Following single proteins at work in living cells

June 23, 2017

Scientists at the Max Planck Institute for Terrestrial Microbiology have developed a novel approach for photoactivated localization microscopy (PALM) using a recently described new photoconversion mechanism called primed conversion. The new method promises to bring novel insights into the spatial organization of cells and their macromolecules. The results were recently published in the journal Angewandte Chemie.

All cells are spatially highly organized with various macromolecules dynamically localizing to specific subcellular addresses. An important technique for investigating this organization relies on super-resolution fluorescence microscopy using the PALM technique. Here, the fluorescence of a specific group of fluorescent proteins, the so-called photoactivatable or photoconvertible fluorescent proteins, is actively controlled by illumination with UV light. While UV light causes no adverse effects in fixed samples, it is a cause of phototoxicity in experiments with living cells, e.g. in single-particle tracking PALM (sptPALM) studies.

Single-molecule trajectories of RNA polymerase dynamics in living E. coli cells obtained over 6 minutes of sptPALM imaging using either UV light (left) or primed conversion (488/730 nm light, right) for super-resolution imaging.
Cell shapes are marked by dashed outlines, solid outlines highlight exemplary cells (red dead, orange paused, green undisturbed) which are also marked in the following brightlight images. The brightlight images depict cell growth after sptPALM over several hours. Whereas UV-imaged cells are highly affected (left), cells imaged by primed conversion (right) continue to grow normally. Scale bars 1 μm (sptPALM), 5 μm (brightlight).

In 2015, the group of Prof. Dr. Pantazis at ETH Zürich described a novel mechanism of photoconversion called primed conversion for the Dendra2 protein. Normally, Dendra2 undergoes green-to-red photoconversion after exposure to UV light. However, with the new method Dendra2 could be converted from its initial green fluorescence to red by a combination of blue and near-infrared light instead of UV light. “In our experiments using PALM we often track single fluorescently labelled proteins using the sptPALM. This means that we have a huge problem with cells dying during the experiments. So, when we read this paper we were wondering whether primed conversion could be applied in super-resolution microscopy with sptPALM” explains Dr. Endesfelder.

Now, the research group of Dr. Endesfelder at the Max Planck Institute for Terrestrial Microbiology in Marburg addressed this new mechanism in more detail. In their recently published paper, the scientists show that primed conversion can be used successfully in live-cell sptPALM. In comparison to UV-mediated photoconversion, primed conversion-induced phototoxicity is greatly reduced when imaging the dynamics of single proteins in Escherichia coli cells. In fact, cells imaged by primed conversion-sptPALM continued to grow without any negative effects whereas growth of cells exposed to UV-sptPALM imaging almost stopped. Moreover, the Endesfelder group demonstrated that mainly one single amino acid residue, more specifically arginine 66 that interacts with the chromophore, controls the ability of fluorescent proteins to undergo green-to-red primed conversion. Using this finding, the Endesfelder group modified the popular fluorescent proteins Eos, KikGR and pcDronpa to gain the ability to undergo primed conversion.

Further reading

B. Turkowyd, A. Balinovic, D. Virant, H. G. Gölz Carnero, F. Caldana, M. Endesfelder, D. Bourgeois, U. Endesfelder. A general mechanism of photoconversion of green-to-red fluorescent proteins based on blue and infrared light reduces phototoxicity in live-cell single-molecule imaging, Angew. Chem. Int. Ed. 2017, 10.1002/anie.201702870.

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