In recent years, overexpression-based approaches have been developed to monitor the spatiotemporal localization and activity of various intracellular factors in real time. These approaches often rely on the immunofluorescence labeling of fixed cells and exogenous overexpression of fluorescently tagged proteins to visualize cellular processes and structures in live cells. Although a combination of these approaches was used to garner understanding about the dynamics of transcription regulation, they cannot be used to precisely track non-tagged native transcription factors or detect the disappearance and/or appearance of post translational modifications (PTMs) at a high resolution in the nuclear environment of living cells. Exogenous protein expression and limited quantum yield of fluorescent protein tags have limited the use of these approaches in the detection of endogenous cellular factors in live cells. Although certain methods have been developed to overcome these limitations, the methods either require specialized know-how and/or equipment, suffer from low efficiency, and/or inflict harm on live cells. Thus, there is an increased need to develop a powerful tool that can provide insights into the dynamic behavior of endogenously expressed proteins in live cells.
In a new research paper published in Journal of Cell Biology, scientists at Institut de Génétique et de Biologie Moléculaire et Cellulaire (France): Sascha Conic, Veronique Fischer, Vincent Heyer, Bernardo Reina San Martin, Julien Pontabry, Mustapha Oulad-Abdelghani, Nacho Molina, and László Tora in collaboration with Dominique Desplancq and Etienne Weiss from Ecole Supérieure de Biotechnologie de Strasbourg and Université de Strasbourg (France) developed a simple and versatile antibody-based imaging approach (VANIMA) for the accurate tracking and localization of endogenous nuclear factors in live cells. They demonstrated that distinct biological stimuli at a nanometer scale cause dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX). Alexia Ferrand from University of Basel (Switzerland), Kishore Babu from Nanyang Technological University (Singapore) and Graham Wright from the Institute of Medical Biology (Singapore) contributed also to this research.
The research team selected the largest subunit of RNA Polymerase II (RPB1) as the endogenous nuclear target protein. The authors observed that the labeled anti-RPB1 monoclonal antibody recognized and bound to the RPB1 target protein in the cytoplasm, and was piggybacked into the nucleus of the cell. Moreover, the transduced labeled anti-RPB1 mAb can bind to transcribing Pol II on the chromatin and the electroporated mAb remained bound to its target. In addition, they observed that the labeled mAbs or their corresponding labeled antigen-binding (Fab) fragments were able to label the endogenous transcription factors in live cells.
In their study, the transduced Fabs freely diffused and were able to bind to PTMs in the chromatin of live cell nuclei, almost all the labeled anti-γH2AX Fab stayed bound to the chromatin, and the anti-RPB1, -TBP, or -TAF10, antibodies tested did not significantly inhibit newly synthetized mRNA transcription, cell cycle progression, cell proliferation, or induce apoptosis. The authors observed that their approach reveal the changes in individual nuclear structures, where transcription factors or specific PTMs accumulate and uncovered the novel dynamic behaviors of transcription factors and PTM events of certain histone proteins (i.e. H2AX) in real time.
In a nutshell, Sascha Conic and colleagues demonstrated their new technique of fluorescently labeled antibodies or antibody fragments can be used for the conventional and super-resolution imaging and tracking of endogenous factors in live cells. This novel method can uncover the dynamic behavior of transcription factors and posttranslational modifications in the nucleus of single live cells. Their findings hopefully will enhance our knowledge and understanding of cellular delivery of therapeutic antibodies and their use in the treatment of various diseases.
Conic, S., Desplancq, D., Ferrand, A., Fischer, V., Heyer, V., Martin, B.R.S., Pontabry, J., Oulad-Abdelghani, M., Babu N.K., Wright, G.D., Molina, N., Weiss, E., and Tora, L. Imaging of native transcription factors and histone phosphorylation at high resolution in live cells, Journal of Cell Biology 217: 4 (2018) 1537–1552Go To Journal of Cell Biology