Figure 4.
Figure 4. Characterization of myosin II dependence of vinculin recruitment to adhesions. (A) Immunolocalization in human foreskin fibroblasts (HFF1) of vinculin (Vcl; green) and paxillin (Pxn; red) in untreated (control) or 20 µM blebbistatin (Blebb)-treated cells. Bar, 10 µm. Merged, magnified images of boxed regions are shown in the third column. Bar, 2 µm. n = number of blebbistatin-treated cells/number of control cells. (B) Effects of blebbistatin on adhesion localization of paxillin and vinculin in the noted cell types expressed as the ratio of mean fluorescence intensities within segmented adhesions of blebbistatin-treated relative to control cells. Mean ratios are shown above each plot. *, P < 0.02. Error bars indicate 95% confidence interval of the mean. (C) Area of individual adhesions from paxillin immunostaining in MEF cells in untreated control cells at specific times after treatment with 20 µM blebbistatin (15, 30, and 60 min) or washout of 20 µM blebbistatin into control media (15, 30, and 60 min w/o). Mean adhesion areas (micrometer squared) are shown next to each box plot. Red symbols indicate the outliers at more than three interquartiles; blue symbols indicate a −95% confidence interval of the mean. n = number of adhesions. (D) Immunolocalization of paxillin (red) and vinculin (green) and fluorescent phalloidin staining of actin filaments in cells after treatment (Treat) for 60 min and washout (blebbistatin w/o) for 15, 30, and 60 min of 20 µM blebbistatin. Merged images are shown in the fourth column, and boxed regions are magnified in the fifth column. Bars: (fourth column) 10 µm; (fifth column) 2 µm. (E) Images from a time-lapse dual-color TIRF series of EGFP-paxillin (GFP-Pxn) and mCherry vinculin (mCherry-Vcl) during adhesion formation and growth in a migrating MEF cell. Time is shown in seconds. Bar, 2 µm. (F) Normalized fluorescent protein intensity (green, paxillin; red, vinculin) in the adhesion shown in E. Horizontal lines show the value of two times the standard deviation of the normalized background fluorescence (2× SD Vcl or Pxn Bckg); note that this is higher for mCherry because it is much dimmer than EGFP. Arrows indicate the time when the intensity rose above these values. The time difference between arrows indicates lag time between the accumulation of EGFP-paxillin and mCherry-vinculin at the adhesion (Pxn-Vcl lag).

Characterization of myosin II dependence of vinculin recruitment to adhesions. (A) Immunolocalization in human foreskin fibroblasts (HFF1) of vinculin (Vcl; green) and paxillin (Pxn; red) in untreated (control) or 20 µM blebbistatin (Blebb)-treated cells. Bar, 10 µm. Merged, magnified images of boxed regions are shown in the third column. Bar, 2 µm. n = number of blebbistatin-treated cells/number of control cells. (B) Effects of blebbistatin on adhesion localization of paxillin and vinculin in the noted cell types expressed as the ratio of mean fluorescence intensities within segmented adhesions of blebbistatin-treated relative to control cells. Mean ratios are shown above each plot. *, P < 0.02. Error bars indicate 95% confidence interval of the mean. (C) Area of individual adhesions from paxillin immunostaining in MEF cells in untreated control cells at specific times after treatment with 20 µM blebbistatin (15, 30, and 60 min) or washout of 20 µM blebbistatin into control media (15, 30, and 60 min w/o). Mean adhesion areas (micrometer squared) are shown next to each box plot. Red symbols indicate the outliers at more than three interquartiles; blue symbols indicate a −95% confidence interval of the mean. n = number of adhesions. (D) Immunolocalization of paxillin (red) and vinculin (green) and fluorescent phalloidin staining of actin filaments in cells after treatment (Treat) for 60 min and washout (blebbistatin w/o) for 15, 30, and 60 min of 20 µM blebbistatin. Merged images are shown in the fourth column, and boxed regions are magnified in the fifth column. Bars: (fourth column) 10 µm; (fifth column) 2 µm. (E) Images from a time-lapse dual-color TIRF series of EGFP-paxillin (GFP-Pxn) and mCherry vinculin (mCherry-Vcl) during adhesion formation and growth in a migrating MEF cell. Time is shown in seconds. Bar, 2 µm. (F) Normalized fluorescent protein intensity (green, paxillin; red, vinculin) in the adhesion shown in E. Horizontal lines show the value of two times the standard deviation of the normalized background fluorescence (2× SD Vcl or Pxn Bckg); note that this is higher for mCherry because it is much dimmer than EGFP. Arrows indicate the time when the intensity rose above these values. The time difference between arrows indicates lag time between the accumulation of EGFP-paxillin and mCherry-vinculin at the adhesion (Pxn-Vcl lag).

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