Endogenous and exogenous paxillin, Hic-5 and vinculin colocalize in integrin-mediated adhesions of varying size.

<p>(A) Immunofluorescence images of NIH 3T3 cells spread on 2D fibronectin for 4hrs and stained for paxillin (top panels), Hic-5 (middle panels) and vinculin (bottom panels). Masks indicate that the endogenous proteins localize to all adhesions (<1 μm² to >10 μm²). Blue line indicates the cell edge as determined by F-actin staining. Representative images of cells co-expressing (B) mRFP-paxillin and (C) mRFP-Hic-5 with vinculin-YFP. Line profiles indicate fluorophore-tagged protein colocalization in all adhesion structures. (D) Representative image and line profiles of mRFP-paxillin and zyxin-YFP colocalization in adhesions. (E) Average Pearson's Correlation analyses indicate a high level of colocalization of exogenous proteins. Error bars are standard errors of the mean.</p

Paxillin is immobilized in adhesions upon Rac1 inhibition through an increased interaction with actopaxin.

<p>(A) Representative images of YFP-actopaxin-expressing NIH 3T3 cells used for FRAP analyses. YFP-actopaxin exhibits robust adhesion localization but a reduction in non-adhesion localization upon Rac1 inhibition. (B) Compiled FRAP recovery curves for YFP-actopaxin in the presence of wtRac1 or N17Rac1. Error bars represent standard error of the mean and n = 3 individual experiments. (C) Immobile fraction quantitation for YFP-actopaxin with wtRac1, N17Rac1 and ±50 μM Rac1inhibitor. Error bars are standard error of the mean from a minimum of n = 3 individual experiments. (D) Images and (E) quantitation of YFP-actopaxin and mRFP-paxillin FRET in adhesion contacts upon Rac1 inhibition. Error bars are standard error of the mean and are from all adhesions from a minimum of 11 cells from 3 individual experiments. * = p<0.05.</p

Rac1 inhibition specifically affects the dynamics of paxillin in adhesions.

<p>(A) Images of FRAP time series of GFP-paxillin-expressing NIH 3T3 cells ±50 μM Rac1 inhibitor. Inset are pseudo-colored images of the adhesions undergoing FRAP analysis highlighting a reduction in the GFP-paxillin recovery. (B) Compiled fluorescence recovery curves for GFP-paxillin in adhesions from n = 3 individual experiments ± 50μM Rac1 inhibitor and (C) with wtRac1 or N17Rac1. (D) Quantitation of the t1/2 of fluorescence recovery and immobile fraction for GFP-paxillin and vinculin-YFP ±50 μM Rac1inhibitor. (E) Quantitation of the t1/2 of fluorescence recovery and immobile fraction for GFP-paxillin and vinculin-YFP with wtRac1 or N17Rac1. FRAP analyses reveal a significant increase in the immobile fraction of GFP-paxillin, but not vinculin-YFP in cells with decreased Rac1 activity. Error bars are standard error of the mean. * = p<0.05.</p

Paxillin and Hic-5 are within FRET proximity of vinculin in adhesions.

<p>(A) Raw images of NIH 3T3 cells expressing the donor (vinculin-YFP) and acceptor (mRFP-paxillin) pair used for FRET calculations before and after acceptor photobleaching. The lower panel images highlight the complete photobleaching of the acceptor protein. (B) Mask image of the vinculin-YFP image indicating the variation of adhesion contact area. (C) FRET efficiency image of mRFP-paxillin and vinculin-YFP with zoomed images, indicating FRET in both small and large adhesions. (D) Raw images of NIH 3T3 cells expressing the donor (vinculin-YFP) and acceptor (mRFP-Hic-5) pair used for FRET calculations. (E) FRET efficiency image of mRFP-Hic-5 and vinculin-YFP with zoomed images indicating FRET in both small and large adhesions. (F) Quantitation of average percentage FRET efficiency of indicated FRET pairs in adhesions with small, focal complex-like areas (<1 μm²) and larger more mature adhesions (>1 μm²). Error bars are standard errors of the mean and are compiled from analysis of all adhesions from a minimum of 9 cells from 3 individual experiments (measurements were made from 450 to 2500 individual adhesions). Statistical analyses are relative to the mRFP and vinculin-YFP FRET control unless otherwise indicated, *  = p<0.05 and *** = p<0.0005. (G) Representative image of a vinculin-YFP-expressing NIH 3T3 cell with adhesions of (a) <1 μm² and (b) 1 to 10 μm² indicating an increase in measured FRET efficiency between mRFP-paxillin and vinculin-YFP in the smaller focal complex-like adhesions. Dashed blue line indicates the cell edge.</p

