Limits...
Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed.

Gardel ML, Sabass B, Ji L, Danuser G, Schwarz US, Waterman CM - J. Cell Biol. (2008)

Bottom Line: In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress.We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity.Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

Show MeSH

Related in: MedlinePlus

Traction stresses across the cell front. (A) Immunofluorescence of paxillin (red), serine-19–phosphorylated myosin II light chain marking activated myosin II (blue), and phalloiden staining of F-actin (green). Locations of lamellipodium, lamellipodium base, and lamella are indicated; distal and proximal directions are defined. (B) GFP-paxillin (inverted contrast) with traction stress vectors superimposed (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1). (C) Heat-scale plot of traction stress magnitude; segmented FAs indicated by black outlines (Video 2). White lines delineate boundaries between lamellipodium (LP), FAs, and cell body (CB). (D) Box plots of traction stresses in the lamellipodium, FAs, cell body, and background (BG) measured directly outside the cell. Box and whisker plots in all figures indicate the 25% (lower bound), median (middle line), and 75% (upper bound) nearest observations within 1.5 times the interquartile range (whiskers), 95% confidence interval of the median (notches) and near (+) and far (0) outliers. (E) FSM image of x-rhodamine F-actin with F-actin velocity vectors superimposed (Video 1). (F) Box plots of F-actin speed in the lamellipodium (LP), FAs, and cell body (CB). (G) Histograms of the F-actin speed across the entire cell front (left) and in areas of highest (>95%) traction stresses (right). (H) Heat scale plot of directional coupling, cosine of angle θ, between F-actin velocity and traction stress vectors; segmented FAs indicated by black outlines. Bars: (A) 10 μm; (B, C, and E) 3 μm; (H) 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2600750&req=5

fig1: Traction stresses across the cell front. (A) Immunofluorescence of paxillin (red), serine-19–phosphorylated myosin II light chain marking activated myosin II (blue), and phalloiden staining of F-actin (green). Locations of lamellipodium, lamellipodium base, and lamella are indicated; distal and proximal directions are defined. (B) GFP-paxillin (inverted contrast) with traction stress vectors superimposed (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1). (C) Heat-scale plot of traction stress magnitude; segmented FAs indicated by black outlines (Video 2). White lines delineate boundaries between lamellipodium (LP), FAs, and cell body (CB). (D) Box plots of traction stresses in the lamellipodium, FAs, cell body, and background (BG) measured directly outside the cell. Box and whisker plots in all figures indicate the 25% (lower bound), median (middle line), and 75% (upper bound) nearest observations within 1.5 times the interquartile range (whiskers), 95% confidence interval of the median (notches) and near (+) and far (0) outliers. (E) FSM image of x-rhodamine F-actin with F-actin velocity vectors superimposed (Video 1). (F) Box plots of F-actin speed in the lamellipodium (LP), FAs, and cell body (CB). (G) Histograms of the F-actin speed across the entire cell front (left) and in areas of highest (>95%) traction stresses (right). (H) Heat scale plot of directional coupling, cosine of angle θ, between F-actin velocity and traction stress vectors; segmented FAs indicated by black outlines. Bars: (A) 10 μm; (B, C, and E) 3 μm; (H) 5 μm.

Mentions: For simultaneous measurement of F-actin dynamics and traction on the ECM at FAs, we developed high-resolution traction force microscopy (Dembo et al., 1996) compatible with quantitative fluorescent speckle microscopy (FSM; Danuser and Waterman-Storer, 2006). We used migrating PtK1 epithelial cells because their F-actin organization and dynamics are well characterized (Fig. 1 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1; Ponti et al., 2004). Cells expressing GFP-fused paxillin (GFP-paxillin) to mark FAs (Webb et al., 2004) and microinjected with x-rhodamine–conjugated actin were plated on 10–20-μm-thick compliant (1.5–6 kPa) fibronectin-coated polyacrylamide (PAA) substrates that were embedded with a high density of far-red fluorescent 40-nm spheres (∼10 spheres/μm2). Time-lapse images of GFP-paxillin, x-rhodamine–actin, and spheres were acquired by spinning-disk confocal microscopy (Video 1). Cellular traction stress (force/area) vector fields were reconstructed from cell-induced bead displacements using either boundary element or Fourier transform traction cytometry methods to provide ∼1.5-μm spatial resolution (Fig. 1 B and Video 2; Sabass et al., 2008).


Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed.

Gardel ML, Sabass B, Ji L, Danuser G, Schwarz US, Waterman CM - J. Cell Biol. (2008)

Traction stresses across the cell front. (A) Immunofluorescence of paxillin (red), serine-19–phosphorylated myosin II light chain marking activated myosin II (blue), and phalloiden staining of F-actin (green). Locations of lamellipodium, lamellipodium base, and lamella are indicated; distal and proximal directions are defined. (B) GFP-paxillin (inverted contrast) with traction stress vectors superimposed (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1). (C) Heat-scale plot of traction stress magnitude; segmented FAs indicated by black outlines (Video 2). White lines delineate boundaries between lamellipodium (LP), FAs, and cell body (CB). (D) Box plots of traction stresses in the lamellipodium, FAs, cell body, and background (BG) measured directly outside the cell. Box and whisker plots in all figures indicate the 25% (lower bound), median (middle line), and 75% (upper bound) nearest observations within 1.5 times the interquartile range (whiskers), 95% confidence interval of the median (notches) and near (+) and far (0) outliers. (E) FSM image of x-rhodamine F-actin with F-actin velocity vectors superimposed (Video 1). (F) Box plots of F-actin speed in the lamellipodium (LP), FAs, and cell body (CB). (G) Histograms of the F-actin speed across the entire cell front (left) and in areas of highest (>95%) traction stresses (right). (H) Heat scale plot of directional coupling, cosine of angle θ, between F-actin velocity and traction stress vectors; segmented FAs indicated by black outlines. Bars: (A) 10 μm; (B, C, and E) 3 μm; (H) 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600750&req=5

fig1: Traction stresses across the cell front. (A) Immunofluorescence of paxillin (red), serine-19–phosphorylated myosin II light chain marking activated myosin II (blue), and phalloiden staining of F-actin (green). Locations of lamellipodium, lamellipodium base, and lamella are indicated; distal and proximal directions are defined. (B) GFP-paxillin (inverted contrast) with traction stress vectors superimposed (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1). (C) Heat-scale plot of traction stress magnitude; segmented FAs indicated by black outlines (Video 2). White lines delineate boundaries between lamellipodium (LP), FAs, and cell body (CB). (D) Box plots of traction stresses in the lamellipodium, FAs, cell body, and background (BG) measured directly outside the cell. Box and whisker plots in all figures indicate the 25% (lower bound), median (middle line), and 75% (upper bound) nearest observations within 1.5 times the interquartile range (whiskers), 95% confidence interval of the median (notches) and near (+) and far (0) outliers. (E) FSM image of x-rhodamine F-actin with F-actin velocity vectors superimposed (Video 1). (F) Box plots of F-actin speed in the lamellipodium (LP), FAs, and cell body (CB). (G) Histograms of the F-actin speed across the entire cell front (left) and in areas of highest (>95%) traction stresses (right). (H) Heat scale plot of directional coupling, cosine of angle θ, between F-actin velocity and traction stress vectors; segmented FAs indicated by black outlines. Bars: (A) 10 μm; (B, C, and E) 3 μm; (H) 5 μm.
Mentions: For simultaneous measurement of F-actin dynamics and traction on the ECM at FAs, we developed high-resolution traction force microscopy (Dembo et al., 1996) compatible with quantitative fluorescent speckle microscopy (FSM; Danuser and Waterman-Storer, 2006). We used migrating PtK1 epithelial cells because their F-actin organization and dynamics are well characterized (Fig. 1 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1; Ponti et al., 2004). Cells expressing GFP-fused paxillin (GFP-paxillin) to mark FAs (Webb et al., 2004) and microinjected with x-rhodamine–conjugated actin were plated on 10–20-μm-thick compliant (1.5–6 kPa) fibronectin-coated polyacrylamide (PAA) substrates that were embedded with a high density of far-red fluorescent 40-nm spheres (∼10 spheres/μm2). Time-lapse images of GFP-paxillin, x-rhodamine–actin, and spheres were acquired by spinning-disk confocal microscopy (Video 1). Cellular traction stress (force/area) vector fields were reconstructed from cell-induced bead displacements using either boundary element or Fourier transform traction cytometry methods to provide ∼1.5-μm spatial resolution (Fig. 1 B and Video 2; Sabass et al., 2008).

Bottom Line: In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress.We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity.Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

Show MeSH
Related in: MedlinePlus