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).