Rac1 inhibition or RhoA activation promotes mRFP-Hic-5 and vinculin-YFP FRET in adhesions.

<p>(A) Western blots of donor and myc-tagged Rac1 and RhoA mutant expression. (B) Representative images of mRFP-Hic-5 and vinculin-YFP FRET in adhesions of cells expressing wtRac1, N17Rac1, V14RhoA or V12Rac1 constructs. FRET image zooms highlight representative FRET pattern of cells as indicated. (C) Quantitation of the FRET efficiency between mRFP-Hic-5 and vinculin-YFP in all adhesions and (D) average adhesion area in the presence of Rac1/RhoA activation mutants. Error bars represent standard error of the mean and are calculated from a minimum of 10 cells from 3 individual experiments. (E) Quantitation of the FRET efficiency between mRFP-Hic-5 and vinculin-YFP and (F) average adhesion area in the presence of 50 μM Rac1 inhibitor. Error bars are standard error of the mean from a minimum of 13 cells from 4 individual experiments. * = p<0.05, ** = p<0.005 and *** = p<0.0005. (G) Western blots of vinculin-YFP and Hic-5 coimmunoprecipitation. A small but consistent increase in the interaction of vinculin-YFP with both endogenous and fluorophore-tagged Hic-5 was seen upon Rac1 inhibition. Data is representative of n = 4 individual experiments. (H) Images and line profile of mRFP-Hic-5 localization to stress fibers upon Rac1 inhibition.</p

Vinculin activation state spatially regulates its interaction with paxillin and Hic-5.

<p>(A) Schematic of vinculin highlighting the distinct domains and associated binding partners as well as mutant constructs used for FRET experiments. (B) Western blot of vinculin-YFP (FRET donor) mutant protein expression in mRFP-paxillin-expressing NIH 3T3 cells. (C) Representative images and (D) quantitation of vinculin-YFP activity mutant's FRET efficiency with mRFP-paxillin. Error bars are standard error of the mean from a minimum of 12 cells from 3 individual experiments. Statistical analyses are relative to the vinculin880-YFP mutant, which lacks the paxillin interacting domain. (E) Western blot of vinculin-YFP (FRET donor) mutant protein expression in mRFP-Hic-5-expressing NIH 3T3 cells. (F) Quantitation and (G) representative images of mRFP-Hic-5 FRET with vinculin-YFP mutants. * = p<0.05 and *** = p<0.0005.</p

Vinculin preferentially interacts with Hic-5 rather than paxillin in 3D adhesion contacts.

<p>(A) Images of NIH 3T3 cells spread on 2D fibronectin (FN) or 3D cell-derived matrix (CDM) for 4hrs stained for paxillin/Hic-5 (green), fibronectin (blue) and F-actin (red). (B) Average Pearson's Correlation analyses of mRFP-paxillin or mRFP-Hic-5 with vinculin-YFP in 3D adhesions reveals no significant difference in colocalization. (C) Quantitation and representative images of (D) mRFP-paxillin and (E) mRFP-Hic-5 FRET efficiency with vinculin-YFP in 3D adhesions demonstrating FRET between vinculin-YFP and mRFP-Hic-5 but not mRFP-paxillin in 3D adhesions. Error bars are standard error of the mean and are calculated from a minimum of 15 cells from 3 individual experiments.</p

The interaction between paxillin and vinculin in adhesions is spatially regulated by the activity of the RhoGTPases.

