Direct Imaging of Protein-Specific Methylation in Mammalian Cells.

The abundant post-translational modification methylation alters a protein's function, stability, and/or localization. Its malfunctions are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now their usage was limited to cell lysates. Here, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and Förster resonance energy transfer (FRET) detected by fluorescence lifetime imaging (FLIM) microscopy. Using this approach, we succeeded in imaging methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines. To further demonstrate its potential we visualized the localization-dependent methylation state of Foxo1 in the cellular context.