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First published online 7 March 2006
doi: 10.1242/jcs.02846

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ER-bound PTP1B is targeted to newly forming cell-matrix adhesions

¹ Instituto de Investigaciones Biotecnológicas, IIB-INTECH, Universidad de San Martín, 1650 San Martín, Buenos Aires, Argentina
² Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA

View larger version (33K): [in a new window]   Fig. 1. The substrate-trapping PTP1B DA localizes to punctate structures at cell margins. Cells were plated on fibronectin-coated coverslips, fixed and examined by wide-field fluorescence microscopy. (A) PTP1B null cells were transfected with GFP-wild-type PTP1B or (B) GFP-PTP1B DA and visualized 20 hours later. The arrows indicate punctate structures (B, inset). Note the absence of punctate structures in A. (C) PTP1B null cells stably reconstituted with PTP1B DA were stained with monoclonal anti-PTP1B followed by Alexa Fluor 488-conjugated secondary antibody. Note that the punctate structures look larger because of the signal amplification by the antibodies. Bar, 20 µm. (D) Representative western blot of equivalent amounts of soluble protein prepared from PTP1B null cells (lane 1); PTP1B null cells transiently transfected with GFP-wild type PTP1B (lane 2); GFP-PTP1B DA (lane 3); or stably reconstituted with PTP1B DA (lane 4). The endogenous levels of PTP1B in a non related fibroblast cell line is also shown (lane 5). The blot was probed with anti-PTP1B.

View larger version (52K): [in a new window]   Fig. 2. PTP1B DA puncta co-localize with proteins associated with cell-matrix adhesion sites and the cytoskeleton. PTP1B null cells stably expressing PTP1B DA (A,B) or PTP1B null cells transiently transfected with GFP-PTP1B DA (C,D), or GFP-PTP1B DA/QA (E) were plated on fibronectin-coated coverslips and examined 24 hours later. (A) Cells double-stained with a polyclonal anti-PTP1B and a monoclonal (hVIN-1) anti-vinculin followed by secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 568, respectively. Shown is one confocal section (0.5 µm) at the cell-matrix interface. (B) Cells stained with anti-PTP1B and anti-paxillin followed by secondary antibodies conjugated with Alexa Fluor 568 and Alexa Fluor 488, respectively. (C) Cells stained with rhodamine-phalloidin. Dotted line indicates the boundary of the cell. (D) Cells stained with anti-tubulin (DM1 A), followed by a secondary antibody conjugated with Alexa Fluor 568. (E) Cells stained with anti-PTP1B followed by a secondary antibody conjugated with Alexa Fluor 568. In all cases, arrows indicate punctate structures. Note that punctate structures localize at the distal tips of vinculin and paxillin (A,B,E), as well as at the tips of actin (C) and microtubules (D). Bars, (A,B,E) 20 µm; (C,D) 10 µm.

View larger version (90K): [in a new window]   Fig. 3. PTP1B puncta co-localize with Src and Src-pY418 at cell margins. PTP1B null cells were transfected with GFP-PTP1B DA and examined 20 hours later by fluorescence microscopy (A-G). Cells were labeled with anti-Src (A-F) or anti-Src-pY418 (G) followed by a secondary antibody conjugated with Alexa Fluor 568. Note the co-localization (yellow) at large punctate structures formed at the peripheral ER network (C), or in small punctate structures at individual ER tubules at the cell margins (F) Arrows indicate puncta. Note that a subpopulation of PTP1B DA puncta is coincident with the phospho-active form of Src (G). Bars, (A-C) 10 µm; (D-G) 15 µm. (H) The formation of Src-PTP1B complexes was analyzed by immunoprecipitation. PTP1B null cells (-/-), and cells stably expressing PTP1B wild type (Wt) or PTP1B DA (DA) were lysed and immunoprecipitated with anti-PTP1B. The amount of PTP1B and Src in the lysate (input) and in the immunoprecipitates was detected with the respective antibodies (Blot).

