Measuring Membrane Protein Dynamics

3T3 cells stably expressing GBP-TM-VSVG-mScarlet were spread on fibronectin-coated glass-bottom dishes, and the transmembrane construct was then clustered with B(c)-GFP and A(d) as described above, before mitotic stalling was induced with 30 nM nocodazole for 12 h. The Flipper-TR probe (Spirochrome, see Colom et al.²² (link)) was then added (2 μM final in L15-20 mM HEPES medium) and Flipper-TR fluorescence lifetime imaging was performed on a setup comprising a Zeiss LSM710 stand, a 63X NA 1.4 oil objective, a Zeiss 710 confocal scanner head and Time-Correlated Single Photon Counting (TCSPC) hardware from Picoquant. A 470 nm pulsed laser (Picoquant), operating at 40 MHz was used to excite the probe, and detection was performed on a gated PMA hybrid 40 detector (Picoquant) behind a 600/50 bandpass filter (Semrock). SymPhotime 2.0 software (Picoquant) was used for data analysis. Flipper-TR fluorescence lifetime was fitted to a dual exponential model. The intensity-weighted average lifetime in a Regions of Interest (ROI) encompassing either the array-containing membrane (assessed by GFP signal), or regions of the membrane devoid of arrays was then measured, followed by averaging over several cells.