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First published online 12 December 2006
doi: 10.1242/jcs.03314

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A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel

View larger version (66K): [in this window] [in a new window]   Fig. 1. Paxillin is tyrosine phosphorylated in focal complexes (FXs) and focal adhesions (FAs), but not in fibrillar adhesions (FBs). (A) Porcine aortic endothelial cells (PAECs) were fixed and double-stained for paxillin and phospho (Y118) paxillin. Arrows indicate an FA and arrowheads an FB. Note the high level of phosphorylated paxillin in FXs and FAs, and its absence in FBs. The intensity profile of paxillin (blue line) or phosphorylated paxillin (pink line) along a line one-pixel wide, spanning both an FA and an FB, is shown. (B) PAEC staining showing the co-localization of paxillin and tensin in both FA and FB. Bars, 10 µm (A); 5 µm (B).

View larger version (56K): [in this window] [in a new window]   Fig. 2. Paxillin tyrosine phosphorylation is associated with both assembly and turnover of adhesions, whereas dephosphorylation correlates with adhesion stability. Time-lapse movies of cells expressing YFP-paxillin were analyzed by temporal ratio imaging (A) or by temporal autocorrelation analysis (B). At the end of the movie, cells were fixed and stained for phosphorylated paxillin, revealing the relationship between dynamics and phosphorylation state (C). Dividing intensity values of each pixel by the intensity in the same pixel 6 minutes earlier, created the temporal ratio image (A), which is presented in color, according to the look-up table. In general, red hues denote an increase in intensity or the appearance of new structures, blue hues denote structures that disappeared or decreased in intensity, and yellow marks unchanged pixels. Arrows indicate dynamic FAs, and arrowheads stable FBs. Inserts are enlarged four times. Similar movies were used to perform autocorrelation analysis for FBs and FAs separately (B). The area and intensity of chosen adhesions in the first frame of a movie were correlated with each consecutive frame. Data from ten movies is displayed, along with smoothing spline fits (=1000, FAs in red, FBs in blue). (C) Dynamics, presented by a three-color temporal overlay (left column); the phosphorylation status of paxillin, presented by a ratio image (middle column); and an intensity line profile of selected adhesions on a merged image (right column). Examples for three situations are presented: (i) assembly of FXs at the leading edge; (ii) disassembly in `treadmilling' adhesions and (iii) a stable FA. Note that assembling FXs have the same intensity of phosphopaxillin as the focal adhesion, but only one-third of the intensity of paxillin (i). Note the shift in intensity profiles between paxillin and phosphopaxillin in the `treadmilling' FA (right arrow) compared with a more stable adhesion (left arrow) (ii). Note the 30-40% decrease in phosphopaxillin intensity in the stable FA (iii). Arrowheads point to the cell center. Bars, 5 µm (A); 2 µm (C).

