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. 2013 Nov 18;110(49):E4723–E4732. doi: 10.1073/pnas.1312540110

Fig. 8.

Fig. 8.

The localization of RacE(G20V) is regulated by chemoattractant gradients in the absence of the actin cytoskeleton. (A) WT cells coexpressing GFP-RacE(G20V) and PHcrac-RFP were treated with Latrunculin A and placed in a cAMP gradient. The arrow indicates the direction of the gradient. (B) WT cells expressing GFP-RacE(G20V) were treated with Latrunculin A, placed in a cAMP gradient, and observed by fluorescence microscopy at the indicated time points. The direction of the gradient was changed at 1 and 3.5 min by repositioning a micropipette filled with cAMP. White dots indicate the position of the micropipette tip. (C) WT cells coexpressing GFP-RacE(G20V) and PHcrac-RFP were treated with Latrunculin A and placed in a cAMP gradient. Images were taken before (Upper) and ∼2 min after (Lower) the addition of the PI3 kinase inhibitor LY294002 (20 µM). White dots indicate the position of the cAMP source. (D) WT cells expressing GFP-RacE(G20V) or GFP-RacE(G20V, T43A) were observed by fluorescence microscopy in a cAMP gradient in the absence of Latrunculin A. The concentration of cAMP was higher on the upper side of each image. (E) The ratio of GFP fluorescence on the plasma membrane facing lower (FL) and higher (FH) concentrations of cAMP was determined (FL/FH) in WT cells expressing GFP-RacE(G20V) or GFP-RacE(G20V, T43A) in the presence of Latrunculin A. Values represent the mean ± SEM. More than 12 cells were examined for each. ***P < 0.001. (F) WT cells expressing GFP-RhoA or GFP-RhoA(Q63L) were placed in chemoattractant gradients in the absence of Latrunculin A. The concentration of cAMP was higher on the left side of each image. (G) The ratio of GFP fluorescence on the plasma membrane (FPM) and in the cytoplasm (FCYT) was determined (FPM/FCYT). Values represent the mean ± SEM. More than five cells were examined for each. *P < 0.05. (H) WT cells expressing GFP-human RhoA or GFP-human RhoA(Q63L) were placed in a cAMP gradient in the presence of Latrunculin A. FL/FH was determined as described above. Values represent the mean ± SEM. Twelve cells were examined for each. **P < 0.01.