Prof Lynne Regan, Dr Mathew Horrocks and their teams have developed a novel super-resolution technique for live cell imaging using reversible peptide-protein interactions. Image The technique offers a simple way to study proteins in living cells and has many advantages over traditional labelling techniques in which fluorescent labels are directly fused to a protein of interest and become irreversibly photobleached during an imaging experiment. Traditional microscopy is limited by the diffraction limit of light. To study cellular processes at higher resolution requires sparse labelling of fluorescent molecules, so that individual fluorescent molecules are separated in space and/or time. Some techniques, like photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), use photoswitchable fluorophores to achieve sparse labelling – but these bleach over time. Another technique called Point accumulation for imaging in nanoscale topography (PAINT) achieves sparse labelling using the transient binding of fluorescent molecules to a molecule of interest. DNA-PAINT accomplishes this reversible binding by attaching complementary DNA strands to the fluorophore and indirectly to the protein of interest, for example via a DNA-tagged nanobody. However, DNA-PAINT cannot be used inside living cells, because cells must be fixed and permeabilized to enable the DNA-tagged nanobody and the DNA-tagged fluorophore to enter the cell. Regan and Horrocks combined their expertise in protein-protein interactions and super-resolution microscopy to overcome this limitation, working with their group members Curran Oi, Zoe Gidden, Louise Holyoake, and Owen Kantelberg to develop LIVE-PAINT. LIVE-PAINT involves reversible binding of a protein (fused to a fluorescent protein) to a small peptide (fused to the protein of interest). The fluorescent protein complex can move around the cell until it is transiently bound to its complementary peptide, which results in a transient fluorescence localization. Because the cell is imaged continuously, the localizations that are acquired at different times can be summed to create the super-resolution image. LIVE-PAINT does not rely on sequential photoswitching of fluorophores or need special instrumentation. Photobleaching is far less of a problem, as any bleached fluorescent proteins diffuse away and can be replaced by unbleached proteins: the sample can then be imaged for longer. The protein of interest behaves more naturally because it is not directly fused to a bulky fluorescent protein. Regan, Horrocks and colleagues developed LIVE-PAINT by imaging the proteins Cdc12, actin, and cofilin in live yeast with a resolution as low as 20 nm and also use their labelling technique to observe the dynamic movement of cofilin. They are keen to collaborate with any researchers who think LIVE-PAINT may be a useful approach in their own biological system. The team are currently setting up LIVE-PAINT in mammalian cells, working with the UK Centre for Mammalian Synthetic Biology at the University of Edinburgh, who are experts in genome manipulation. You can read a highlight of the paper here https://prelights.biologists.com/highlights/live-paint-super-resolution-... Pre-print paper here https://www.biorxiv.org/content/10.1101/2020.02.03.932228v2.full This article was published on 2024-06-17
