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Observing protein activity in the nucleus of living human cells

Illustration: Three dimensional representation of a cell nucleus after electroporating the cells with fluorescently labeled antibodies binding to RNA Polymerase II (green spheres) visualized using 3D SIM super-resolution microscopy. 

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

Conic S(1)(2)(3)(4), Desplancq D(5), Ferrand A(6), Fischer V(1)(2)(3)(4), Heyer V(1)(2)(3)(4), Reina San Martin B(1)(2)(3)(4), Pontabry J(1)(2)(3)(4)(7), Oulad-Abdelghani M(1)(2)(3)(4), Babu N K(8), Wright GD(9), Molina N(1)(2)(3)(4), Weiss E(10), Tora L(11)(2)(3)(4)(8).

J Cell Biol April 2, 2018


Feb. 13, 2018

Visualize without disturbing structures and processes taking place in the living cell nucleus is now possible thanks to an innovative technique developed by Laszlo Tora’s team at the IGBMC (CNRS/Inserm/Unistra) in close collaboration with Etienne Weiss at the ESBS Research Institute (CNRS/Unistra). This method allows to introduce, efficiently and without damaging the cells, antibodies labelled with small fluorescent molecules. In the cytoplasm of cells, these fluorescent antibodies bind to their antigens or targets. When the target is a nuclear protein, antibodies are transported with them into the nucleus, allowing researchers to locate and track the movements of this nuclear protein with high accuracy and in real time. The results of this study were published on February 12th, 2018 in the Journal of Cell Biology

 

Protein detection by immunofluorescence is a technique that has been widely used for many years, where proteins or their post translational modifications are identified with antibodies coupled to microscopically detectable fluorescent compounds. The disadvantage of this process is that the cells need to be fixed and permeabilized, which may damage them, to allow the entry of labelled antibodies. Furthermore, the need to fix cells does not allow the observation of the progression of a biological mechanism over time.

 

In this new study, researchers from Laszlo Tora’s and Etienne Weiss's teams have developed an ingenious method that allows them to observe chromatin-based mechanisms in the nucleus of living cells, without altering the observed processes. This technique allows the introduction of any antibody labelled with fluorescent compounds that bind specifically to nuclear factors into the cells by using an electric micro-shock that does not modify cells physiology. When these antibodies are in the cytoplasm, they bind to the nuclear factors produced in this cell compartment. Since these factors are naturally transported to the nucleus to exert their function, the nuclear factors linked antibodies are taken to the nucleus. "The antibodies, which bind to these newly synthesized factors, are transported in the nucleus of the cell like small backpacks and, as they are marked with fluorochromes of any color, they will send a signal which allows us to observe the nuclear factors to which they are linked", explains Laszlo Tora. In addition, this visualization of nuclear factors in living cells is very effective because each antibody can be labelled with five to seven fluorescent molecules. The treated cells were observed with a confocal microscope and, in collaboration with the Basel Imaging Centre, with a very high-resolution microscope, the 3D-SIM. Precise observation of proteins that have nuclear activity during the cell cycle allows to see their dynamics and motion in the nucleus of living cells. Researchers have also shown that this strategy makes it possible to detect in real time the appearance and management of DNA damages in the nucleus by detecting a post translational modifications. By using an antibody fragment that has the same properties as a complete antibody and targets a major actor in triggering DNA repair (gammaH2AX), researchers were able to reveal areas in the nucleus where cell DNA is damaged by a substance causing genome changes, called genotoxic agent.

 

Given the large number of antibodies available on the market, this simple and innovative method will undoubtedly provide new information about nuclear proteins and a better understanding of their behavior in transmitting and maintaining the integrity of genetic information.

 

This study was funded by the ANR, ERC and the Regional League Against Cancer.

Imprimer Envoyer

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