Figure 7.
Synchronized paxillin recruitment to loose paxillin-enriched island clusters detected by the live-cell dSTORM. (A and B) Spatially non-homogeneous paxillin recruitment in time found by dSTORM of live MEF cells. Typical dSTORM image sequences of HMSiR-labeled Halo-paxillin on the basal PM of a live MEF (A) and a fixed MEF (B) are shown every 10 s (data acquisition of 2,500 frames at 250 Hz; top rows). The bottom rows show the expanded images of the regions in squares in A superimposed by the contours of FAs (white) and paxillin-enriched islands (green) determined by the Voronoï segmentation analysis. For the circles in the expanded images of the live MEF in A, see the legend of C. (C) The bottom-left image in A (0–10-s expanded image superimposed by the Voronoï diagram) is further expanded. Here, we placed seven 250-nm diameter circles, which are the plausible loose clusters of paxillin-enriched islands. These seven circles are included in the image sequences in the bottom row in A to show the spatially heterogeneous time-dependent changes of the local fluorescent paxillin concentration. Even outside of the detected paxillin-enriched islands, the FA is enriched in paxillin, which might exist in islands enriched in other FA proteins and/or as smaller paxillin oligomers and monomers. (D) Paxillin recruitment might take place synchronously in each loose cluster, suggesting that the loose island cluster functions as the unit for recruiting paxillin. The figure shows the time-dependent changes of the percentages of paxillin localizations in each circle (loose cluster) shown in C, relative to the total paxillin localizations, during each 10-s sequence in the entire FA. The number of each graph refers to each numbered circle in C. (E and F) Recruited single paxillin molecules frequently undergo rapid movements on the FA membrane, which might induce synchronous paxillin recruitment to paxillin-enriched islands within the loose island cluster. Compare the results obtained in live (E) and fixed (F) MEF cells. The trajectories in the figure show those of newly detected paxillin molecules of four frames and longer (for ≥16 ms; data acquisition at 250 Hz) observed during a 2-s dSTORM data acquisition period (500 frames) to obtain the superimposed 0- to 10-s dSTORM images, which are the same as those shown in A and B (images on the extreme left). The trajectory color is changed every 0.5 s in the 2-s image frame sequence.

Synchronized paxillin recruitment to loose paxillin-enriched island clusters detected by the live-cell dSTORM. (A and B) Spatially non-homogeneous paxillin recruitment in time found by dSTORM of live MEF cells. Typical dSTORM image sequences of HMSiR-labeled Halo-paxillin on the basal PM of a live MEF (A) and a fixed MEF (B) are shown every 10 s (data acquisition of 2,500 frames at 250 Hz; top rows). The bottom rows show the expanded images of the regions in squares in A superimposed by the contours of FAs (white) and paxillin-enriched islands (green) determined by the Voronoï segmentation analysis. For the circles in the expanded images of the live MEF in A, see the legend of C. (C) The bottom-left image in A (0–10-s expanded image superimposed by the Voronoï diagram) is further expanded. Here, we placed seven 250-nm diameter circles, which are the plausible loose clusters of paxillin-enriched islands. These seven circles are included in the image sequences in the bottom row in A to show the spatially heterogeneous time-dependent changes of the local fluorescent paxillin concentration. Even outside of the detected paxillin-enriched islands, the FA is enriched in paxillin, which might exist in islands enriched in other FA proteins and/or as smaller paxillin oligomers and monomers. (D) Paxillin recruitment might take place synchronously in each loose cluster, suggesting that the loose island cluster functions as the unit for recruiting paxillin. The figure shows the time-dependent changes of the percentages of paxillin localizations in each circle (loose cluster) shown in C, relative to the total paxillin localizations, during each 10-s sequence in the entire FA. The number of each graph refers to each numbered circle in C. (E and F) Recruited single paxillin molecules frequently undergo rapid movements on the FA membrane, which might induce synchronous paxillin recruitment to paxillin-enriched islands within the loose island cluster. Compare the results obtained in live (E) and fixed (F) MEF cells. The trajectories in the figure show those of newly detected paxillin molecules of four frames and longer (for ≥16 ms; data acquisition at 250 Hz) observed during a 2-s dSTORM data acquisition period (500 frames) to obtain the superimposed 0- to 10-s dSTORM images, which are the same as those shown in A and B (images on the extreme left). The trajectory color is changed every 0.5 s in the 2-s image frame sequence.

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