Table 1.
Lifetime and FRET efficiency of eGFP- and eGFP-tagged Vpr in living cells
| Nuclear envelope | Cytoplasm | Nucleus | Whole Cell | |||||
| E(%) | τ(ns) | E(%) | τ(ns) | E(%) | τ(ns) | E(%) | τ(ns) | |
| eGFP | - | - | - | 2.50 (± 0.01) | - | 2.50 (± 0.01) | - | 2.50 (± 0.01) |
| Vpr-eGFP | - | 2.36 (± 0.01) | - | 2.40 (± 0.01) | - | 2.41 (± 0.01) | - | 2.39 (± 0.01) |
| eGFP-Vpr | - | 2.47 (± 0.01) | - | 2.46 (± 0.01) | - | 2.47 (± 0.01) | - | 2.47 (± 0.01) |
| Vpr-eGFP+mCherry | - | 2.41 (± 0.02) | - | 2.42 (± 0.01) | - | 2.42 (± 0.01) | - | 2.42 (± 0.01) |
| Vpr-eGFP+Vpr-mCherry | 27 | 1.72 (± 0.02) | 23 | 1.86 (± 0.03) | 19 | 1.95 (± 0.03) | 23 | 1.85 (± 0.03) |
| Vpr-eGFP+mCherry-Vpr | 17 | 1.95 (± 0.02) | 14 | 2.06 (± 0.02) | 13 | 2.09 (± 0.02) | 15 | 2.02 (± 0.03) |
| eGFP-Vpr+mCherry | - | 2.43 (± 0.01) | - | 2.43 (± 0.01) | - | 2.43 (± 0.02) | - | 2.43 (± 0.01) |
| eGFP-Vpr+Vpr_mCherry | 13 | 2.14 (± 0.03) | 9 | 2.25 (± 0.03) | 6 | 2.32 (± 0.02) | 9 | 2.25 (± 0.03) |
| eGFP-Vpr+mCherry-Vpr | 13 | 2.14 (± 0.03) | 7 | 2.28 (± 0.03) | 6 | 2.31 (± 0.02) | 8 | 2.28 (± 0.03) |
The fluorescence lifetimes (τ) of eGFP alone or linked to the Vpr C-terminus are the average values (+/- standard deviation) for 10 to 35 cells. For each cell, measurements were performed at the nuclear envelope, in the nucleus and in the cytoplasm. The FRET efficiency (E) is related to the distance between the two chromophores and is calculated from the lifetime ratio with and without the acceptor using equation (2). The whole cell E and τ values represent the average values calculated over the entire cell.