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FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells.

Hu YL, Lu S, Szeto KW, Sun J, Wang Y, Lasheras JC, Chien S - Sci Rep (2014)

Bottom Line: The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front.To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation.The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA [2] Institute of Engineering in Medicine University of California, San Diego, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Cell migration requires the fine spatiotemporal integration of many proteins that regulate the fundamental processes that drive cell movement. Focal adhesion (FA) dynamics is a continuous process involving coordination between FA and actin cytoskeleton, which is essential for cell migration. We studied the spatiotemporal relationship between the dynamics of focal adhesion kinase (FAK) and paxillin at FAs in the protrusion of living endothelial cells. Concurrent dual-color imaging showed that FAK was assembled at FA first, which was followed by paxillin recruitment to the FA. By tracking and quantifying FAK and paxillin in migrating cells, the normalized FAK/Paxillin fluorescence intensity (FI) ratio is > 1 (≈ 4 fold) at cell front, ≈ 1 at cell center, and < 1 at cell rear. The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front. To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation. The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front.

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The results of time correlation analysis of independent experiments on thirteen migrating cells (206 individual FAs) yielded a time shift of 2.62 ± 0.27 min (mean ± s.e.m.) between GFP-FAK and mCherry-paxillin, which is significantly different from 0 (n = 206, p < 0.001).
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f4: The results of time correlation analysis of independent experiments on thirteen migrating cells (206 individual FAs) yielded a time shift of 2.62 ± 0.27 min (mean ± s.e.m.) between GFP-FAK and mCherry-paxillin, which is significantly different from 0 (n = 206, p < 0.001).

Mentions: To determine the time difference between the assemblies of FAK and paxillin at nascent FAs at cell front, the image series of thirteen migrating cells were measured and analyzed (Fig. 3A), including the cell reported in Figs. 1 and 2. The FAK and paxillin images were captured simultaneously at each time point for the quantification of time differences between FAK and paxillin dynamics at nascent (assembling) FAs in cell front. A pair of image frames in which FAK and paxillin attained their maximum values at each FA (labeled with numbers in Fig. 3B) were selected and identified as the final frames (Fig. 3B), and the FAK and paxillin intensities at the FA were tracked backward from the final frames. The pair of frames that attained the minimum values of FAK and paxillin were selected as their respective initial frames. Fig. 3C shows the time courses of the FI values of FAK and paxillin for four of these FAs (No. 4, 6, 15, and 20 in Fig. 3B). Time correlation analysis for these four and all the other FAs marked in 3B was used to deduce the time shift between FAK (green) and paxillin (red) at each of the 22 FAs marked, as well as the mean curve for all 22 FAs in that same cell (Fig. 3D). Fig. 4 shows the combined results of such analysis on all thirteen cells, with a total of 206 FAs. The results indicate that FAK is assembled ahead of paxillin in the nascent FAs at the cell front, leading by 2.62 ± 0.27 min (mean ± s.e.m.).


FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells.

Hu YL, Lu S, Szeto KW, Sun J, Wang Y, Lasheras JC, Chien S - Sci Rep (2014)

The results of time correlation analysis of independent experiments on thirteen migrating cells (206 individual FAs) yielded a time shift of 2.62 ± 0.27 min (mean ± s.e.m.) between GFP-FAK and mCherry-paxillin, which is significantly different from 0 (n = 206, p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4129417&req=5

f4: The results of time correlation analysis of independent experiments on thirteen migrating cells (206 individual FAs) yielded a time shift of 2.62 ± 0.27 min (mean ± s.e.m.) between GFP-FAK and mCherry-paxillin, which is significantly different from 0 (n = 206, p < 0.001).
Mentions: To determine the time difference between the assemblies of FAK and paxillin at nascent FAs at cell front, the image series of thirteen migrating cells were measured and analyzed (Fig. 3A), including the cell reported in Figs. 1 and 2. The FAK and paxillin images were captured simultaneously at each time point for the quantification of time differences between FAK and paxillin dynamics at nascent (assembling) FAs in cell front. A pair of image frames in which FAK and paxillin attained their maximum values at each FA (labeled with numbers in Fig. 3B) were selected and identified as the final frames (Fig. 3B), and the FAK and paxillin intensities at the FA were tracked backward from the final frames. The pair of frames that attained the minimum values of FAK and paxillin were selected as their respective initial frames. Fig. 3C shows the time courses of the FI values of FAK and paxillin for four of these FAs (No. 4, 6, 15, and 20 in Fig. 3B). Time correlation analysis for these four and all the other FAs marked in 3B was used to deduce the time shift between FAK (green) and paxillin (red) at each of the 22 FAs marked, as well as the mean curve for all 22 FAs in that same cell (Fig. 3D). Fig. 4 shows the combined results of such analysis on all thirteen cells, with a total of 206 FAs. The results indicate that FAK is assembled ahead of paxillin in the nascent FAs at the cell front, leading by 2.62 ± 0.27 min (mean ± s.e.m.).

Bottom Line: The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front.To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation.The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA [2] Institute of Engineering in Medicine University of California, San Diego, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Cell migration requires the fine spatiotemporal integration of many proteins that regulate the fundamental processes that drive cell movement. Focal adhesion (FA) dynamics is a continuous process involving coordination between FA and actin cytoskeleton, which is essential for cell migration. We studied the spatiotemporal relationship between the dynamics of focal adhesion kinase (FAK) and paxillin at FAs in the protrusion of living endothelial cells. Concurrent dual-color imaging showed that FAK was assembled at FA first, which was followed by paxillin recruitment to the FA. By tracking and quantifying FAK and paxillin in migrating cells, the normalized FAK/Paxillin fluorescence intensity (FI) ratio is > 1 (≈ 4 fold) at cell front, ≈ 1 at cell center, and < 1 at cell rear. The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front. To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation. The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front.

Show MeSH
Related in: MedlinePlus