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Fig. 8. PTP1B DA/t280 co-localizes preferentially with paxillin-containing foci in lamellae. PTP1B null cells were co-transfected with GFP-PTP1B DA/t281 and mRFP-paxillin, GFP-PTP1B DA/t280 and DsRed-zyxin, or GFP-paxillin and DsRed-zyxin. 20 hours post-transfection cells were fixed and analyzed by fluorescence microscopy (A,B), or replated on fibronectin-coated coverslips and analyzed by time-lapse fluorescence confocal microscopy 24 hours later (C-E). Numbers over the frames indicate the time elapsed. Note that most PTP1B DA/t280 foci in lamellar extensions contain paxillin but not zyxin (A, arrows). Approximately 93% of PTP1B DA/t280 foci contain paxillin (B, bar 2) and about 35% also contain zyxin (B, bar 1). Most paxillin foci also contain PTP1B DA/t280 (B, bar 3). Results shown are the mean ± s.d. of three independent experiments (n=30). Time-lapse analysis reveals that the ratio of fluorescence intensities for GFP-PTP1B DA/t280 and mRFP-paxillin (indicated by the yellow color) does not change significantly over time. New foci at extending lamellae (C, arrows), and foci that disassemble at retracting regions of the cell (C, arrowheads) maintain constant relative fluorescence intensities of PTP1B and paxillin. In contrast to paxillin, zyxin and PTP1B DA/t280 do not consistently overlap (D, arrows). New PTP1B DA/t280-rich foci are usually devoid of zyxin (D, arrows). Foci at retracting sites frequently incorporate zyxin before disassembling (revealed by a change of color from green or yellow to red, arrowheads). Co-localization of paxillin and zyxin is partial (E). Foci at extending parts of the cell are rich in paxillin foci (arrows) and foci in retracting lamellae change from paxillin-rich (green or yellow) to zyxin-rich (red). Bars, (A) 20 µm; (C-E) 30 µm.

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