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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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Dynamics of FAK and paxillin in a live EC transfected with GFP-FAK (green) and mCherry–paxillin (red).(A) FAK and paxillin in the cell. The three boxed regions are cell front (B), center (C) and rear (D), with enlarged views shown in panels (B), (C) and (D), respectively. B–D show the results for FAK, paxillin, and FAK + Paxillin at 0, 30 and 60 min. In these ratio photos, superimposition of GFP-FAK (green) and mCherry-paxillin (red) images appears as yellow. During the 60-min of cell migration, there are many more GFP-FAK clusters than mCherry-paxillin at cell front (B), but they are more similar in numbers at cell center (C), and rear (D). The corresponding Supplementary Movies are S4–6 for B, S7 for C, and S8 D. Scale bars: A = 20 μm, B = 15 μm, C = 4.5 μm and D = 12 μm. (E) FI ratio images of FAK/paxillin in the same cell as 1A at 0 (left) and 60 min (right). The cell contour at 60 min is outlined in solid white, whereas the initial cell contour (0 min) is outlined with dashed white. Pseudo-color for the FAK/paxillin FI ratio value ranges from low (blue) to high (red) (ROIs in both panels are cyan for cell front, green for center, and pink for rear) (Supplementary Movie S9). Scale bar = 18 μm. (F) Time courses (over 60-min) of FAK/paxillin FI ratio at the three ROIs as shown in Figs. 1E. FI ratios of FAK/paxillin at cell front, center and rear were obtained from eleven migrating cells, each with seven time points. The bar graphs represent mean ± s.e.m. (F1) ROIs moving with the cell migration. (F2) ROIs staying at fixed positions (0-time).
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f1: Dynamics of FAK and paxillin in a live EC transfected with GFP-FAK (green) and mCherry–paxillin (red).(A) FAK and paxillin in the cell. The three boxed regions are cell front (B), center (C) and rear (D), with enlarged views shown in panels (B), (C) and (D), respectively. B–D show the results for FAK, paxillin, and FAK + Paxillin at 0, 30 and 60 min. In these ratio photos, superimposition of GFP-FAK (green) and mCherry-paxillin (red) images appears as yellow. During the 60-min of cell migration, there are many more GFP-FAK clusters than mCherry-paxillin at cell front (B), but they are more similar in numbers at cell center (C), and rear (D). The corresponding Supplementary Movies are S4–6 for B, S7 for C, and S8 D. Scale bars: A = 20 μm, B = 15 μm, C = 4.5 μm and D = 12 μm. (E) FI ratio images of FAK/paxillin in the same cell as 1A at 0 (left) and 60 min (right). The cell contour at 60 min is outlined in solid white, whereas the initial cell contour (0 min) is outlined with dashed white. Pseudo-color for the FAK/paxillin FI ratio value ranges from low (blue) to high (red) (ROIs in both panels are cyan for cell front, green for center, and pink for rear) (Supplementary Movie S9). Scale bar = 18 μm. (F) Time courses (over 60-min) of FAK/paxillin FI ratio at the three ROIs as shown in Figs. 1E. FI ratios of FAK/paxillin at cell front, center and rear were obtained from eleven migrating cells, each with seven time points. The bar graphs represent mean ± s.e.m. (F1) ROIs moving with the cell migration. (F2) ROIs staying at fixed positions (0-time).

Mentions: Fig. 1A shows the image of a cell with FAK- and paxillin-FAs. The boxed areas B (cell front), C (cell center) and D (cell rear) are magnified in Figs. 1B, 1C, and 1D, respectively; each of these magnified photo panels show the images for FAK, paxillin and their FI ratio at 0, 30, and 60 min. The results indicate that the dynamics of FAK-FAs and paxillin-FAs are different at the front (Fig. 1B; Supplementary Movie S4–6), center (Fig. 1C; Supplementary Movie S7), and rear of the cell (Fig. 1D; Supplementary Movie S8). Fig. 1E shows the FAK/paxillin FI ratio (pseudocolored) in this cell (same cell as 1A) at 0 (left) and 60 min (right) with regions of interest (ROIs) identified and tracked along the cell movement. The FIs of both FAK and paxillin were quantified at the three ROIs (i.e., cell front, center, and rear) of eleven migrating cells. The 60-min time course of the FAK/paxillin FI ratio in the three ROIs is expressed in two ways: In Fig. 1F1, the ROIs being tracked and measured are moved with the cell, whereas in Fig. 1F2 the ROIs are fixed at their 0-time positions in the field of view without moving with the cells. All results are normalized to the 0-min value at cell center for each cell. When the ROIs are moved with the cell (Fig. 1F1), the ratio values in all three regions do not vary significantly with time; the ratio is > 1 at cell front (mean ± s.e.m.; 3.79 ± 0.15), ≈1 at cell center (0.88 ± 0.04), and < 1 at cell rear (0.56 ± 0.02). When the ROI is fixed at 0-time position (Fig. 1F2), however, the FI ratio at the initial cell front decreases as cell moves forward, but there are little changes in the ROIs fixed at initial cell center and rear. These data show that the intensity of FAK-FAs is much higher than paxillin-FAs at the protrusive region of cell front. At cell center and rear, there are significantly less differences between FAK and paxillin at FAs in migrating cells. Such analysis was performed on eleven cells (Fig. 1F), and the results confirmed our visual observations that the FAK/paxillin FI ratio was highest at cell front, intermediate at cell center, and lowest at cell rear.


