JCS03314 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Table 2 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Differential phosphorylation of paxillin and correlation between phosphorylation and adhesion dynamics in REF52 cells. Rat Embryonic Fibroblasts (REF52) stably expressing YFP-paxillin were followed in time-lapse movies, and at the end of the movies they were fixed and stained for phosphorylated paxillin (Y118). Here, an overlay image showing the two last frames of a movie (last time point in yellow and 5 minutes earlier in blue) is presented alongside a ratio image of the same cell showing the relative level of paxillin phosphorylation in different adhesions. Note the high phosphorylation of FXs at the bottom right corner and dephosphorylated paxillin in FB in the upper left. Enlarged inserts show two growing FA (right), in which paxillin is highly phosphorylated next to three stable adhesions (left) in which it is not. Bar, 5 μm.

• Supplemental Figure 2 -

  Fig. S2. Paxillin phosphorylation mutants change the adhesive phenotype of NIH3T3 fibroblasts. NIH3T3 fibroblasts were transfected with either the phosphomimetic (Y2E) or non-phosphorylatable (Y2F) mutants of YFP-paxillin. Similarly to their effect in PAEC, Y2E-paxillin induced the formation of FX and abolished FB, and Y2F-paxillin had the opposite effect. Bar, 5 μm.

• Supplemental Figure 3 -

  Fig. S3. FAK does not localize to fibrillar adhesions and co-localizes with phospho-paxillin. (A) Double staining for FAK and phospho-paxillin (Y118) shows almost complete co-localization of the two proteins. (B) Double staining of endothelial cells for FAK and tensin demonstrates that FAK is almost completely absent from fibrillar adhesions. Bar, 5 μm.

• Movie 1 -

  Movie 1. Paxillin exits FAs and enters FBs. Movie of YFP-paxillin, taken at 2-minute intervals for 30 minutes, shows paxillin leaving the inner end of a FA and moving along a straight line. This movement, which most likely is along the actin stress fiber, forms a FB.

• Movie 2 -

  Movie 2. Adhesion dynamics of wild-type, phosphomimetic and non-phosphorylatable mutants of YFP-paxillin. One hour long (5-minute interval) time-lapse movies, show the differential dynamics of adhesion structures in wild-type (left) and mutant YFP-paxillin. Note the rapid translocation of FA in the phospho-mimetic (Y2E, middle) mutant and the relative stability of adhesions in the non-phosphorylatable (Y2F, right) mutant.

• Movie 3 -

  Movie 3. FAK increases FA dynamics downstream of paxillin phosphorylation. One hour long (5-minute interval) time-lapse movies show that co-expression of FAK with wild-type paxillin increases FA dynamics (left). Co-expression of FAK with Y2E-paxillin (middle) or Y2F-paxillin (right) does not change the dynamics any further.