View larger version (94K): [in a new window]   Fig. 4. PTP1B puncta co-localize with FAK in fixed and living cells. (A-C) PTP1B null cells stably expressing PTP1B DA double labeled with anti-FAK and anti-PTP1B, followed by secondary antibodies conjugated with Alexa Fluor 568 (A) and Alexa Fluor 488 (B), respectively. Note the high overlap of both proteins at the cell margins (arrows). (D) PTP1B null cells co-transfected with mRFP-PTP1B DA and GFP-FAK analyzed by time-lapse confocal microscopy after 24 hours. Note that most PTP1B DA puncta also contain FAK (in yellow), however, in instances of non co-localization, FAK has an elongated distribution. Arrows indicate puncta containing both PTP1B DA and FAK that move or disappear from the plane of focus without changing their relative fluorescence. Arrowheads indicate puncta that fuse or split. Bars, 20 µm.

View larger version (93K): [in a new window]   Fig. 5. PTP1B DA puncta do not co-localize with endocytic markers, but do co-localize with ER markers and require intact microtubules. PTP1B null cells stably expressing PTP1B DA were transfected with different endocytic markers: (A) the dominant-negative mutant GFP-dynamin K44A; (B) GFP-Rab 5; (C) GFP-Rab 7; (D) GFP-caveolin 1. Cells were fixed and stained with anti-PTP1B followed by a secondary antibody conjugated with Alexa Fluor 568. (E-G) PTP1B null cells were transfected with GFP-PTP1B DA (E) and stained with anti-calnexin antibody (F) followed by a secondary antibody conjugated with Alexa Fluor 568. The merged image is shown in G. PTP1B null cells were transfected with GFP-PTP1B DA in the absence (H) or presence (I) of Nocodazole (0.5 µM). After 20 hours, cells were fixed and stained with rhodamine-phalloidin. Note that the punctate structures (arrows in H) are not longer visible in the presence of nocodazole (I). Bars, (A-G) 20 µm; (H,I) 25 µm.

View larger version (76K): [in a new window]   Fig. 6. PTP1B DA puncta develop from ER tubules extending over cell-matrix adhesion foci. PTP1B null cells were co-transfected with GFP-PTP1B DA and mRFP-paxillin, and 24 hours later were replated for 30 minutes on fibronectin-coated coverslips. Cells were immediately analyzed by time-lapse confocal microscopy. Different regions of a cell in selected frames of a time-lapse sequence are shown (A-C). Numbers over the time-lapse series indicate minutes elapsed. Arrows indicate punctate structures that develop from ER tubules at the tips of new paxillin foci. Yellow arrows in B indicate a punctate structure that forms and disappears, whereas the paxillin foci remain. Note that the punctate structures become larger after the arrival of additional ER tubules (arrowheads, 120 minutes in A; 26 minutes in B; 114 minutes in C). Bar, 10 µm. See also Movie 1 in supplementary material.

View larger version (91K): [in a new window]   Fig. 7. PTP1B structural requirements for formation of cell-matrix adhesion site puncta. PTP1B null cells were transfected with GFP-PTP1B DA/PA (A,B), GFP-PTP1B DA/YF (C,D) and GFP-PTP1B DA/t280 (E,F) and fixed 20 hours later. Cells stained with anti-FAK (A,C) or anti-vinculin (B,D,F), followed by secondary antibodies conjugated with Alexa Fluor 568. Note that PTP1B DA/PA and PTP1B DA/YF form typical punctate structures (A-D, arrows) whereas PTP1B DA/t280 has lost the punctate distribution and appears throughout the entire area of the cell-matrix adhesion foci (arrows in E and F). Bar, 20 µm.

View larger version (57K): [in a new window]   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.

View larger version (83K): [in a new window]   Fig. 9. Lamellar extensions in cells lacking PTP1B are unstable and contain immature cell-matrix sites. PTP1B null cells (null) and cells reconstituted with wild-type PTP1B (+wt) were transfected with GFP-vinculin or GFP-paxillin and analyzed by time-lapse confocal microscopy. Note that PTP1B null cells produce lamellar extensions and assemble vinculin and paxillin foci (arrows); however, the lamellae quickly retract (arrowheads). By contrast, cells expressing wild-type PTP1B extend lamellae and assemble vinculin and paxillin foci that are persistent in time (arrows). Bar: 30 µm. The percentage of paxillin foci that also contain zyxin in PTP1B null cells and cells stably expressing wild-type PTP1B is shown in the histogram in the bottom, right panel. Results shown are the mean ± s.d. of three independent experiments (n=44 PTP1B null cells; n=39 cells expressing wild-type PTP1B). The number of cell-matrix sites per cell ranged from 50-100.