Defence mechanism discovered in cells that may be preventing effective gene therapy
19 Jan 2023
- Jake Hepburn



Through fluorescence and phosphorescence lifetime imaging, scientists have recently discovered a peculiar defence mechanism in cells that may be preventing effective gene therapy.

Scientist using a pipette in a fume hood to fill vials with liquid.

This defence mechanism is the ability of the cell to begin to repair itself when it is exposed to certain conditions, e.g., when the double DNA-strand is damaged, proteins aggregate at the damage to initiate its own repair. The aggregation and recruitment of several proteins may be causing the environment around the damaged DNA to become extra viscous. This could explain why gene therapies have been unsuccessful in some cases,

This discovery came after Ellen Clancy, a placement student completing an MSci in Biological Science with the University of Birmingham, joined the Octopus Group​ at the Science and Technology Facilities Council's (STFC) Central Laser Facility (CLF) as a professional placement student, to study the dimerization of the Ku protein and its role in DNA damage and repair.

Ellen Clancy, completing an MSci in Biological Science with the University of Birmingham, said:

“This is a fantastic result that proves the exceptional potential of fluorescent proteins and chemical probes to monitor intracellular viscosity. This achievement provides a deeper understanding of the cellular processes and changes following DNA damage and repair which may influence the use of future therapy and drug treatment. I look forward to the impact this will have. I am proud to have been a member of the Octopus Group working on this project using the amazing microscope facilities at STFC CLF during my placement year."

Understanding cell behaviour through fluorescence and phosphorescence lifetime imaging

We demonstrate here the ability to determine cell viscosity using fluorescence and phosphorescence lifetime techniques using standard green fluorescent protein (GFP) technology and platinum chemical probes that are tolerated by cells.

Fluorescence Lifetime Imaging microscopy (FLIM) is a method used in physical and life science to study proteins, organelles, cells, or tissues. Samples emit lower energy light than those absorbed in a process called fluorescence or phosphorescence. We can precisely measure the average time that the molecule spends as it enters an excited state before emitting photons (fluorescent light) before returning to its ground state, usually lasting nanoseconds – a Fluorescence Lifetime.​

The image produced by FLIM represents the fluorescence lifetime of the molecule's environment providing a unique quantitative contrast to the image.

Phosphorescence Lifetime Imaging microscopy (PLIM) is not too dissimilar from FLIM, but only that it images the phosphorescence as opposed to the fluorescence, and that the lifetime is measured up to milliseconds as the process happen via a forbidden route.

The cell behaviour discovered

The findings show that cells respond to damage such as two breaks (very close together) in a DNA strand or other environmental changes such as photo-toxicity by altering and significantly increasing their cytoplasmic and nuclear viscosity. Interestingly, findings have also shown that undamaged cells next to a damaged one show increased viscosity.

The rapid phosphorylation of H2A.X (a specific histone protein) – known as g -H2AX foci – is used as a clear indication of a double strand break in a DNA double helix, for example, from radiation. It also acts as an early signal in the DNA-damage response that is required to recruit a multitude of repair proteins. Researchers speculate that these foci of non-membrane aggregates or biomolecular condensates may contribute to the viscosity changes that was observe.

However, there are limits to the cells ability to repair itself. If the damage to the cell is too severe, the cell will initiate a process called apoptosis-programmed cell death. Once initiated, the cell will die. On many occasions, this process is missing in cancer cells, allowing them to grow excessively. It is thought that the increase in cellular viscosity in damaged cells may be a mechanism to minimise the damage spread and to stop it initiating its own death.

Professor Stanley W. Botchway, UKRI-STFC Fellow and Research Lead at CLF Octopus, says:

“Our ability to visualise, interrogate and control complex biological systems rely on technologies that provide non-invasive monitoring of bio-molecular processes in living cells and in real time. This work demonstrates the power of advanced time resolved imaging on the nano-to-micro-second time scales provided by FLIM and PLIM to show how the fluidity of cell nucleus changes in adverse conditions such as DNA damage. Our results have the potential to explain how cells protect their nuclei and how drugs may or may not work in the complex cell nucleus. The unique National Facilities of STFC such as the CLF Octopus makes such a research possible. "

Future research on our ne​w understanding

The new findings may explain the reason why gene therapy doesn't work so well- the difficulties in getting introduced DNA to function, since the cell might be protecting its nucleus by increasing the cellular nuclear viscosity in attempts to defend its nucleus. It may also explain why some drugs may be so slow to act.

Despite not currently understanding why this happens, the data opens new perspectives and future studies on the processes that may be affected by the changes in cellular nuclear viscosity, in circumstances such as protein transcription, gene therapy, and drug action in the cytoplasm and nucleus.

Read the publication to find out more. 

Contact: Hepburn, Jake (STFC,RAL,COMMS)