Figure 4.

Comparison of the apparent lateral membrane diffusion coefficient and apparent plasma membrane dissociation time of Cys1–GFP in response to the addition of PMA, PC-PLC, or DiC8. Fluorescence recovery after photobleaching was used to determine the diffusion coefficient and dissociation time of Cys1–GFP bound to the plasma membrane after either PMA, PC-PLC, or DiC8 addition. A small region of the plasma membrane was photobleached using a short laser pulse (8 ms), and sequential images were recorded every 330 ms for PC-PLC and DiC8 addition and every 1.5 s for PMA addition. (A) Example of four images of a cell expressing Cys1–GFP and stimulated with PC-PLC. The images shown were recorded immediately before and 0.33, 2, and 6 s after the photobleaching pulse. The plasma membrane bleach spot is indicated by the arrow. (B) Comparison of the recovery in fluorescence intensity of Cys1–GFP at the center of the bleach spot. (C) In each series of images, the one-dimensional fluorescence intensity profiles along the plasma membrane were measured as a function of time and each profile was fit by a Gaussian function. (D) Calculated relative increase in the square radius of each Gaussian profile as a function of time for three typical cells. Data for cells stimulated with PMA, PC-PLC, and DiC8 are shown. The apparent lateral plasma membrane diffusion coefficients are proportional to the slope of each linear fit (dy/dt = 4 × D). (E) Calculated membrane dissociation time courses for Cys1–GFP localized to the plasma membrane by PMA, PC-PLC or DiC8 addition.