Supplemental Material

This article contains the following supporting material:

• Supplemental Material.pdf
• Video1fig1.avi (1.74 MB) - Visualization of PH_Crac-GFP membrane translocation in responses of a uniform cAMP stimulation. A cell expressing PH_Crac-GFP (green) is stimulated with 10 nM cAMP (red). Images were captured at 0.9 s intervals and are replayed at 5 frames/s
  
  
• Video2fig2.avi (6 MB) - Visualization of heterotrimeric G-protein dissociation in a single cell (right) upon a uniform cAMP stimulation.
  A cell, expressing PH_Crac-GFP (left) is used to monitor the cAMP stimulation. The addition of 1 μM cAMP caused the shaken images of both cells. Upon stimulation, the CFP fluorescence signal increased (Video2fig2.avi) and the YFP signal decreased (Video3fig2.avi) around the entire membrane of the G cell (right). PH_Crac-GFP transiently translocated from the cytosol to the plasma membrane of the PH cell (left), which can be seen in both videos. Images were captured at 1.1s intervals and are replayed at 5 frames/s.
  
  
• Video3fig2.avi (6.18 MB)
• Video4.avi (7.72 MB) - Simultaneously visualizing heterotrimeric G-protein dissociation (left) and PH_Crac-GFP translocation (right) upon cAMP stimulation. 1 μM cAMP was added 2 seconds after the second frame. Following stimulation, the CFP fluorescence signal increased around the entire membrane of the left cell (red signal increase). PH_Crac-GFP transiently translocated from the cytosol to the plasma membrane of the left cell (yellow signals). The confocal images were processed by the program?s 2.5D function of LSM 510META and photoshop software. Time is indicated in video4.mov.
  
  
• Video5fig4.avi (9.37 MB) - PH_Crac-GFP translocation (green) in response to an acute exposure to a cAMP gradient (red). A pulse of cAMP (red) was released from the right side of the frames. Frames were captured at 785 ms intervals and are replayed at 5 frames/s.
  
  
• Video6fig6.avi (8.70 MB) - Dynamics of PH_Crac-GFP translocation (green) in a cell suddenly exposed to a static cAMP gradient (red). Images were captured at 2.5s intervals and are replayed at 5 frames/second.
  
  
• Video7suppfig1.avi (5 MB) - PH_Crac-GFP translocation in multiple cells in response to a uniform cAMP stimulation. cAMP (10 nM) was uniformly applied to the cell chamber in the seventh frame of the movie. Images were captured at 0.62s intervals and are replayed at 5 frames/second.
  
  
• Video8.avi (2.19 MB) - Asymmetrical translocation of PH_Crac-GFP in multiple cells in response to a coming wave of cAMP. A pulse of cAMP (red) was released. Images were captured at 3.9s intervals and are replayed at 5 frames /s.
  
  
• Video9.avi (10.4 MB) - Simultaneously visualizing cAMP gradient and chemotaxis of cells expressing PH_Crac-GFP. A microinjector, which is linked to a Femtojet with constant pressure (Pc=70 and Pi=70), injects a constant and small volume of a mixture of cAMP and Alxea594 into a one-well cell chamber with total buffer volume of 6 ml, to establish a constant circular gradient (red). When the micropipette is moved from one position to another, the gradient remains almost constant. Cells move toward the center of the gradient. Images were captured at 3.9s intervals and are replayed at 5 frames/s.
  

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