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)

Dynamics of FAK and paxillin in a live EC transfected with GFP-FAK (green) and mCherry–paxillin (red).(A) FAK and paxillin in the cell. The three boxed regions are cell front (B), center (C) and rear (D), with enlarged views shown in panels (B), (C) and (D), respectively. B–D show the results for FAK, paxillin, and FAK + Paxillin at 0, 30 and 60 min. In these ratio photos, superimposition of GFP-FAK (green) and mCherry-paxillin (red) images appears as yellow. During the 60-min of cell migration, there are many more GFP-FAK clusters than mCherry-paxillin at cell front (B), but they are more similar in numbers at cell center (C), and rear (D). The corresponding Supplementary Movies are S4–6 for B, S7 for C, and S8 D. Scale bars: A = 20 μm, B = 15 μm, C = 4.5 μm and D = 12 μm. (E) FI ratio images of FAK/paxillin in the same cell as 1A at 0 (left) and 60 min (right). The cell contour at 60 min is outlined in solid white, whereas the initial cell contour (0 min) is outlined with dashed white. Pseudo-color for the FAK/paxillin FI ratio value ranges from low (blue) to high (red) (ROIs in both panels are cyan for cell front, green for center, and pink for rear) (Supplementary Movie S9). Scale bar = 18 μm. (F) Time courses (over 60-min) of FAK/paxillin FI ratio at the three ROIs as shown in Figs. 1E. FI ratios of FAK/paxillin at cell front, center and rear were obtained from eleven migrating cells, each with seven time points. The bar graphs represent mean ± s.e.m. (F1) ROIs moving with the cell migration. (F2) ROIs staying at fixed positions (0-time).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Dynamics of FAK and paxillin in a live EC transfected with GFP-FAK (green) and mCherry–paxillin (red).(A) FAK and paxillin in the cell. The three boxed regions are cell front (B), center (C) and rear (D), with enlarged views shown in panels (B), (C) and (D), respectively. B–D show the results for FAK, paxillin, and FAK + Paxillin at 0, 30 and 60 min. In these ratio photos, superimposition of GFP-FAK (green) and mCherry-paxillin (red) images appears as yellow. During the 60-min of cell migration, there are many more GFP-FAK clusters than mCherry-paxillin at cell front (B), but they are more similar in numbers at cell center (C), and rear (D). The corresponding Supplementary Movies are S4–6 for B, S7 for C, and S8 D. Scale bars: A = 20 μm, B = 15 μm, C = 4.5 μm and D = 12 μm. (E) FI ratio images of FAK/paxillin in the same cell as 1A at 0 (left) and 60 min (right). The cell contour at 60 min is outlined in solid white, whereas the initial cell contour (0 min) is outlined with dashed white. Pseudo-color for the FAK/paxillin FI ratio value ranges from low (blue) to high (red) (ROIs in both panels are cyan for cell front, green for center, and pink for rear) (Supplementary Movie S9). Scale bar = 18 μm. (F) Time courses (over 60-min) of FAK/paxillin FI ratio at the three ROIs as shown in Figs. 1E. FI ratios of FAK/paxillin at cell front, center and rear were obtained from eleven migrating cells, each with seven time points. The bar graphs represent mean ± s.e.m. (F1) ROIs moving with the cell migration. (F2) ROIs staying at fixed positions (0-time).
Mentions: Fig. 1A shows the image of a cell with FAK- and paxillin-FAs. The boxed areas B (cell front), C (cell center) and D (cell rear) are magnified in Figs. 1B, 1C, and 1D, respectively; each of these magnified photo panels show the images for FAK, paxillin and their FI ratio at 0, 30, and 60 min. The results indicate that the dynamics of FAK-FAs and paxillin-FAs are different at the front (Fig. 1B; Supplementary Movie S4–6), center (Fig. 1C; Supplementary Movie S7), and rear of the cell (Fig. 1D; Supplementary Movie S8). Fig. 1E shows the FAK/paxillin FI ratio (pseudocolored) in this cell (same cell as 1A) at 0 (left) and 60 min (right) with regions of interest (ROIs) identified and tracked along the cell movement. The FIs of both FAK and paxillin were quantified at the three ROIs (i.e., cell front, center, and rear) of eleven migrating cells. The 60-min time course of the FAK/paxillin FI ratio in the three ROIs is expressed in two ways: In Fig. 1F1, the ROIs being tracked and measured are moved with the cell, whereas in Fig. 1F2 the ROIs are fixed at their 0-time positions in the field of view without moving with the cells. All results are normalized to the 0-min value at cell center for each cell. When the ROIs are moved with the cell (Fig. 1F1), the ratio values in all three regions do not vary significantly with time; the ratio is > 1 at cell front (mean ± s.e.m.; 3.79 ± 0.15), ≈1 at cell center (0.88 ± 0.04), and < 1 at cell rear (0.56 ± 0.02). When the ROI is fixed at 0-time position (Fig. 1F2), however, the FI ratio at the initial cell front decreases as cell moves forward, but there are little changes in the ROIs fixed at initial cell center and rear. These data show that the intensity of FAK-FAs is much higher than paxillin-FAs at the protrusive region of cell front. At cell center and rear, there are significantly less differences between FAK and paxillin at FAs in migrating cells. Such analysis was performed on eleven cells (Fig. 1F), and the results confirmed our visual observations that the FAK/paxillin FI ratio was highest at cell front, intermediate at cell center, and lowest at cell rear.

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