PhotoChromic HYdrogen PERoxide biosensors for super-resolution imaging of hydrogen peroxide microenvironments

Project Information

PCHYPERs

Grant agreement ID: 101030525

DOI

10.3030/101030525

Project closed

EC signature date 19 March 2021

Start date 1 November 2021

End date 31 October 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 178 320,00

EU contribution

€ 178 320,00

Coordinated by

KATHOLIEKE UNIVERSITEIT LEUVEN
Belgium

Periodic Reporting for period 1 - PCHYPERs (PhotoChromic HYdrogen PERoxide biosensors for super-resolution imaging of hydrogen peroxide microenvironments)

Reporting period: 2021-11-01 to 2023-10-31

Summary of the context and overall objectives of the project

Hydrogen peroxide (H2O2) is a cellular second messenger that is deliberately produced and is key for a healthy cellular metabolism. An adequate amount of H2O2 production and delivery is required to maintain normal cellular and tissue functions, including neural development, wound healing, and innate immune response, with an imbalance compromising such functions and potentially leading to disease.

Most of the advancements on H2O2 signaling have occurred thanks to the development and use of genetically-encoded fluorescent biosensors, which connect the presence of H2O2 to a fluorescence signal that can be easily recorded on a fluorescence microscope. However, most H2O2 signaling events happen at the nanoscale, which is not possible to visualize with current biosensors, as they are not compatible with super-resolution microsopy techniques that allow for nanoscale imaging. Such lack of tools impede us to get a full understanding how these H2O2 signaling events work.

The goal of this action “PhotoChromic HYdrogen PERoxide biosensors for super-resolution imaging of hydrogen peroxide microenvironments” (PCHYPERs) is to develop variants of current hydrogen peroxide biosensors that are compatible with nanoscale imaging microscopy techniques. The developed H2O2 biosensors will allow monitoring hydrogen peroxide signaling events at their natural nanoscale.

The PCHYPERs action is divided into 3 objectives: Generate the nanoscale imaging compatible biosensors, known as PCHYPERs (objective 1), apply the PCHYPERs in a cellular setting (objective 2), and determine super-resolution hydrogen peroxide gradients using the PCHYPERs (objective 3). We successfully generated green fluorescent PCHYPERs (objective 1), which we were able to express in cells and obtain super-resolution images of hydrogen peroxide (objective 2). Whilst objective 3 could not be achieved in this action, due to delays in work packages related to objectives 1 and 2, this action has generated tools that allow the visualization of physiologically relevant hydrogen peroxide signaling events.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The research work was carried out through 3 work packages (WP) although there was no time left for the last one, due to delays in WP1 and WP2. The WP1 consisted in generating the photochromic variants of the ancestral hydrogen peroxide biosensors, which was successful for the green PCHYPERs. This was not the case for the red PCHYPERs, as the modifications rendered the fluorescent protein component more sensitive to bleaching by hydrogen peroxide, the very molecule we were trying to monitor. The data concerning this phenomenon was recently published at the International Journal of Biological Macromolecules, and we described the mechanism on how this could be happening. For WP2, we managed to achieve super-resolution images of the PCHYPERs expressed in tubulin fibers, and we have established a dual PCHYPER-local hydrogen peroxide generator system to generate and monitor local hydrogen peroxide waves.

Besides the research work, I have organized, in collaboration with other researchers, 3 international scientific conferences (Gordon Research Seminar on Thiol-Based Redox Regulation and Signaling, Shine On Symposium, Leuven Biosensor Symposium), and organized a public exhibition highlighting the beauty of fluorescence microscopy in research (Shine On). This public exhibition was held at the entrance of the Leuven public library and had interactive material (poster of fluorescence microscopy pictures and a video on how a fluorescence microscope is used) so anyone passing by the library could see it. We also had an interactive session with high school students, who were amazed at how fluorescence microscopy was done in the lab.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project has obtained photochromic hydrogen peroxide biosensors, some of them are compatible with super-resolution imaging by PCSOFI. This is a turning point in understanding hydrogen peroxide signaling at greater detail in the cell. Adequate hydrogen peroxide production and delivery is needed to keep normal cellular functions, including neural development, wound healing, and innate immune response, with an imbalance compromising such functions and potentially leading to disease. As most of hydrogen peroxide signaling happens in the spatial scale that is only compatible with super-resolution techniques, the PCHYPERs are the only tools that allow for the moment to visualize physiologically relevant hydrogen peroxide signaling events. In addition, there is general very little progress in obtaining fluorescent biosensors that are compatible with super-resolution imaging, meaning that the PCHYPERs are also a tool breakthrough in the biosensor field.

Super-resolution image of tubulin-associated PCHYPER7