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Fluctuations of intracellular forces during cell protrusion.

Ji L, Lim J, Danuser G - Nat. Cell Biol. (2008)

Bottom Line: Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s.Maximal F-actin assembly was observed about 20 s after maximal edge advancement.On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
We present a model to estimate intracellular force variations from live-cell images of actin filament (F-actin) flow during protrusion-retraction cycles of epithelial cells in a wound healing response. To establish a mechanistic relationship between force development and cytoskelal dynamics, force fluctuations were correlated with fluctuations in F-actin turnover, flow and F-actin-vinculin coupling. Our analyses suggest that force transmission at focal adhesions requires binding of vinculin to F-actin and integrin (indirectly), which is modulated at the vinculin-integrin but not the vinculin-F-actin interface. Force transmission at focal adhesions is colocalized in space and synchronized in time with transient increases in the boundary force at the cell edge. Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s. Maximal F-actin assembly was observed about 20 s after maximal edge advancement. On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

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Prediction of intracellular forces from F-actin network flow in Fig. 1. (a) Predicted force vectors (cyan: boundary forces; red: domain forces). Polygons I - IV highlight regions with different force characteristics (see text). (b) Flow field (yellow vectors) that would be produced by the predicted forces. Vectors are overlaid onto the color-coded speed map of the measured network flow. Insert: Comparison of the calculated (yellow) and measured (green) flow indicating the noise filtering during force reconstruction. (c) Cone rule to separate the contributions from adhesion and contraction to the predicted domain force. (d) Distribution of angles between predicted force vectors and measured flow vectors. (e) Classification of predicted forces following the cone rule. In adhesion-dominant regions the magnitude of the adhesion force component is color-coded. Positions with significant contributions from both adhesion and contraction (mixed zone) are indicated by grey dots. Insets e-I and e-II show regions of mixed adhesion and contraction forces, respectively. Mixed forces are further decomposed into a contraction component (blue) and adhesion component (magenta). Scale bar in a: 10 μm.
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Figure 2: Prediction of intracellular forces from F-actin network flow in Fig. 1. (a) Predicted force vectors (cyan: boundary forces; red: domain forces). Polygons I - IV highlight regions with different force characteristics (see text). (b) Flow field (yellow vectors) that would be produced by the predicted forces. Vectors are overlaid onto the color-coded speed map of the measured network flow. Insert: Comparison of the calculated (yellow) and measured (green) flow indicating the noise filtering during force reconstruction. (c) Cone rule to separate the contributions from adhesion and contraction to the predicted domain force. (d) Distribution of angles between predicted force vectors and measured flow vectors. (e) Classification of predicted forces following the cone rule. In adhesion-dominant regions the magnitude of the adhesion force component is color-coded. Positions with significant contributions from both adhesion and contraction (mixed zone) are indicated by grey dots. Insets e-I and e-II show regions of mixed adhesion and contraction forces, respectively. Mixed forces are further decomposed into a contraction component (blue) and adhesion component (magenta). Scale bar in a: 10 μm.

Mentions: F-actin flow fields were recorded with multi-fluorophore speckles, allowing the measurement of F-actin flow gradients over sub-micron distances17. Using the continuum mechanical model and numerical force inference discussed in Supplementary Notes 4 and 5 we predicted maps of intracellular force transients (Fig. 2a). These maps indicate on a relative scale force variations between different cellular locations and between time-points. Inference of absolute force levels would require measurements of the elastic properties of lamellipodial and lamellar F-actin structures. No method exists to accomplish this at the length scale of flow gradients. Nevertheless, relative force levels were sufficient to examine the modulation of contraction, adhesion and boundary forces during protrusion and retraction events.


Fluctuations of intracellular forces during cell protrusion.

Ji L, Lim J, Danuser G - Nat. Cell Biol. (2008)

Prediction of intracellular forces from F-actin network flow in Fig. 1. (a) Predicted force vectors (cyan: boundary forces; red: domain forces). Polygons I - IV highlight regions with different force characteristics (see text). (b) Flow field (yellow vectors) that would be produced by the predicted forces. Vectors are overlaid onto the color-coded speed map of the measured network flow. Insert: Comparison of the calculated (yellow) and measured (green) flow indicating the noise filtering during force reconstruction. (c) Cone rule to separate the contributions from adhesion and contraction to the predicted domain force. (d) Distribution of angles between predicted force vectors and measured flow vectors. (e) Classification of predicted forces following the cone rule. In adhesion-dominant regions the magnitude of the adhesion force component is color-coded. Positions with significant contributions from both adhesion and contraction (mixed zone) are indicated by grey dots. Insets e-I and e-II show regions of mixed adhesion and contraction forces, respectively. Mixed forces are further decomposed into a contraction component (blue) and adhesion component (magenta). Scale bar in a: 10 μm.
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Related In: Results  -  Collection

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Figure 2: Prediction of intracellular forces from F-actin network flow in Fig. 1. (a) Predicted force vectors (cyan: boundary forces; red: domain forces). Polygons I - IV highlight regions with different force characteristics (see text). (b) Flow field (yellow vectors) that would be produced by the predicted forces. Vectors are overlaid onto the color-coded speed map of the measured network flow. Insert: Comparison of the calculated (yellow) and measured (green) flow indicating the noise filtering during force reconstruction. (c) Cone rule to separate the contributions from adhesion and contraction to the predicted domain force. (d) Distribution of angles between predicted force vectors and measured flow vectors. (e) Classification of predicted forces following the cone rule. In adhesion-dominant regions the magnitude of the adhesion force component is color-coded. Positions with significant contributions from both adhesion and contraction (mixed zone) are indicated by grey dots. Insets e-I and e-II show regions of mixed adhesion and contraction forces, respectively. Mixed forces are further decomposed into a contraction component (blue) and adhesion component (magenta). Scale bar in a: 10 μm.
Mentions: F-actin flow fields were recorded with multi-fluorophore speckles, allowing the measurement of F-actin flow gradients over sub-micron distances17. Using the continuum mechanical model and numerical force inference discussed in Supplementary Notes 4 and 5 we predicted maps of intracellular force transients (Fig. 2a). These maps indicate on a relative scale force variations between different cellular locations and between time-points. Inference of absolute force levels would require measurements of the elastic properties of lamellipodial and lamellar F-actin structures. No method exists to accomplish this at the length scale of flow gradients. Nevertheless, relative force levels were sufficient to examine the modulation of contraction, adhesion and boundary forces during protrusion and retraction events.

Bottom Line: Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s.Maximal F-actin assembly was observed about 20 s after maximal edge advancement.On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
We present a model to estimate intracellular force variations from live-cell images of actin filament (F-actin) flow during protrusion-retraction cycles of epithelial cells in a wound healing response. To establish a mechanistic relationship between force development and cytoskelal dynamics, force fluctuations were correlated with fluctuations in F-actin turnover, flow and F-actin-vinculin coupling. Our analyses suggest that force transmission at focal adhesions requires binding of vinculin to F-actin and integrin (indirectly), which is modulated at the vinculin-integrin but not the vinculin-F-actin interface. Force transmission at focal adhesions is colocalized in space and synchronized in time with transient increases in the boundary force at the cell edge. Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s. Maximal F-actin assembly was observed about 20 s after maximal edge advancement. On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.

Show MeSH
Related in: MedlinePlus