Figure 5.
Ultrafast simultaneous dSTORM (HMSiR-labeled Halo-paxillin) and PALM (mEos3.2-paxillin) in live mEos3.2-paxillin–rescued MEFs reveal that FAs contain paxillin-enriched islands of ≈32 nm in diameter with a median of ≈30 paxillin copies, which are not homogeneously distributed in the FA, but form island-enriched domains (loose island clusters) with a diameter of ≈300 nm.(A) Typical ultrafast dSTORM image of HMSiR-labeled Halo-paxillin expressed on the basal PM of a live MEF (data acquisition at 1 kHz for 10 s, with a total of 10,000 frames) and its expanded image. (B) The Voronoï diagram of the dSTORM image is shown on the left. The contours of the FA (red) and paxillin-enriched islands (green) are shown (see Fig. S2 B). (C) Left: A typical overlaid image of simultaneously recorded ultrafast live-cell dSTORM of HMSiR-labeled Halo-paxillin (magenta) and PALM of mEos3.2-paxillin (green) in an mEos3.2-paxillin-rescued MEF. Data were acquired at 1 kHz for 10 s, with a total of 10,000 frames for both dSTORM and PALM (three figures on the right). The paxillin-enriched islands detected by the Voronoï segmentation analysis of the image on the left (the images are superimposed here) exhibited only partial colocalizations of the paxillin-enriched islands, probably due to limited copy numbers of paxillin in the islands. (D) Distributions of the diameters of paxillin-enriched islands obtained from dSTORM and PALM images using the tessellation analysis. The mean diameters were 50 and 58 nm, respectively, but after the correction for single-molecule localization precisions (Fig. S2 C), the true mean diameter of the islands was estimated to be 32 nm for both the dSTORM and PALM results. (E) Distributions of the number of detections (localizations) of mEos3.2- and HMSiR-Halo–linked paxillin molecules per detected paxillin-enriched island. The additional x-axis scale shows the estimated endogenous paxillin copy number per island in the parental MEFs. (F and G) Auto-correlation (F) and cross-correlation (G) functions calculated for all of the fluorescent spots localized in the detected paxillin-enriched islands (polygons; calculated for each FA and the mean ± SEM was obtained). The best-fit functions are shown in color. (H) Schematic FA model, proposing that the FA consists of islands of paxillin (and other FA proteins) with a mean diameter of ≈32 nm, which are not homogeneously distributed in the FA, and instead form loose clusters of ≈300 nm in mean diameter. The FA region outside the paxillin-enriched islands is also probably enriched in paxillin monomers and oligomers (shown by green shading and smaller dots, respectively).

Ultrafast simultaneous dSTORM (HMSiR-labeled Halo-paxillin) and PALM (mEos3.2-paxillin) in live mEos3.2-paxillin–rescued MEFs reveal that FAs contain paxillin-enriched islands of ≈32 nm in diameter with a median of ≈30 paxillin copies, which are not homogeneously distributed in the FA, but form island-enriched domains (loose island clusters) with a diameter of ≈300 nm. (A) Typical ultrafast dSTORM image of HMSiR-labeled Halo-paxillin expressed on the basal PM of a live MEF (data acquisition at 1 kHz for 10 s, with a total of 10,000 frames) and its expanded image. (B) The Voronoï diagram of the dSTORM image is shown on the left. The contours of the FA (red) and paxillin-enriched islands (green) are shown (see Fig. S2 B). (C) Left: A typical overlaid image of simultaneously recorded ultrafast live-cell dSTORM of HMSiR-labeled Halo-paxillin (magenta) and PALM of mEos3.2-paxillin (green) in an mEos3.2-paxillin-rescued MEF. Data were acquired at 1 kHz for 10 s, with a total of 10,000 frames for both dSTORM and PALM (three figures on the right). The paxillin-enriched islands detected by the Voronoï segmentation analysis of the image on the left (the images are superimposed here) exhibited only partial colocalizations of the paxillin-enriched islands, probably due to limited copy numbers of paxillin in the islands. (D) Distributions of the diameters of paxillin-enriched islands obtained from dSTORM and PALM images using the tessellation analysis. The mean diameters were 50 and 58 nm, respectively, but after the correction for single-molecule localization precisions (Fig. S2 C), the true mean diameter of the islands was estimated to be 32 nm for both the dSTORM and PALM results. (E) Distributions of the number of detections (localizations) of mEos3.2- and HMSiR-Halo–linked paxillin molecules per detected paxillin-enriched island. The additional x-axis scale shows the estimated endogenous paxillin copy number per island in the parental MEFs. (F and G) Auto-correlation (F) and cross-correlation (G) functions calculated for all of the fluorescent spots localized in the detected paxillin-enriched islands (polygons; calculated for each FA and the mean ± SEM was obtained). The best-fit functions are shown in color. (H) Schematic FA model, proposing that the FA consists of islands of paxillin (and other FA proteins) with a mean diameter of ≈32 nm, which are not homogeneously distributed in the FA, and instead form loose clusters of ≈300 nm in mean diameter. The FA region outside the paxillin-enriched islands is also probably enriched in paxillin monomers and oligomers (shown by green shading and smaller dots, respectively).

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