View larger version (62K): [in this window] [in a new window]   Fig. 3. Phosphomimetic and non-phosphorylatable paxillin mutants affect the adhesive phenotype of endothelial cells. Porcine aortic endothelial cells were transfected with phosphomimetic (Y2E) or non-phosphorylatable (Y2F) mutants of YFP-paxillin and with wild-type YFP-paxillin as a control. To verify that the lack of FB in Y2E cells and the lack of FX in Y2F cells was genuine, the transfected cells were stained for tensin (blue) and general phosphotyrosine (PY, red). Bar, 5 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Phosphomimetic paxillin enhances adhesion dynamics and non-phosphorylatable paxillin stabilizes adhesion sites. Time-lapse movies of endothelial cells, expressing wild-type YFP-paxillin or phosphomimetic (Y2E) or non-phosphorylatable (Y2F) mutants, were used to create temporal ratio images (A) and for autocorrelation analysis (B). For each construct, the ratio between two time points, 10 minutes apart, is presented in color code, such that new pixels are red, pixels that disappeared are blue and unchanged pixels are yellow. Note the high proportion of red and blue pixels in Y2E-paxillin and the predominance of yellow pixels in Y2F-paxillin. Inserts are enlarged three times. For each construct, seven movies were used to perform autocorrelation analysis, either on the whole cell or specifically for FA/FB. Autocorrelation is calculated by comparing the adhesions in each time point with time zero. All points are plotted, as well as smoothing spline fits (=1000) as specified to the right. Bar, 5 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Phosphomimetic paxillin inhibits the formation of long stress fibers and fibronectin fibrillogenesis. Cells expressing wild-type or mutant YFP-paxillin were grown for 48 hours and then fixed and stained for F-actin (A) and fibronectin (B). In cells expressing the phosphomimetic mutant (Y2E) there is a conspicuous absence of long actin stress fibers, whereas they are overly abundant in the non-phosphorylatable mutant (Y2F) (A). Similarly, fibronectin fibrillogenesis is impaired in Y2E, and enhanced in Y2F (B). (C) The extent of fibronectin fibrillogenesis was quantified by measuring the average intensity of fibronectin per cell (n=10). Means ± s.d. are shown. Bar, 5 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 6. Paxillin phosphorylation is regulated by mechanical force. Treatment with an actomyosin contractility inhibitor (H7) leads to disassembly of FAs and formation of FXs, as seen in the frames from a time-lapse movie of PAECs expressing YFP-paxillin (top). Preceding the disassembly there is an increase in paxillin phosphorylation levels, as indicated by immunolabeling of cells for paxillin and phosphopaxillin after 1 minute of treatment with H7 (bottom). Single labels are spectrum scaled for intensity, using the same scale for control and treated cells. The ratio image was scaled according to the provided look-up table. Bar, 5 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 7. FAK interacts preferentially with tyrosine phosphorylated paxillin in vitro and in vivo. Purified GST-fusion proteins of wild-type, phosphomimetic (Y2E) or non-phosphorylatable (Y2F) paxillin were used to pull down proteins from equal amounts of endothelial cell extract, followed by western blotting for FAK. A representative blot is shown (A) as well as quantification of the mean (± s.e.) of four separate pull-down experiments (B). Localization of phosphorylated FAK (Y397) was examined by staining in PAECs expressing mutant paxillin alongside non-transfected (N.T) neighboring cells. Images in C are colored according to an intensity scale, as indicated. For convenience of comparison, squares mark adhesions in transfected and non-transfected neighboring cells. (D) Eight transfected or non-transfected cells were segmented and the intensity of pYFAK (means ± s.d.) in each adhesion was calculated. Bar, 5 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 8. FAK mediates the effect of paxillin phosphorylation on adhesion dynamics. Paxillin phosphorylation mutants do not have any effect on adhesion dynamics when expressed in FAK-knockout mouse embryonic fibroblasts (MEFs), whereas they do affect the dynamics in FAK-knockout cells re-expressing FAK (A). Displayed are smoothing spline fits (=100) based on autocorrelation analysis on nine movies for each condition. When FAK is expressed along with wild-type paxillin in PAECs, it substantially increases adhesion dynamics (B). Expression of Y2E-paxillin or Y2F-paxillin along with FAK does not modify the dynamics any further. The time constant for the correlation decay () is given in parentheses, along with the R2 of the fitting spline fit.

View larger version (24K): [in this window] [in a new window]   Fig. 9. Hypothetical model suggesting the involvement of a switch between phosphorylated and non-phosphorylated paxillin and a negative feedback loop with FAK in the regulation of adhesion dynamics. (A) Based on the results in this paper we constructed a working model composed of the following assumptions: (1) Phosphopaxillin is initially recruited following an external stimuli (integrin adhesion). (2) Thereafter its recruitment rate is positively regulated by the existence of both phosphopaxillin and paxillin. (3) FAK recruitment into the adhesion is dependent on phosphopaxillin concentration. (4) Phosphopaxillin disassembles in a FAK-dependent manner where FAK concentration increases the disassembly rate. (5) FAK disassembly occurs at the same rate it assembles, after a short delay. (6) Phosphopaxillin becomes dephosphorylated at a high rate when mechanical force exists and paxillin becomes phosphorylated constantly at a low rate. (7) Paxillin disassembles at a constant rate. (B) The above assumptions were compiled into a set of three simple differential equations (see model in Materials and Methods) and their solutions for the changing concentration of phosphopaxillin (red), FAK (green) and paxillin (blue) are given here in graphical form. Arrows under the time line mark the adhesion stimuli. Gray shading depicts the existence of mechanical force. The change over time in adhesion `area' (sum of paxillin and phosphopaxillin) is presented in the insert. (C) This hypothetical scheme presents the manner by which a paxillin phosphorylation switch and FAK might regulate the formation of the three integrin-mediated adhesion forms: focal complexes (FX), focal adhesions (FA) and fibrillar adhesions (FB).

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