Table 2.
Summary of FCS parameters for GFP tagged NPY receptors.
| Receptor | τD1 |
τD2 |
D |
n | |
|---|---|---|---|---|---|
| μs | % | ms | × 10− 9 cm2 s− 1 | ||
| Y1eGFP | |||||
| Control (5) | 144 ± 5 | 46.3 ± 1.6 | 40.4 ± 2.2 | 2.05 ± 0.15 | 53 |
| NPY (5) | 190 ± 10 | 49.5 ± 2.1 | 59.3 ± 5.1 | 1.64 ± 0.15 | 46 |
| Y1sfGFP | |||||
| Control (16) | 250 ± 9 | 41.6 ± 1.1 | 40.0 ± 1.6 | 2.22 ± 0.15 | 148 |
| NPY (16) | 258 ± 9 | 39.6 ± 1.2 | 60.4 ± 3.4 | 1.48 ± 0.08 | 117 |
| Y16AsfGFP | |||||
| Control (7) | 230 ± 8 | 41.6 ± 1.4 | 33.9 ± 1.8 | 2.45 ± 0.12 | 87 |
| NPY (7) | 244 ± 8 | 42.6 ± 1.2 | 40.0 ± 2.0 | 2.09 ± 0.10 | 95 |
| Y2sfGFP | |||||
| Control (4) | 223 ± 11 | 52.2 ± 0.6 | 37.5 ± 2.2 | 2.15 ± 0.15 | 50 |
| NPY (4) | 265 ± 12 | 51.6 ± 0.6 | 46.4 ± 4.8 | 1.99 ± 0.15 | 47 |
| Y2H155PsfGFP | |||||
| Control (4) | 213 ± 11 | 53.4 ± 0.6 | 31.6 ± 2.1 | 2.55 ± 0.14 | 51 |
| NPY (4) | 260 ± 16 | 53.1 ± 1.3 | 53.0 ± 6.2 | 1.78 ± 0.16 | 35 |
Plasma membrane FCS measurements (2 × 15 s reads, with pre-bleach) were performed on stable transfected 293TR cells inducibly expressing Y receptors, under control conditions or treated with 100 nM NPY. Details are described in Materials and methods, and Fig. 2 provides illustrative examples. Autocorrelation curves were fitted with a two dimensional diffusional model with two dwell time components and a fluorophore blinking component (Zeiss Aim 4.2). The dwell times (mean ± s.e.m.) were attributed to GFP photophysics (τD1) and receptor diffusion (τD2). The proportion, % τD2, refers to the percentage contribution of this component to the autocorrelation curve amplitude, relative to τD1. D was calculated from τD2 measurements from each individual record, before averaging the pooled data shown here. Y1 or Y2 receptor constructs were fused to constructs labelled with sfGFP or enhanced eGFP. n values quoted represent the number of cell records, whilst values in parenthesis to the left indicate the number of experiments performed.