Split-GFP Complementation for Targeting and Imaging Single Molecules in Living Cells

Abstract

Current limitations for studying intracellular dynamics of biomolecules by single molecule fluorescence include the necessity to express a molecule of interest at very low concentration or to target it very specifically with a small number of probes. Inside cells, both conditions are hard to meet. In the first case, very low expression levels of a molecule of interest might influence the cell physiological responses. In the second case, probes with high specificity and very high affinity binding constants for their target are required. To circumvent these issues we introduce the use of split-GFP fusions and their complementation by small synthetic peptides to image the dynamics of single proteins in live cells. Cells expressing the split-GFP fusions are non-fluorescent and a controlled subset of the GFPs can be “lit up” when providing the complementary peptide at different concentrations. With this approach, we imaged the plasma membrane diffusion of single CD4-split GFP and GPI-split GFP proteins in different cell lines. We also demonstrate intracellular single molecule imaging using caveolin-1-split GFP proteins as an example. In all cases, background free tracking of single proteins could be achieved by TIRF microscopy within minutes of complementation. Split-GFP fusions and the development of other split-fluorescent protein variants provide a generic method for multicolor single molecule imaging in living cells even at elevated protein expressions. Split-fluorescent protein fusions also complement the toolbox of intracellular targeting strategies by providing a unique way to verify the specific targeting of molecules introduced in living cells. Toward this aim, we present early efforts at employing split-GFP fusions for addressable targeting of single fluorophores and single quantum dots.