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Using super-resolution microscopy to diagnose disease in a dish

Scientists at the CSIR, in collaboration with colleagues at Institut Pasteur, University College London, Necker Hospital and the Université Paris-Descartes, have identified the first visual proof of complex intra-cellular structures acting as pre-formed regulators of the immune response. The work demonstrates how super-resolution microscopy can provide novel insights into the single-molecule mechanism of genetic disorders, as well as crucial cellular mechanisms.

Until recently, there were no simple approaches to prove the existence of complex intra-cellular structures and therefore, little means of understanding the basic molecular pathways as a stepping stone to unravelling mechanisms of disease. This is largely due to the very basic physical properties of light itself, which cannot be resolved below 200 nm. Since many structures in the cell may be smaller than this, they cannot be visualised with conventional microscopy.

Through extensive research over the past decade, scientists have been able to overcome this problem. The CSIR custom-designed the first – and to date only – super-resolved fluorescence microscope in the country. Using this technology, the researchers have been able to provide the first visual proof of these higher-order structures.

One of the critical pathways in human cells is called the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) signalling cascade. Every cell in the body must have an innate ability to respond to an outside stimulus or attack. Whatever form that signal may take, essentially healthy cells will respond by moving the protein NFkB from the cell body (cytoplasm) to the DNA storage area (the nucleus). There it will activate genes that lead to the cell’s protection. Although extremely simplified, in this way, mankind stays healthy.

A recently proposed theory suggests that the intermediate protein, which links the signal from the outside of the cell to the successful movement of NFkB to the nucleus, might be able to do this by forming complex structures that would be able to act as a finely tuned regulator – waiting for a minimum signal threshold to arise before switching on a green light of activation. The research team used super-resolution microscopy to reveal the complex structure of this intermediate protein in non-stimulated cells and have identified how this structure is held together in cellulo.

 

Partners

Institut Pasteur and the Université Paris-Descartes

Contact Person

Dr Janine Scholefield