Advertisement

Targeted Manipulation/Repositioning of Subcellular Structures and Molecules

  • Kathrin S. Heinz
  • M. Cristina CardosoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)

Abstract

Technical advances in live-cell imaging have made cell biology into a highly dynamic field, allowing the visualization and quantification of complex processes in individual cells and in real time. To follow changes and to specifically manipulate factors potentially involved in processes like DNA replication, transcription or repair, we set up a universal targeting approach, allowing directed manipulation of subcellular structures and molecules therein. This strategy is based on the very strong and specific interaction of GFP and GFP-binding nanobody. We describe in detail how to set up the targeting approach with appropriate controls, as well as how to improve and validate its efficiency and finally provide exemplary applications.

Key words

Green fluorescent protein GFP-binding nanobody Live-cell microscopy Protein–protein interaction Targeted manipulation Targeted repositioning 

Notes

Acknowledgments

We thank H. Leonhardt (LMU-Munich, Germany) for providing plasmids and the C2C12 and MEF cell lines stably expressing RFP-PCNA. We thank Juan Alberto Marchal (University of Jaen, Spain) for the Microtus cabrerae fibroblasts and all present and past members of the laboratory for their contributions over the years. The laboratory of M. Cristina Cardoso is supported by grants of the German Research Foundation (DFG).

References

  1. 1.
    Zolghadr K, Rothbauer U, Leonhardt H (2012) The fluorescent two-hybrid (F2H) assay for direct analysis of protein-protein interactions in living cells. Methods Mol Biol 812:275–282CrossRefGoogle Scholar
  2. 2.
    Herce HD, Deng W, Helma J, Leonhardt H, Cardoso MC (2013) Visualization and targeted disruption of protein interactions in living cells. Nat Commun 4:2660CrossRefGoogle Scholar
  3. 3.
    Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, Backmann N, Conrath K, Muyldermans S, Cardoso MC et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3:887–889CrossRefGoogle Scholar
  4. 4.
    Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7:282–289CrossRefGoogle Scholar
  5. 5.
    Heinz KS, Casas-Delucchi CS, Torok T, Cmarko D, Rapp A, Raska I, Cardoso MC (2018) Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks. Nucleic Acids Res 46:6112–6128CrossRefGoogle Scholar
  6. 6.
    Casas-Delucchi CS, Cardoso MC (2011) Epigenetic control of DNA replication dynamics in mammals. Nucleus 2:370–382CrossRefGoogle Scholar
  7. 7.
    Brero A, Easwaran HP, Nowak D, Grunewald I, Cremer T, Leonhardt H, Cardoso MC (2005) Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation. J Cell Biol 169:733–743CrossRefGoogle Scholar
  8. 8.
    Vissel B, Choo KH (1989) Mouse major (gamma) satellite DNA is highly conserved and organized into extremely long tandem arrays: implications for recombination between nonhomologous chromosomes. Genomics 5:407–414CrossRefGoogle Scholar
  9. 9.
    Peters AH, O’Carroll D, Scherthan H, Mechtler K, Sauer S, Schofer C, Weipoltshammer K, Pagani M, Lachner M, Kohlmaier A et al (2001) Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107:323–337CrossRefGoogle Scholar
  10. 10.
    Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926CrossRefGoogle Scholar
  11. 11.
    Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727CrossRefGoogle Scholar
  12. 12.
    Abbott DW, Chadwick BP, Thambirajah AA, Ausio J (2005) Beyond the Xi: macroH2A chromatin distribution and post-translational modification in an avian system. J Biol Chem 280:16437–16445CrossRefGoogle Scholar
  13. 13.
    Nielsen AL, Oulad-Abdelghani M, Ortiz JA, Remboutsika E, Chambon P, Losson R (2001) Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins. Mol Cell 7:729–739CrossRefGoogle Scholar
  14. 14.
    Cheutin T, McNairn AJ, Jenuwein T, Gilbert DM, Singh PB, Misteli T (2003) Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 299:721–725CrossRefGoogle Scholar
  15. 15.
    Fernandez R, Barragan MJ, Bullejos M, Marchal JA, Martinez S, Diaz de la Guardia R, Sanchez A (2001) Molecular and cytogenetic characterization of highly repeated DNA sequences in the vole Microtus cabrerae. Heredity (Edinb) 87:637–646CrossRefGoogle Scholar
  16. 16.
    Kim JA, Cho K, Shin MS, Lee WG, Jung N, Chung C, Chang JK (2008) A novel electroporation method using a capillary and wire-type electrode. Biosens Bioelectron 23:1353–1360CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Cell Biology and EpigeneticsTechnische Universität DarmstadtDarmstadtGermany

Personalised recommendations