FRET images showing activation of Ras (top panel) and Rap1 (bottom panel) in COS cells in response to treatment with epidermal growth factor for 0, 5 or 30 min (from left to right). A red hue is associated with a high emission ratio and the intensity of this hue correlates with source image brightness. Image reproduced with permission from Nature © (2001) Macmillan Magazines Ltd.

There's a time and place for most things — and intracellular reactions are no exception. Reporting in Nature, Matsuda and colleagues have shown that Ras and Rap1 are activated in a spatio-temporal manner in cells using fluorescent resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) techniques.

The key to their findings was the design of a protein comprising H-Ras, the Ras-binding domain of Raf (Raf RBD), and yellow- and cyan-emitting mutants of the green fluorescent protein, YFP and CFP, respectively. Intramolecular interactions between active, GTP-bound Ras and the Raf RBD bring YFP and CFP into close proximity, thereby increasing FRET. A probe for Rap1 was also made by substituting Rap1 for Ras. The proteins were designated Raichu-Ras and Raichu-Rap1 (Raichu standing for Ras and interacting protein chimeric unit). Co-expressing Raichu-Ras or Raichu-Rap1 with specific guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) effectively showed the increase and decrease in FRET, as did activating and inhibiting mutants of Ras and Rap1 substituted in the Raichu proteins.

Next the authors measured the activation of Ras and Rap1 in response to epidermal growth factor (EGF) in live COS cells. Using dual-emission microscopy to measure the emission intensities from CFP and YFP, FRET images were generated. EGF-induced activation of Ras occurred at the peripheral plasma membrane, and was lower at the sites of cell–cell contact — probably due to increased local Ras GAP activity. By contrast, EGF led to Rap1 activation at the internal perinuclear region. As a Raichu-Rap1 mutant lacking a farnesyl moiety was not activated by EGF, and pretreating the Raichu-Rap1-expressing cells with an inhibitor of clathrin-mediated endocytosis prevented Rap1's activation, it seems that Rap1 is activated at intracellular membrane compartments by internalization of the EGF receptor.

In PC12 cells, prolonged activation of Ras by nerve growth factor (NGF) stimulates neurite outgrowth. Early activation of Raichu-Ras occurred in the cell body, but at later time points active Ras persisted only in the extending neurites. This is consistent with the proposal that survival responses are mediated by activation of the NGF receptor, TrkA, at the cell surface, whereas differentiation occurs in response to TrkA in endosomes. Again, by contrast, active Rap1 was seen only in the intracellular region of these cells.

Is the sustained activation of Ras and the low activity of Rap1 in neurites a cause of retaining active Ras or inactive Rap1 here, or is it due to the activities of these two G proteins being determined locally? As the fluorescence intensity of PC12 neurites expressing YFP-Ras or YFP-Rap1 recovered quickly after photobleaching (FRAP technology), this suggests the latter.

The use of this technique to pinpoint active Ras and Rap1 proteins in specific regions of the cell at distinct times clearly also indicates that a large proportion of these proteins is inactive, emphasizing the importance of being in the right place at the right time.