<p>(A) Western blot of vinculin-YFP and myc-tagged Rac1 and RhoA mutant expression in NIH 3T3 cells used for FRET analyses. (B) Representative donor (vinculin-YFP) and FRET efficiency images indicating that in the presence of wild type (wt) or active (V12Rac1) Rac1, paxillin and vinculin are within FRET proximity. In contrast, inhibition of Rac1 (N17Rac1) or activation of RhoA (V14RhoA) results in a loss of adhesion-localized FRET. FRET image zooms highlight representative FRET pattern of cells as indicated. (C) Quantitation of average FRET efficiency of mRFP-paxillin and vinculin-YFP in all adhesions of cells expressing activation mutants of Rac1 and RhoA. (D) Adhesion area calculations for cells expressing mRFP-paxillin and vinculin-YFP with indicated Rac1 and RhoA mutants. Expression of the active V12Rac1 mutant promotes the formation of smaller adhesions, while the dominant negative N17Rac1 or active V14RhoA constructs induce an increase in adhesion area. Error bars are standard error of the mean from cells used for FRET calculations. (E) Quantitation of average FRET efficiency of mRFP-paxillin and vinculin-YFP in all adhesions of cells in the presence of vehicle (dH₂0) or 50 μM Rac1 inhibitor (NSC23766). (F) Quantitation of average adhesion area in cells treated with vehicle or 50 μM Rac1 inhibitor. Error bars are standard error of the mean and values calculated from all adhesions from a minimum of 10 cells from 3 individual experiments. * = p<0.05, ** = p<0.005 and *** = p<0.0005. (G) Representative images of NIH 3T3 cells displaying increased vinculin-YFP cytosol/membrane localization indicating positive FRET in both adhesion contacts and areas outside integrin-mediated adhesions. Treatment with 50 μM Rac1 inhibitor decreased FRET in adhesion contacts with cytosolic/membrane FRET still observed. White lines highlight vinculin-YFP-positive adhesion areas. (H) Western blots indicating an increase in endogenous vinculin recruitment to the membrane-enriched fraction of cells treated with 50 μM Rac1 inhibitor. (I) Representative Western blots indicating no effect of the Rac1 inhibitor on the ability of endogenous paxillin and vinculin to coimmunoprecipitate, n = 3 individual experiments.</p

LIM Domains Target Actin Regulators Paxillin and Zyxin to Sites of Stress Fiber Strain

<div><p>Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber. Previous work showed the LIM-domain adaptor protein, zyxin, is essential for repair and stabilization of these sites. Using live imaging, we show paxillin, another LIM-domain adaptor protein, is also recruited to stress fiber strain sites. Paxillin recruitment to stress fiber strain sites precedes zyxin recruitment. Zyxin and paxillin are each recruited independently of the other. In cells lacking paxillin, actin recovery is abrogated, resulting in slowed actin recovery and increased incidence of catastrophic stress fiber breaks. For both paxillin and zyxin, the LIM domains are necessary and sufficient for recruitment. This work provides further evidence of the critical role of LIM-domain proteins in responding to mechanical stress in the actin cytoskeleton. </p> </div

Paxillin and zyxin do not require the other for recruitment to stress fiber strain sites.

<p>Kymograph (A) and average intensity plot (B) of paxillin-GFP recruitment and actin thinning at stress fiber strain sites in zyxin-/- fibroblasts showing paxillin recruitment in the absence of zyxin. The rate of paxillin recruitment at strain sites (C) is faster in cells lacking zyxin when compared to zyxin rescued cells. Kymograph (D) and intensity plot (E) of zyxin-GFP recruitment and actin thinning at stress fiber strain sites in paxillin-/- fibroblasts showing zyxin recruitment occurs in the absence of paxillin. The rate of zyxin recruitment (F) is not significantly different in paxillin-/- cells when compared to paxillin rescued cells.</p