al in 2012 [1] and is now commercially available (4Dcell, an Elveflow spin-off). (Learn more on microfluidics for cell confinement). It allows to maintain cells in a microfluidic channel and to apply controlled external forces using a pressure generator. The schematics of this confiner as highlighted by 4Dcell on their website is shown in figure 2.

Mesenchymal or skin epidermis stem cells (CellInTec HPEKp // ATCC CRL-1629 // ATCC CCL-121) were seeded in the confiner and various compression levels were applied to the cells thanks to a dual pressure controller. This specific -900mbars / 1000mbars configuration of the Elveflow OB1 allowed to apply negative pressure and trigger compression.

Similar to the moderate 20% to high 40% uniaxial stretch, 30% compression with the confiner resulted in nuclear deformation in the form of nuclear flattening. Both deformations triggered the calcium-dependent H3K9me3 heterochromatin response.  Authors further identified H3K9me3 loss as the driver of nuclear softening and thus required for DNA damage protection under mechanical stress. 

Cells and tissue stretching

Stretching assays have been performed following Faust et al. 2011 [2] and Noethel et al. 2018 paper [3]. This custom-built uniaxial cell stretcher is based on 4 cm^2 PDMS elastomers where cells and tissues can be seeded or attached, respectively. Elastomers are then clamped in dedicated chambers and the entire system is submitted to uniaxial stretch.

Key findings

DNA damage is prevented in stem cells through short term nuclear softening and long term monolayer reorientation

Nuclear deformation (elongation in the case of stretch, flattening in the case of compression)  allowed  global softening of the nuclei by reducing the amount of peripheral heterochromatin.

Transient loss of heterochromatin did not elicit large transcriptional effects as changes occurred mostly in non-coding regions of the genome. In other words, nuclei could smartly respond to mechanical stresses by modifying their physical properties in order to prevent DNA damage while keeping homeostatic transcriptional activity and thus maintain basal cell state. 

Altogether, these findings uncover that nuclei respond rapidly to mechanical stress to prevent DNA damage by reducing lamina-associated heterochromatin at the nuclear periphery close to the nuclear envelope. 

This response relies on nuclear deformation driven release of calcium from the endoplasmic reticulum, which is continuous with the outer nuclear membrane, which is a part of the nuclear envelope.

On longer timescales tissues respond to mechanical stress by re-orienting their cytoskeleton to dissipate forces from cell to cell while at the same time restoring baseline heterochromatin.

Nava, Miroshnikova and colleagues showed that F-actin re-oriented perpendicularly to stretching stress forces to mitigate the mechanical stress propagation from cell to cell. This supracellular slower response relies on cell-cell contacts to prevent nuclear tension. Authors confirmed this slower response at the tissue level on mice skin explants.

You can also read this related content on mechanical cell compression.

References