Fig. 2.

Measuring the lateral mobility of sfGFP-tagged Y receptors and Y receptor-arrestin BiFC complexes by FCS. A schematic diagram of the FCS method is shown in (A), together with three illustrations of FCS recordings and analysis from 293TR Y1sfGFP cells under control conditions (B) or pretreated with 100 nM NPY (C) for 15 min at 37 °C, and from HEK Y1 A2 cells pretreated with 100 nM NPY for 60 min at 37 °C (D). A confocal z-scan, located on the cell nucleus in x–y, identified the upper and lower plasma membrane (LM, UM), shown by the peaks in the top left insets for B–D. Following a 15 s prebleach, fluorescence intensity fluctuations were recorded at 22 °C from the confocal volume positioned on the upper membrane (one 15 s read illustrated in top right graphs for B–D), using the same laser power in all cases. The main graphs illustrate the resultant autocorrelation curves, and below, the fit deviation from a two dimensional, 2 component diffusional model (see also Materials and methods). From this model the dwell time for the receptor species was estimated, and thus the indicated value for diffusion co-efficient D was derived on the basis of the calibrated confocal volume. Pooled data for these and other parameters are presented in Figs. 3 and 7, and also Tables 2 and 3.