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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 immunostaining images of an EC stained with FAK, paxillin and actin filaments.(A) The fluorescence photomicrographs show FAK immunostained with anti-FAK (left column), F-actin stained with rhodamine-phalloidin (middle column), and merging of FAK (green) and F-actin (red) images, with a yellow color for co-localization of FAK and actin filaments (right column). FAK and actin filaments are shown to be co-localized. FAK plaques are present at the points where stress fibers end and some FAK plaques are present at cell periphery (arrows). Actin filaments show staining weakly (up arrow). Scale bar = 6 μm. (B) Fluorescence photomicrographs show FAK (left column) and paxillin (middle column) immunfluorescence stained with anti-FAK and anti-paxillin antibodies, respectively. Merging of FAK (green) and paxillin (red) images (right column), with a yellow color for co-localization of FAK and paxillin. At the cell periphery, there are many more FAK plaques than paxillin plaques (arrows). Scale bar = 10 μm.
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f6: The immunostaining images of an EC stained with FAK, paxillin and actin filaments.(A) The fluorescence photomicrographs show FAK immunostained with anti-FAK (left column), F-actin stained with rhodamine-phalloidin (middle column), and merging of FAK (green) and F-actin (red) images, with a yellow color for co-localization of FAK and actin filaments (right column). FAK and actin filaments are shown to be co-localized. FAK plaques are present at the points where stress fibers end and some FAK plaques are present at cell periphery (arrows). Actin filaments show staining weakly (up arrow). Scale bar = 6 μm. (B) Fluorescence photomicrographs show FAK (left column) and paxillin (middle column) immunfluorescence stained with anti-FAK and anti-paxillin antibodies, respectively. Merging of FAK (green) and paxillin (red) images (right column), with a yellow color for co-localization of FAK and paxillin. At the cell periphery, there are many more FAK plaques than paxillin plaques (arrows). Scale bar = 10 μm.

Mentions: To validate the results obtained on live cells using exogenous fusion proteins of FAK and actin filaments, we applied laser scanning confocal microscopy to visualize the endogenous FAK and actin filaments localization in fixed cells. Fig. 6A shows the fluorescence images of FAK and actin filaments, as well as their merged images. These pictures show the presence of FAK immunostaining as plaques in the fixed cell at cell periphery (arrows). Actin filaments show strong staining with the formation of extensive arrays of stress fibers (SFs), with weak staining at cell periphery. In the merged pictures, FAK spots are positioned at the ends of the stress fibers, and some FAK spots are positioned at the cell periphery with actin filaments (arrows). At the cell periphery, actn filament staining is very weak (up arrow). These immunostaining results are consistent with the live cell images (Fig. 5; Supplementary Movies S16), showing GFP-FAK may be associated with actin filaments at the cell leading edge.


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 immunostaining images of an EC stained with FAK, paxillin and actin filaments.(A) The fluorescence photomicrographs show FAK immunostained with anti-FAK (left column), F-actin stained with rhodamine-phalloidin (middle column), and merging of FAK (green) and F-actin (red) images, with a yellow color for co-localization of FAK and actin filaments (right column). FAK and actin filaments are shown to be co-localized. FAK plaques are present at the points where stress fibers end and some FAK plaques are present at cell periphery (arrows). Actin filaments show staining weakly (up arrow). Scale bar = 6 μm. (B) Fluorescence photomicrographs show FAK (left column) and paxillin (middle column) immunfluorescence stained with anti-FAK and anti-paxillin antibodies, respectively. Merging of FAK (green) and paxillin (red) images (right column), with a yellow color for co-localization of FAK and paxillin. At the cell periphery, there are many more FAK plaques than paxillin plaques (arrows). Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The immunostaining images of an EC stained with FAK, paxillin and actin filaments.(A) The fluorescence photomicrographs show FAK immunostained with anti-FAK (left column), F-actin stained with rhodamine-phalloidin (middle column), and merging of FAK (green) and F-actin (red) images, with a yellow color for co-localization of FAK and actin filaments (right column). FAK and actin filaments are shown to be co-localized. FAK plaques are present at the points where stress fibers end and some FAK plaques are present at cell periphery (arrows). Actin filaments show staining weakly (up arrow). Scale bar = 6 μm. (B) Fluorescence photomicrographs show FAK (left column) and paxillin (middle column) immunfluorescence stained with anti-FAK and anti-paxillin antibodies, respectively. Merging of FAK (green) and paxillin (red) images (right column), with a yellow color for co-localization of FAK and paxillin. At the cell periphery, there are many more FAK plaques than paxillin plaques (arrows). Scale bar = 10 μm.
Mentions: To validate the results obtained on live cells using exogenous fusion proteins of FAK and actin filaments, we applied laser scanning confocal microscopy to visualize the endogenous FAK and actin filaments localization in fixed cells. Fig. 6A shows the fluorescence images of FAK and actin filaments, as well as their merged images. These pictures show the presence of FAK immunostaining as plaques in the fixed cell at cell periphery (arrows). Actin filaments show strong staining with the formation of extensive arrays of stress fibers (SFs), with weak staining at cell periphery. In the merged pictures, FAK spots are positioned at the ends of the stress fibers, and some FAK spots are positioned at the cell periphery with actin filaments (arrows). At the cell periphery, actn filament staining is very weak (up arrow). These immunostaining results are consistent with the live cell images (Fig. 5; Supplementary Movies S16), showing GFP-FAK may be associated with actin filaments at the cell leading edge.

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