Targeted Manipulation/Repositioning of Subcellular Structures and Molecules
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 wordsGreen fluorescent protein GFP-binding nanobody Live-cell microscopy Protein–protein interaction Targeted manipulation Targeted repositioning
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).