Visualization of Endogenous Transcription Factors in Single Cells Using an Antibody Electroporation-Based Imaging Approach
In this chapter, we describe an antibody electroporation-based imaging approach that allows for precise imaging and quantification of endogenous transcription factor (i.e., RNA Polymerase II) distributions in single cells using 3D structured illumination microscopy (3D-SIM). The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies into living cells and the specific binding of these antibodies to the targeted factors. Our step-by-step protocol describes the procedure of the labeling of the specific antibodies, their electroporation into living cells, the sample preparation and 3D-SIM imaging as well as the postimaging analyses of the labeled endogenous transcription factors to obtain information about their nuclear distribution as well as their function. This protocol can be applied to a plethora of endogenous nuclear factors by using target specific noninhibiting antibodies.
Key wordsAntibodies Antibody delivery Single cells Endogenous proteins RNA polymerase II Transcription Imaging Nucleus Factor distribution 3D structured illumination microscopy (3D-SIM)
This work was supported by funds from CNRS, INSERM, University of Strasbourg, Ligue Régionale contre le Cancer (CCIRGE-BFC) (to EW), by the European Research Council (ERC) Advanced grant (ERC-2013-340551, Birtoaction) (to LT) and a grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02 (to IGBMC).
- 7.Cassimeris L, Guglielmi L, Denis V, Larroque C, Martineau P (2013) Specific in vivo labeling of tyrosinated alpha-tubulin and measurement of microtubule dynamics using a GFP tagged, cytoplasmically expressed recombinant antibody. PLoS One 8(3):e59812. https://doi.org/10.1371/journal.pone.0059812CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Freund G, Desplancq D, Stoessel A, Weinsanto R, Sibler AP, Robin G, Martineau P, Didier P, Wagner J, Weiss E (2014) Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells. J Mol Recognit 27(9):549–558. https://doi.org/10.1002/jmr.2378CrossRefPubMedGoogle Scholar
- 12.Teng KW, Ishitsuka Y, Ren P, Youn Y, Deng X, Ge P, Belmont AS, Selvin PR (2017) Labeling proteins inside living cells using external fluorophores for microscopy. eLife 5. https://doi.org/10.7554/eLife.20378
- 13.Courtete J, Sibler AP, Zeder-Lutz G, Dalkara D, Oulad-Abdelghani M, Zuber G, Weiss E (2007) Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA. Mol Cancer Ther 6(6):1728–1735. https://doi.org/10.1158/1535-7163.MCT-06-0808CrossRefPubMedGoogle Scholar
- 14.Conic S, Desplancq D, Ferrand A, Fischer V, Heyer V, Reina San Martin B, Pontabry J, Oulad-Abdelghani M, Babu NK, Wright GD, Molina N, Weiss E, Tora L (2018) Imaging of native transcription factors and histone phosphorylation at high resolution in live cells. J Cell Biol 217(4):1537–1552. https://doi.org/10.1083/jcb.201709153CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Freund G, Sibler AP, Desplancq D, Oulad-Abdelghani M, Vigneron M, Gannon J, Van Regenmortel MH, Weiss E (2013) Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells. MAbs 5(4):518–522. https://doi.org/10.4161/mabs.25084CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Conic S, Desplancq D, Tora L, Weiss E (2018) Electroporation of labeled antibodies to visualize endogenous proteins and posttranslational modifications in living metazoan cell types. Bio Protoc 8(21). https://doi.org/10.21769/BioProtoc.3069
- 17.Desplancq D, Freund G, Conic S, Sibler AP, Didier P, Stoessel A, Oulad-Abdelghani M, Vigneron M, Wagner J, Mely Y, Chatton B, Tora L, Weiss E (2016) Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells. Exp Cell Res 342(2):145–158. https://doi.org/10.1016/j.yexcr.2016.03.003CrossRefPubMedGoogle Scholar