Figure 1.
Stress fiber strain sites recruit paxillin.
(A) The localization of paxillin is not restricted to focal adhesions. Time lapse micrographs taken at 10 second intervals of live mouse fibroblasts expressing paxillin-mApple and Lifeact-GFP (A and B) or zyxin-GFP and paxillin-mApple (D and E). Kymograph (B) and average intensity plotted over time (C) of boxed regions from kymograph (B) showing recruitment of paxillin and concurrent thinning actin. (D) Still frames showing overlap of paxillin and zyxin at a strain site. Kymographs (E) showing progression of zyxin and paxillin recruitment in the white bracketed region of (D) over time. Intensity plot (F) of the boxed regions on the kymograph. Scale bar=10um.
Figure 2.
Paxillin and zyxin do not require the other for recruitment to stress fiber strain sites.
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.
Figure 3.
Paxillin aids stabilization of strain sites by altering actin dynamics independently of the zyxin.
Comparison of normalized average paxillin and zyxin fluorescent intensity at strain site over time (A). Line scans through the strain site (B) show the spatial distribution of paxillin and zyxin fluorescence intensity (AIU) at strain sites. (C) Comparison of the catastrophic SF break frequency in paxillin null and paxillin rescued cells. (D) The actin recovery rate, as expressed by the slope of a line fit to actin intensity curve, in strain sites that do not break. (E) Western blot showing paxillin RNAi knocked down in zyxin null cells. (F) The actin recovery rate in cells lacking zyxin and with paxillin knock down. (G) Kymograph analysis of fluorescent labeled SF in zyxin null with paxillin knockdown cells. Note the SFSS slowly pull apart without repairing, but they rarely undergo catastrophic breaks within the imaging period.
Figure 4.
Paxillin does not stabilize stress fiber strain sites through integrin signaling.
Micrographs of fixed fibroblasts labeled for paxillin interacting protein FAK in conjunction zyxin-GFP, and Lifeact-mApple (A), and linescans through either a focal adhesion (B), or stress fiber strain site (C). FAK is present in focal adhesions, but is not found at stress fiber strain sites while zyxin localizes to both sites. Micrographs of fixed fibroblasts labeled for phospo-paxillin Y118 in conjunction zyxin-GFP, and Lifeact-mApple (D). Phospho-paxillin Y118 labels focal adhesions (E) but not strain sites (F). Similarly, phospho-tyrosine immunolocalization in conjunction zyxin-GFP, and Lifeact-mApple (G) detected phopho-tyrosine activation at focal adhesions (H), but not at strain sites (I). Scale bar=10um.
Figure 5.
The LIM domains of paxillin and zyxin are necessary and sufficient for recruitment to SFSS.
We generated GFP tagged truncated zyxin, ZyxLO (A) and mApple tagged truncated paxillin, PaxLO (B), containing the LIM domains of each protein. ZyxLO was expressed in zyxin-/- cells along with full length zyxin-mCherry (C). Time lapse imaging of ZyxLO and full-length zyxin with kymograph analysis (D) and average SFSS fluorescent intensity/time (E), show ZyxLO was recruited to SFSS in synchrony with full length zyxin. LIM-domain deleted N-terminal mutant zyxin, ZyxNT (A), did not show any strain site localization (F). PaxLO was expressed in paxillin-/- cells, along with full length paxillin-GFP. When compared to full length paxillin, PaxLO localized less to focal adhesions, and was significantly more concentrated on stress fibers (G). PaxLO was recruited to stress fibers along with paxillin (H and I). LIM-domain deleted PaxNT showed no strain site localization (J). Scale bar=10um.