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Fig. 6. Ligand-stimulated complex formation between SHP-1 and TrkA-Ros in living COS7 cells. (A) Left panel: fluorescence lifetime image of a COS7 cell coexpressing TrkA-Ros-ECFP and EYFP-SHP1-SH2. Amplitude weighted mean fluorescence lifetimes are encoded by color as specified in the right panels. Right panel: distribution of mean fluorescence lifetimes over the entire cell. (B) Lifetime image of the cell shown in A 40 minutes after stimulation with 100 ng/ml NGF. (C) Subsequent recording of the same cell after photobleaching of the acceptor (EYFP) with the Ar 514 nm line. ECFP fluorescence lifetimes recovered to control values. (D,E) COS7 cells expressing TrkA-Ros-ECFP and EYFPSHP1-C455S before (D) and after (E) treatment with 100 ng/ml NGF for 20 minutes. Images were recorded by confocal LSM in the EYFP channel. Note that a fraction of the SHP1 derivative is enriched at the plasma membrane. Similar translocations were seen in 8 of 10 analyzed cells, but not upon mock-treatment or in cells expressing SHP1 variants with inactivated N-terminal SH2 domain.





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