Journal: The Journal of Cell Biology

Article Title: Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

doi: 10.1083/jcb.200906012

Figure Lengend Snippet: Vinculin is stably bound in adhesions. EGFP fusion proteins localized to adhesions were subjected to FRAP. (A) Sample fluorescence recovery curves for vinculin, α-actinin, talin 1, paxillin, FAK, and zyxin in single adhesions. Note different x-axis scales in left and right plots; fits are shown as solid lines. (B) Half-times of fluorescence recovery. Means are shown above each plot. Vcl, vinculin; Actn, α-actinin; Pxn, paxillin; Tln, talin 1; Zyx, zyxin. n = number of adhesions.

Article Snippet: The EGFP conjugates of paxillin and paxillin mutants were based on the original sequence for avian paxillin-EGFP ( ) but were generated by synthesis (Blue Heron Biotechnology) and included mutations rendering dead an internal translation site of avian paxillin as described previously ( ; ).

Techniques: Stable Transfection, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

doi: 10.1083/jcb.200906012

Figure Lengend Snippet: 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).

Article Snippet: The EGFP conjugates of paxillin and paxillin mutants were based on the original sequence for avian paxillin-EGFP ( ) but were generated by synthesis (Blue Heron Biotechnology) and included mutations rendering dead an internal translation site of avian paxillin as described previously ( ; ).

Techniques: Control, Fluorescence, Immunostaining, Staining, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

doi: 10.1083/jcb.200906012

Figure Lengend Snippet: Paxillin Y31/118 phosphorylation is sufficient for promoting paxillin–vinculin interaction and labile vinculin recruitment to adhesions. (A) Immunoblot analysis of lysates of untreated (control) and cells treated with 20 µM blebbistatin (Blebb) or with 20 µM blebbistatin and 100 µM Na 3 VO 4 using antibodies specific to paxillin (Pxn), pY31 paxillin, pY397FAK, or FAK. (B) Comparison of cells treated with 20 µM blebbistatin or 20 µM blebbistatin and 100 µM Na 3 VO 4 and immunolabeled with antibodies to paxillin (red) and vinculin (Vcl; green). Bar, 2 µm. (C) Effects of Na 3 VO 4 on vinculin localization in adhesions of blebbistatin-treated cells, shown (also in G and H) as the ratio of mean fluorescence intensities within segmented adhesions of 20 µM blebbistatin-treated cells relative to non–blebbistatin-treated cells in the presence and absence of additional Na 3 VO 4 . The mean fluorescence ratio is shown above each plot. (D) Antipaxillin IPs from lysates of untreated control or cells treated with either 20 µM blebbistatin, 100 µM Na 3 VO 4 , and 20 µM blebbistatin or 100 µM Na 3 VO 4 alone followed by analysis by PAGE and immunoblotting with antibodies to vinculin, paxillin, or PY epitopes. (E–I) EGFP-conjugated paxillin (Pxn-GFP wt) or paxillin bearing mutations of tyrosines 31 and 118 to phenylalanines (Pxn Y31/118F) or glutamic acids (Pxn Y31/118E) were expressed in MEFs and either treated with 20 µM blebbistatin or not. (E) Images of cells expressing EGFP-conjugated paxillin mutants (green) and immunolocalization of vinculin (red) or PY epitopes (P-Tyr; red). Right columns show (also in F) merged, magnified images of the boxed regions Bar, 2 µm. (F) Images of EGFP-conjugated Y31/118E paxillin (green) and immunolocalization of zyxin (Zyx; red) or α-actinin (Actn; red) in cells treated with 20 µM blebbistatin. (G) Effects of blebbistatin on adhesion localization of vinculin and paxillin in cells overexpressing wild type (WT) or mutant (Y31/118E) paxillin-EGFP. (H) Effects of blebbistatin on adhesion localization of zyxin, α-actinin, and paxillin in cells overexpressing Y31/118E paxillin-EGFP. (I) FRAP analysis of mCherry-vinculin. mCherry-vinculin was expressed alone (control) or together with EGFP conjugates of wild-type or Y31/118E paxillin in untreated cells or cells treated with 20 µM blebbistatin (+Blebb), and FRAP was performed of the mCherry vinculin fraction in adhesions. Half-times of mCherry vinculin fluorescence recovery. Means are shown above each plot. n = number of adhesions. (J) Immunoblots of lysates of blebbistatin-treated (B) or untreated (C) cells that had been mock transfected or transfected with the EGFP-paxillin mutants and probed with antibodies to paxillin, pY31 paxillin, or tubulin. (K) Anti-GFP immunoprecipitates from MEFs expressing EGFP-tagged paxillins and either untreated (C) or treated with 20 µM blebbistatin (B) and probed by immunoblotting with antibodies to vinculin and GFP. Quantification of blots is shown below. WB, Western blot. *, P < 0.02. Error bars indicate 95% confidence interval of the mean. (C, G, and H) n = number of blebbistatin-treated cells/number of control cells.

Article Snippet: The EGFP conjugates of paxillin and paxillin mutants were based on the original sequence for avian paxillin-EGFP ( ) but were generated by synthesis (Blue Heron Biotechnology) and included mutations rendering dead an internal translation site of avian paxillin as described previously ( ; ).

Techniques: Phospho-proteomics, Western Blot, Control, Comparison, Immunolabeling, Fluorescence, Expressing, Mutagenesis, Transfection