Lanthanide-Based Imaging of Protein–Protein Interactions in Live Cells

Abstract

In order to deduce the molecular mechanisms of biological function, it is necessary to monitor changes in the sub-cellular location, activation and interaction of proteins within living cells in real time. Förster resonance energy transfer (FRET)-based biosensors that incorporate genetically encoded, fluorescent proteins permit high spatial resolution imaging of protein–protein interactions or protein conformational dynamics. However, non-specific fluorescence background often obscures small FRET signal changes, and intensity-based biosensor measurements require careful interpretation and several control experiments. These problems can be overcome by using lanthanide (Tb(III) or Eu(III)) complexes as donors and green fluorescent protein (GFP) or other conventional fluorophores as acceptors. Essential features of this approach are the long-lifetime (~ms) luminescence of Tb(III) complexes and time-gated luminescence microscopy. This allows pulsed excitation followed by a brief delay that eliminates nonspecific fluorescence before detection of Tb(III)-to-GFP emission. The challenges of intracellular delivery, selective protein labeling, and time-gated imaging of lanthanide luminescence are presented, and recent efforts to investigate the cellular uptake of lanthanide probes are reviewed. Data is presented showing that conjugation to arginine-rich, cell penetrating peptides (CPPs) can be used as a general strategy for cellular delivery of membrane impermeable lanthanide complexes. A heterodimer of a luminescent Tb(III) complex, Lumi4, linked to trimethoprim (TMP) and conjugated to nonaarginine via a reducible disulfide linker rapidly (~10 min) translocates into the cytoplasm of Maden Darby canine kidney cells from culture medium. With this reagent, the intracellular interaction between GFP fused to FK506 binding protein 12 (GFP-FKBP12) and the rapamycin binding domain of mTOR fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) was imaged at high signal-to-noise ratio with fast (1–3 s) image acquisition using a time-gated luminescence microscope. The data reviewed and presented here show that lanthanide biosensors enable fast, sensitive and technically simple imaging of protein-protein interactions in live cells.

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

“…In recent years, investigators have made several efforts to develop emissive Tb(III) and Eu(III) complexes for similar types of intracellular imaging and sensing applications (4)(5)(6)(7)(8)(9). These efforts sought to leverage two key features of lanthanide luminophores: 1) millisecond-scale excited-state lifetimes, and 2) multiple narrow (<10 nm, half-maximal) emission bands that span the visible spectrum.…”

“…Numerous studies have reported both time-gated and steady-state imaging of responsive lanthanide sensors in living cells, including sensors for Zn 2þ , bicarbonate, pH, and singlet oxygen (21)(22)(23)(24). Lanthanide biosensors may be designed as responsive probes that exhibit an analyte-dependent change in emission intensity or lifetime (6), or as FRET-based systems that use Tb(III) or Eu(III) complexes as donors and FPs, organic dyes, or even quantum dots as acceptors (8,25). The lanthanidebased FRET approach can detect interactions between donor-labeled and acceptor-labeled proteins (via a so-called dual-chain biosensor) or conformational changes of a single protein labeled with both a lanthanide donor and a fluorescent acceptor (via a single-chain biosensor).…”

“…In either case, changes in the donor/acceptor distance alter the energy transfer efficiency, and an increase in FRET manifests as an increase in the long-lifetime, donor-sensitized acceptor emission intensity and a concomitant decrease in the donor emission. Using such an approach, our lab has shown that interactions between Tb(III)-labeled proteins and green FP (GFP) fusions can be imaged in living cells by time-gated detection of Tb(III)/GFP-sensitized emission (8,26).…”

Near-white light luminescent material from Eu(III) complexes encapsulated in silica/PMMA matrices

“…[1][2][3]. The organic ligands can not only well protect the lanthanide ions from vibrational coupling but also increase the light absorption cross section, which makes LnCs more efficient emission compared to single lanthanide ions [4].…”

“…The solvent anhydrous ethanol (EtOH) and N,N 0 -Dimethylformamide (DMF) were used after desiccation with anhydrous calcium chloride. The organic polymer poly (methyl methacrylate) (PMMA) was synthesized in situ according to the previous report [1]. Eu 2 O 3 were purchased from Shanghai Yuelong Company.…”